Neuroscience PDF

NEUROANATOMY Be able to describe the nervous system’s macroscopic anatomy including major core areas and pathways  Be able to describe blood supply to the brain and fluid circulation The veins that bring blood to the brain are a. carotis interna and a. vertebralis. The distribut...

NEUROANATOMY Be able to describe the nervous system’s macroscopic anatomy including major core areas and pathways  Be able to describe blood supply to the brain and fluid circulation The veins that bring blood to the brain are a. carotis interna and a. vertebralis. The distribution is made by a. cerebri anterior and posterior, coming from a. carotis interna and a. posterior, coming from a. vertebralis The ventricles are a series of interconnected, fluid-filled spaces that lie in the core of the forebrain and brainstream. They are filled with cerebrospinal fluid (CSF). It will flow from the lateral ventricles into the third, that will go through the midbrain and into the 4th, that will narrow to form the canal of spinal cord. The 4th also has perforations to allow the flow to the subarachnoid space. Be able to describe the principles that control axon growth and synapse formation A neuron that is being formed has a cell body, an axon and a growth cone that guides the direction of growth. The growth cone is a transient state that is only present in developing neurons. They are made by microtubules (the axon) and then the growth cone has filopodia, which are a dynamic structure that can retract and move thanks to an actin / myosin meshwork. The filipodiums will move and form based on attractive and repulsive cues, which are chemical substances that drive the growth cone in a specific direction. Attractive cues will cause the assembly in that direction, repulsive, will cause disassembly, all of these by signaling that cause the cytoskeletal remodeling. Types of axon guidance cues: Contact-mediated repulsion Contact-mediated attraction These are non-diffusible cues, like sempahorins that will bind to plexin (repulsive) and neuropilin (attractive), and then act on rho/ GAPs, they can have a attractive or repulsive effect. A final class of non-diffusible axon guidance molecules includes a large family of ephrin ligands and their tyrosine kinase receptors (Eph receptors, or Ephs) that constitute a cell–cell recognition code in a variety of tissues (see Figure 23.4D). In the developing nervous system, immature axons use ephrins and Eph receptors to recognize appropriate path ways for growth as well as appropriate sites for synaptogene sis (see below). Although identified as ligands and receptors, the binding of ephrins with Ephs can initiate “reverse” signaling via the ephrins, which can interact with cytoplasmic protein kinases. Ephrins and Ephs activate a variety of signaing pathways and, depending on the nature of signal trans duction, can be either growth-promoting or growth- limiting. To limit axon growth, the extracellular domain of an ephrin ligand can be proteolytically cleaved, or Eph receptors can be removed via selective endocytosis, thus terminating signaling. Chemoattraction Chemorepulsion These two are mediated by diffusible signals like neutrin (chemoattractant), that binds to receptor DCC or slit (repulsive) that binds to robo and then these intracellularly acts on Rho/ GAPs that remodel actin. The commissural axon is going to have to cross the floor plate on ventral midline, by expressing receptors for netrin (attractant) , after it is going to express receptors for slit and semaphoring (repellent). Slit will be in the floor plate and semaphoring in the wall of the neural tube ensuring that the axon travels in between this space to the brain. Basically, how does an axon find its target? Axon guidance cues ➔ stimulate receptors on growth cone ➔ intracellular signaling cascade that locally alters cytoskeleton ➔ growth cone altered behavior ➔ axon directed to correct target. Trophic factors In the absence of synaptic partners, the axons and dendrites of developing neurons typically atrophy and often die. This dependency is referred to as trophic interaction, and is based on signaling molecules produced by target cells in small quantities, neurotrophic factors. They regulate differentiation, growth, and ultimately survival. Specific for neurons and neuronal cell targets. The amount of this survival factor adjusts the number of nerve cells to size of target. Some regulate synaptic plasticity, others regeneration and repair, others cell survival, other signals for apoptosis or degeneration and others inhibit neurite outgrowth. CELLULAR NEUROBIOLOGY  Be able to describe the function and importance of glial cells and neurons Neurons Neurons have a cell body, called a soma, from with you get various dendrites, that are like spines and that are input structures(electric signal converted to chemical signal) , you also have the axon, which is the output structure (offers a electrical