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Electrophysiology is the branch of physiology that study the ion flows in biological tissues and refers to the techniques of the electrical recordings that enable the measurements of these flows. These techniques allow us to investigate cell physiology by measuring the changes in membrane potentials (Vm) and ionic currents in cells. Vm and ionic currents depend on subcellular and molecular structures including: Ion channels Synapses neurotransmitters Second messengers, protein kinases, etc… These elements are the basis of fundamental processes of the nervous system such as: The onset and propagation of action potentials Synaptic integration Synaptic plasticity and memory Main electrophysiology techniques: Electrophysiological studies can be applied to in vitro or in vivo preparations o cell cultures o brain and cerebellar slices o animal 1) Intracellular recordings using thin tip electrodes (sharp) - minimally invasive approach - accurate detection of membrane potential changes 2) Extracellular recordings of field potentials - non-invasive approach - measurement of field potentials 3) Patch-clamp recordings - invasive approach (limit) - accurate measure of membrane currents variations - direct access to the cytoplasm Electrophysiological recordings, what do we measure? potential (mV) potential (mV) Stim. EXTRACELLULAR Field Field potential 0.5 mV (represent the aggregate activity of small populations of