signal that will be converted into chemical signal by the postsynaptic cell). The axon is long and it is where the signal propagates. In order to be fast, you have myelin, which has interruptions that are important to recharge the electric signal (Node of Ranvier). Myelin is produced by oligodendrocytes. Types Bipolar neurons: These neurons have two extensions: one axon and one dendrite. They are relatively rare and are primarily found in sensory systems, such as in the retina of the eye and in the olfactory system (smell). Unipolar neurons: These neurons have a single extension that splits into two branches. Unipolar neurons are typically found in invertebrates, but in humans, they are often used to describe sensory neurons in the peripheral nervous system. Pseudounipolar neurons: Most sensory neurons are pseudounipolar, which means they only have one axon which is split into two branches. Multipolar neurons: Motor neurons have the most common type of ‘body plan’ for a nerve cell - they are multipolar, each with one axon and several dendrites. Glial cells Maintain the ionic mileu of neurons, they modulate the rate of nerve cell signal propagation and the synaptic action, they also provide a scaffold for some aspect of neural development (remove pathogens), and aid (or impede) recovery from neural injury, they provide a interphase between the brain and immune system and facilitate the connective flow of interstitial fluid through the brain during sleep. They are also important for glucose sensing. Astrocyte: maintain the chemical and ionic environment for neuronal signaling and participate in synapse formation by regulating synapse formation. Astrocytes have specialized extensions called astrocytic endfeet that surround blood vessels in the CNS, important for BBB, homeostasis, transport of nutrients and ions and blood flow control. Metabolic support. Contact with nodes of Ranvier. Interact bidirectionally with Oligodendrocyte Precursor Cell, oligodendrocytes and microglia. Oligodendrocytes. Most proliferative cell of the CNS. Generate myelin. OPC interact with many other cell types, particularly in disease. Make contact with nodes of Ranvier, receive synapses from axons and regulate synaptic function. Dynamic regulation of myelin production – fine tuning of neural circuit function. Organize axonal domains including nodes of Ranvier. Metabolic support to axons. Facilitate ion homeostasis (e.g. uptake of K+ ions) – action potential conductance. In the periphery they are Schwann cells. Microglia: resident immune cells of the CNS (macrophages), they regulate synaptic pruning (the name that the process of eliminating dendritic spines when not contacted in  development), clear apoptotic neurons and interact with multiple CNS cell types in health and disease. - Be able to describe and explain the passive properties of nerve cells and understand how they affect incoming signals Differences between signals: Receptor potentials: signals generated by sensory organs and their size depends on how strong the stimulus is. Action potentials: These are all-or-nothing electrical signals that travel along the axon to transmit information over long distances. Occur only when the membrane potential reaches a threshold due to sufficient depolarization. Have a fixed amplitude. APs occur only if the combined SPs (from multiple synapses) reach the threshold. APs are the output signals of the neuron, sending information to other cells. Synapse potentials: These are graded signals that occur at the synapse when a neuron receives input from another neuron. Are caused by the release of neurotransmitters from the presynaptic neuron, which open ion channels in the postsynaptic neuron. Have a variable amplitude depending on factors like: The amount of neurotransmitter released, The number of synapses activated, The strength of the synaptic connections. They can be excitatory (EPSPs) (moving the membrane potential closer to threshold) or inhibitory (IPSPs) (moving it further away). SPs act as the input signals for the neuron, deciding whether the membrane potential reaches the threshold for firing an action potential. Action potentials are responsible for long-range transmission of information within the nervous system and allow the nervous system to transmit information to its target organs. You can inject currents in the cell, if this is makes the membrane potential more negative (hyperpolarization), nothing major happens. They do not require any unique property of neurons and therefore called passive electrical responses. But if the membrane depolarizes (more positive) to a certain level, the threshold, an action potential happens. Importantly, the amplitude of the action potential is independent of the magnitude of the current used to evoke it; that is, larger currents do not elicit larger action potentials. If the amplitude or duration of the stimulus current is increased sufficiently, multiple action potentials. It follows, therefore, that the intensity of a stimulus is encoded in the frequency of action potentials rather than in their amplitude. Ion transporters and channels are responsible for ionic movements across the membrane. Transporters create ion concentration differences by actively transporting ions against their chemical gradients. Channels take advantage of these concentration gradients, allowing selected ions to move, via diffusion, down their chemical gradients. The negative membrane potential arises primarily because positively charged potassium ions (K⁺) leave the cell through leak channels. This movement creates an imbalance of charge: There is a high concentration of K⁺ inside the cell and a low concentration outside and K⁺ naturally diffuses out of the cell down this concentration gradient. As K⁺ leaves, it leaves behind negatively charged molecules (e.g., proteins) that cannot cross the membrane. This creates an electrical gradient, making the inside of the cell more negative relative to the outside. At some point, the electrical force pulling K⁺ back into the cell (due to the negative interior) equals the force of K⁺ diffusing out. This balance creates the resting membrane potential (~-70 mV in neurons).Thus, the negative membrane potential is mostly due to the efflux of K⁺ and the inability of negatively charged molecules inside the cell to leave. The proof that the resting potential is determined by potassium concentration gradient comes from the injection of different concentration of this, which depolarizes the cell a lot, unlike with sodium ions. The electrical potential generated across the membrane at electrochemical equilibrium—the equilibrium potential—can be predicted by a simple formula called the Nernst equation. This relationship is generally expressed as Nernst's Law (or Nernst Equation) is used to calculate the equilibrium potential for each individual ion based on its concentration gradient across the membrane. V = I * R; R = 1/λ Ohm's Law (V = I * R): V is the voltage (potential difference) across a membrane, I is the current (flow of charge), R is the resistance (opposition to the flow of current). R = 1/λ (where λ is the space constant): λ (the space constant) is a measure of how far an electrical signal can travel along a neuron before it decays significantly. It depends on the membrane resistance and the internal resistance of the neuron. A larger λ means a signal can travel farther before dissipating. Goldman-Hodgkin-Katz (GHK) Equation The resting membrane potential is calculated using the GHK equation, which considers the equilibrium potentials of multiple ions and their relative permeabilities: Where: Pion is the permeability of the ion (K⁺, Na⁺, or Cl⁻), The concentrations of ions are typically given in mM (millimolar). Passive membrane properties  Temporal properties - Membrane time constant = tau. Refers to the time that passes to charge the membrane to 63% of the maximum voltage or until it discharges to 37%. It is a property of the membrane. Temporal summation is the process by which multiple electrical signals (postsynaptic potentials) occurring at the same synapse over time combine to influence the postsynaptic neuron. Whether this leads to an action potential depends on the timing and strength of the inputs. Here's the distinction between long and short time summation => Long time constant: PSPs decay slowly → better temporal summation over longer time intervals. If the time between signals is shorter than the decay time of the membrane potential, the effects "pile up." => Short τ → PSPs decay quickly → effective summation only with high-frequency inputs. The total effect may not reach the action potential threshold unless inputs are strong or sustained. tao = Resistance (R) * Capacitor (1/G). Capacitor depends on surface as well as resistance so it will cancel this effect. If the neurons have similar specific conductance and capacitance, the time- constants will be equal regardless of the diameter. Capacitance is the ability of a system to store electrical charge. In the context of a neuronal membrane, membrane capacitance refers to the ability of the lipid bilayer to store charge on either side of the membrane. Spatial properties Dendrites are passive electrical cables. Dendrites can be conceptualized as passive electrical cables in that they transmit electrical signals, such as postsynaptic potentials, toward the cell body without actively boosting or regenerating the signal.: Voltage Decay: As the signal propagates through the dendrite, it attenuates due to resistance and capacitance within the dendritic membrane and the surrounding cytoplasm. This is described by the cable theory. Time and Space Constants: The degree of signal attenuation depends on the length constant (λ) and the time constant (τ): The length constant determines how far the signal can travel before it decays significantly. It is the distance until the signal is decreased to 37%. It is given by: Where Rm refers to membrane resistance and Ri or Ra to axial resistance. So a higher diameter, can keep the charge longer. Directional Propagation: Dendrites generally transmit signals passively toward the soma unless there are active mechanisms like dendritic spiking. This model assumes a lack of active properties (e.g., voltage-gated ion channels), which are often present in dendrites but may still play a secondary role compared to passive cable properties in some cases. - Be able to describe the active properties of nerve cells and the most important functions of ion channels Specialized membrane proteins that transverse the cell membrane and conduct ions. They recognize and select among specific ions. The flux of ions is passive and the direction is determined by electro-chemical driving force, not the channel. Non-gated: always open, they leak channels and maintain the resting membrane potential. Gated ion channels: open and close in response to a specific stimulus, the transition between closed and open states is called gating. They are in charge of synaptic and action potential. They can be: ligand – gated (transmitter substance and cause a synaptic transmission), phosphorylation-gated (cause an 2nd messenger mediated synaptic transmission), voltage-gated (they generate an action potential) and stretch or pressure gated (they generate receptor potential in mechano-receptors). The action potential is maintained throughout the entire axon, which means that there is an active current flow. A 65 mV depolarization of the 0 Inward −1 3 4 0 Capacitive current Delayed outward current Transient inward current 1 2 Time (ms) 3 4 membrane potential also produces a brief capacitive current, which is followed by a longer lasting but transient phase of inward current and a delayed but sustained outward current. Steps of an Action Potential with Added Details 1. Resting Potential (-70 mV): The neuron is stable, maintained by the sodium-potassium pump (3 Na⁺ out, 2 K⁺ in) and leak channels. Inside is negative relative to the outside. 2. Threshold (-55 mV): A stimulus causes depolarization; voltage-gated Na⁺ channels open if the threshold is reached. 3. Depolarization (+30 mV): Na⁺ influx (sodium enters) rapidly makes the inside of the cell positive. Voltage-gated Na⁺ channels open in a positive feedback loop, amplifying the depolarization. 4. Repolarization: Voltage-gated Na⁺ channels inactivate, stopping sodium influx. Voltage-gated K⁺ channels open, allowing K⁺ efflux (potassium exits), restoring the inside's negative charge. 5. Early Hyperpolarization (Repolarization Overshoot): As K⁺ continues to leave, the membrane potential briefly becomes more negative than resting potential (~-80 mV). This is due to slow closing of the K⁺ channels. 6. Afterhyperpolarization: This extended period of hyperpolarization ensures the neuron is less excitable and prepares it for the next action potential. Membrane potential returns to the resting state (-70 mV) with the help of the sodium-potassium pump and K⁺ leak channels. Refractory Periods These periods ensure that the action potential moves unidirectionally and controls how frequently action potentials can occur. Absolute Refractory Period: Occurs during depolarization and most of repolarization. The voltage-gated Na⁺ channels are either open or inactivated, so a new action potential cannot be generated regardless of the stimulus strength. Relative Refractory Period: Occurs during the afterhyperpolarization phase. Voltage-gated K⁺ channels are still open, and the membrane is hyperpolarized. A stronger-than-normal stimulus is required to generate another action potential because the membrane potential is further from threshold. How does the signal travel? 1. Depolarization at One Point: The opening of voltage-gated Na⁺ channels at one location causes local depolarization. This depolarization spreads to adjacent areas of the membrane. 2. Wave of Depolarization: The positive charge inside the axon from Na⁺ influx triggers nearby voltage-gated Na⁺ channels to open, causing the action potential to move along the axon. 3. Refractory Period Ensures Directionality: After depolarization, Na⁺ channels inactivate, creating a refractory period. This ensures the action potential only moves forward, not backward. 4. Saltatory Conduction in Myelinated Axons: In myelinated neurons, the action potential jumps between Nodes of Ranvier, speeding up conduction. Passive Current Flow Passive current refers to the movement of ions within the cytoplasm and across the membrane in response to the local depolarization. When an action potential occurs: The Na⁺ influx at one region of the membrane creates a local depolarization. This depolarization spreads passively to adjacent regions of the axon, reducing the voltage difference and bringing nearby areas closer to threshold. Passive current flow is fast but decays with distance (as described by cable theory). Active Current Flow Active current flow involves the opening of voltage-gated ion channels to regenerate the action potential. When the passive depolarization reaches the threshold in a neighboring region, voltage- gated Na⁺ channels open, causing an influx of Na⁺ and initiating a new action potential there. This regeneration prevents the signal from fading and ensures that it can travel long distances. Initiation and Spread (Passive): The initial depolarization spreads passively to neighboring segments. Regeneration (Active): Voltage-gated channels in the neighboring segments open in response to the passive depolarization, actively regenerating the action potential. - Be able to describe the presynaptic mechanism for transmitter release at the molecular level and the main classes of transmitters including their synthesis. On a basic level, an action potential causes the opening of calcium channels and the influx of these cause the application of transmitters, agonists, and antagonists. Types: (they can coexist and be co-expressed in the same) Small molecule neurotransmitter Neurotransmitters are located in vesicles in the axon, that will fuse with the membrane and be released. After being used, they will become the precursors and be recycled, entering the cell again, and an enzyme, which is synthesized in the soma and has travelled through the axon, will convert it into the NT, that can enter the vesicle again. A lot drugs act at the level of accumulation of NTs in the vesicles. How does the vesicle work? In the membrane there will be a V-type ATPase that using ATP will introduce protons, that can the be exchanged by the NTs, that goes in. This tranporter can be targeted by some drugs - Acetylcholine: in the terminal, the glucose will be transformed into pyruvate and then in the mitochondria to acetylCoA that can then react with choline and through a reaction mediated by choline acetyl-transferase form acetylcholine that will enter vesicles. After being released it will be, in the synapse, be degraded into acetate ad choline and then a transporter will absorb the choline so that it can be reused. The prolonged presenced of acetylcholine In the synapse will cause desensitization of the receptors, which can ultimately lead to death. - Amino acids (glutamate, GABA, glycine): glutamate. Glutamine will be transformed by glutaminase to glutamate that can then be put into vesicles and then released. Glutamate in the synapse will be reabsorbed by the glial cell that will transform it into glutamine again and then it will be reabsorbed by the presynaptic cell. Prolonged activation causes cell death. - Purines (ATP) - Biogenic amines: catecholamines (dopamine, noradrenaline, adrenaline), serotonin, histamine. Dopamine is synthesized by the substantia nigra and ventral area. Parkinson is caused by the degeneration of neurons here.  Peptide neurotransmitters: High molecular weight. They are synthesized as propetides in the ER and then mature in the Golgi (soma) and then leave as a vesicle travelling through the axon until the terminal. They can bind to ligand-gated ion channels (they open when the NTs bind) or GPCR (bind to a receptor that has the G protein and the subunit alpha of this one will bind to a effector protein that will open the channel). Ligand-gated ion channels: selective for positively charged ions (excitatory). Nicotine acetylcholine receptor, glutamate receptor. - In an EPSP: how long and the intensity will depend on the number of open channels. The influx in mainly of sodium and depends on the membrane potential and the concentration differences. How does this work: For example, for nicotine acetylcholine receptor, there are subunits that have a negatively inner pore that attracts positive ions (vice versa with negatively charged NTs). Glutamate ionotropic receptors: AMPA and kainite receptor (work as for nicotine acetylcholine receptor), and NMDA receptor. NMDA receptor: selective for sodium but also has a high permeability for calcium. The binding of glutamate does not normally lead to a flow of ion because of magnesium that blocks the passage. Magnesium will only be displaced with voltage, so it will be dependent on depolarization. This is related to long term potentiation. Also involved in LTP.  Selective for negatively charged ions (inhibitory). GABAa receptor, glycine receptor. These are ligand-gated chloride (Cl⁻) ion channels. When GABA binds to GABA-A receptors, the channel opens, allowing Cl⁻ to flow into the neuron. This causes hyperpolarization (makes the neuron’s interior more negative), making it harder for the neuron to reach the threshold for an action potential. Fast-acting and responsible for immediate inhibition. Targets of drugs like benzodiazepines (e.g., Valium) and alcohol, which enhance the inhibitory effect of GABA. 1. The type of receptor they bind to: o Neurotransmitters can interact with multiple receptor types (ionotropic or metabotropic), which may have different effects. o For example, GABA is typically inhibitory because it activates receptors that increase chloride ion (Cl⁻) influx, hyperpolarizing the neuron. o Conversely, glutamate is excitatory as it activates receptors that allow sodium (Na⁺) and calcium (Ca²⁺) influx, depolarizing the neuron. 2. Ion channel selectivity: o Excitatory neurotransmitters usually open channels that allow positively charged ions (like Na⁺ or Ca²⁺) into the neuron, causing depolarization and increasing the likelihood of an action potential. o Inhibitory neurotransmitters open channels for negatively charged ions (like Cl⁻) or promote K⁺ efflux, hyperpolarizing the neuron and reducing action potential likelihood. The molecular charge of the neurotransmitter itself is not the primary determinant of its excitatory or inhibitory role. Heterotrimeric G-protein: many receptors and many effectors. - Be able to describe different types of postsynaptic receptors and signal transduction mechanisms. - Electrical synapse: presynaptic and postsynaptic membrane are united by gap junctions and ions flow through them directly (these are called connexons, that are formed by connexins). Faster, low energy cost, no complex signals, little plasticity (Synapses exhibit minimal changes in response to activity or stimuli), bidirectional direction. - Chemical synapse: This are the ones that use NTs. Slower, High energy cost, highly complex signals, large plasticity, unidirectional direction. 1.- Action potential depolarizes the terminal 2.- Opening of calcium channels 3.- Calcium entry 4.- Fusion of SV with plasma membrane 5.- Release of NT and diffusion over synaptic cleft 6.- Activation of postsynaptic receptors. 7.- Uptake or inactivation of NT This creates a EPSP or IPSPs Storage of NTs: SV: small NTs, in the active zone, and it can fuse in low and high frequency stimulus Large Dense-cored Vesicles (LDV): peptides, outside the AZ, in high frequency stimulus. It will not release to synaptic cleft but the laterals. How do the vesicles fuse? In the vesicle you have synaptobrevin (v-SNARE) and syntaxin and SNAP25 in the target membrane (T-SNAREs). They will together drive the fusion of SVs with the plasma membrane. They will interact, bring the membranes together that will then fuse. The SNARE complex assembly is regulated by synaptotagmin 1, complexin, munc. The SNARE complexes will form a vesicle docks and synaptotagmin will bind. Once Calcium binds to this last protein, the plasma membrane will curve and bring the membranes together, leading to fusion. Using ATP, NSF (an ATPase) will separate the SNARE complex and then synaptobrevin will be recycled. The vesicles will be made with clathrin that will also be recycled. - Be able to describe and explain the properties of synapses and their importance for information processing in the CNS. Synaptic plasticity refers to changes in synaptic strength, can be: - Short-term plasticity: lasts for ms to min, and is based in presynaptic changes. Changes in synaptic strength that are caused by repetitive stimulation. Facilitation: increased NTs release due to presynaptic calcium ions accumulation. Depression: decreased NTs release due to lack of primed vesicles and /or feed-back inhibition. - Long-term plasticity: lasts for more than hours, pre and postsynaptic changes. First discovered in the hippocampus, with glutamate and the processes of AMPA and NMDA receptors. LTP is a process where the connection (synapse) between two neurons becomes stronger with repeated activity. It’s thought to be one of the main mechanisms underlying learning and memory. How does it happen? Here’s a simplified explanation: 1. Baseline communication: o When a presynaptic neuron releases glutamate, it binds to both AMPA and NMDA receptors on the postsynaptic neuron. o At this stage, only AMPA receptors are active because the NMDA receptors are blocked by magnesium (Mg²⁺). 2. High activity triggers LTP: o If the presynaptic neuron releases a lot of glutamate AND the postsynaptic neuron is depolarized (e.g., because of AMPA activation), the magnesium block on the NMDA receptor is removed. o This allows calcium (Ca²⁺) and sodium (Na⁺) to enter the postsynaptic neuron through the NMDA receptor. 3. Calcium signals changes: o The influx of calcium activates signaling pathways inside the postsynaptic neuron. o These pathways cause: ▪ Insertion of more AMPA receptors into the synapse, making the postsynaptic neuron more responsive to glutamate. ▪ Structural changes in the synapse, like growing new dendritic spines, which further strengthen the connection. 4. Result: After LTP, the same amount of glutamate release from the presynaptic neuron causes a bigger postsynaptic response (stronger synapse). Why is this important? LTP is like "saving" information in the brain. When synapses are strengthened, they are more likely to fire together in the future, forming the basis for learning and memory LTP occurs only in the pathway receiving tetanus, not in adjacent unstimulated pathways. One synapse, the one affected will be strengthened and not the rest. Associativity means that a weakly stimulated synapse can undergo LTP if it is active at the same time as a strongly stimulated synapse on the same post-synaptic neuron. How It Works: Imagine two inputs (synapses) onto the same post-synaptic neuron: Synapse 1: Receives a strong, high-frequency stimulus (sufficient to induce LTP). Synapse 2: Receives a weak stimulus on its own, which normally wouldn’t induce LTP. If Synapse 2 is active (releasing neurotransmitter) at the same time as Synapse 1 (when the post- synaptic neuron is depolarized due to strong activity), the NMDA receptors at Synapse 2 can open due to depolarization spreading across the neuron. This allows calcium entry and initiates LTP at Synapse 2, despite the weak stimulus. - Have knowledge about the mechanisms of action of toxins and diseases affecting synaptic transmission, including botulism, tetanus, myasthenia gravis, and nerve gas poisoning. - Botulism: reduced muscle tone, paralysis. Caused by food poisoning. BoTx acts at the neuromuscular junction, primarily inhibiting acetylcholine (ACh) release at motor neurons. This causes flaccid paralysis by preventing muscle contraction. BoTx acts locally in the neuromuscular terminal. - Tetanus: increased muscle tones. Caused by bacteria. TeTx targets interneurons in the central nervous system (CNS), especially inhibitory interneurons that release GABA and glycine. By blocking these neurotransmitters, TeTx causes spastic paralysis due to unchecked excitatory signals. Acts in inhibitory neurons - Myasthenia Gravis (Autoimmune disease): Myasthenia gravis is characterized by the production of antibodies that target and block or destroy acetylcholine receptors (AChRs) on the postsynaptic membrane at the neuromuscular junction.This reduces the number of functional AChRs, impairing synaptic transmission and leading to muscle weakness.Ptosis (drooping eyelids), diplopia (double vision), difficulty swallowing and speaking, and generalized muscle weakness. - Nerve Gas Poisoning: Nerve gases are acetylcholinesterase inhibitors, meaning they prevent the breakdown of acetylcholine in the synaptic cleft. This results in persistent activation of acetylcholine receptors, causing continuous stimulation of muscles and glands, leading to muscle spasms, excessive salivation, and other symptoms. Constricted pupils, muscle twitching, convulsions, respiratory failure, and potentially death due to paralysis of respiratory muscles. - Have knowledge of methods used to register activity in neurons. - Glass microelectrode method: you insert a pipette filled with electrolyte solution into the interior of an axon to measure the difference between outside and inside the cell. They can then measre voltage changes. - Voltage clamp: A voltage clamp is an experimental technique used to measure the ionic currents that flow through a membrane while controlling the membrane potential. The device "clamps" the voltage at a set value and allows researchers to observe the relationship between membrane potential and current. This technique is crucial in understanding the behavior of ion channels, as it helps isolate specific currents by maintaining a constant voltage across the membrane. - Patch clamp technique: allows the registration of the incredibly small electrical currents that passes through a single ion channel. This technique is unique as it records how a single channel alters its shape and controls the flow if current within a timeframe of a few millionths of a second. Contemporary electrophysiology: Passive -- Surface electrode -- Extracellular electrodes Active -- Sharp electrodes --Patch clamp electrodes: can measure activity of single neurons, individual ion channels, synapses, intracellular signaling pathways. Materials: microscope, anti-vibration table, micromanipulators, fine pipettes, chlorinated silver wire, bath electrode, differential amplifier, oscilloscope, perfusion system, specialized software. The primary goal of the patch clamp technique is to measure the ionic currents through single or a few ion channels while maintaining control over the membrane potential. The "patch" refers to a small section of the cell membrane that is isolated for study. The voltage across this membrane patch is controlled, and the current that flows through the membrane can be measured. Patch clamping can be used to: 1. Measure Single-Channel Currents: Detect tiny currents flowing through individual ion channels. 2. Study Channel Conductance: Investigate how the opening and closing of ion channels contribute to cellular function. 3. Characterize Ion Channel Properties: Evaluate factors such as ion selectivity, gating kinetics, and pharmacological sensitivity. 4. Examine Membrane Excitability: Study the electrical activity of excitable cells like neurons and muscle cells. Technique 1. Seal Formation: A glass pipette with a very fine tip (often less than 1 µm) is used to form a high-resistance seal with the membrane. This step is crucial to ensure that currents measured come only from the patch of membrane in the pipette and not from the rest of the cell. 2. Patch Isolation: After a successful seal, the patch of membrane can be excised (cut) from the cell membrane, or the pipette can be kept in contact to measure currents in response to controlled voltage steps. 3. Current Measurement: Once the patch is isolated, the membrane potential is controlled using a feedback system, and the current is measured under various conditions, allowing for the examination of ion channel behavior in detail. 4. Data Analysis: The resulting current-voltage (I-V) curves are used to analyze ion flow through the channels, their conductance, gating mechanisms, and response to drugs or other factors. Types of Patch Clamp Configurations There are several different configurations of the patch clamp technique, each suitable for different experimental needs: 1. Cell-Attached Configuration:  o Purpose: Measures currents through ion channels still embedded in the cell membrane. o Method: The pipette is gently attached to the membrane surface without disrupting the cell membrane. o Application: Used to study channel activity in their native environment, often in living cells. 2. Whole-Cell Configuration: o Purpose: Measures currents from the entire cell, allowing the investigation of ion currents across the whole membrane. o Method: The pipette forms a seal on the membrane, and then gentle suction is applied to break through the membrane, allowing access to the interior of the cell. o Application: Useful for studying the overall cellular properties, such as membrane potential, action potentials, and ion channel function. 3. Inside-Out Configuration: o Purpose: Allows study of the intracellular side of the ion channel while maintaining control over the intracellular environment. o Method: After forming a seal and applying suction to isolate the patch, the membrane patch is pulled out of the pipette, exposing the cytoplasmic side of the membrane to the solution in the bath. o Application: Ideal for studying the regulation of ion channels by intracellular factors like calcium, phosphorylation, or second messengers. 4. Outside-Out Configuration: o Purpose: Allows study of the extracellular side of the ion channel. o Method: After isolating a patch of membrane in the cell-attached configuration, the pipette is pulled away, causing the patch to "re-seal," with the extracellular side now exposed to the bath. o Application: Useful for studying the effects of extracellular ligands, such as neurotransmitters or drugs, on ion channel function. 5. Perforated patch clamp o The perforated patch clamp technique is a variation of the traditional patch clamp that allows for the measurement of ionic currents while maintaining the integrity of the cell membrane. In this configuration, a membrane patch is isolated using a glass pipette, but instead of fully breaking the membrane to gain access to the intracellular environment (as in the whole-cell configuration), small pores are  created in the membrane. This method preserves the physiological properties of the cell better than the whole-cell approach, as it allows the cell to retain its natural ionic composition. Advantages of Patch Clamp 1. Single-Channel Resolution: It enables the study of single ion channels or small groups of channels, providing a high level of resolution. 2. Control over Experimental Conditions: By controlling the voltage and ionic environment, researchers can isolate and analyze specific channel behaviors. 3. Versatility: Patch clamp is adaptable for studying a wide variety of ion channels and cellular contexts. 4. High Sensitivity: The technique can detect extremely small ionic currents, making it sensitive enough to detect the activity of individual ion channels. Applications of Patch Clamp Neuroscience: Studying neuronal action potentials, synaptic transmission, and the effects of drugs on neurotransmitter receptors. Cardiology: Investigating ion channel behavior in cardiac cells to understand arrhythmias or the effects of antiarrhythmic drugs. Pharmacology: Screening for new drugs that modulate ion channel activity, particularly for conditions like epilepsy, pain, or cardiovascular diseases. Biophysics: Understanding the fundamental properties of ion channels, such as gating mechanisms, ion selectivity, and permeability. In summary, the patch clamp technique is a highly refined method that provides invaluable insights into the behavior of ion channels and cellular electrical properties, with various configurations suited to different experimental goals. It remains an essential tool in cell physiology, biophysics, and pharmacology.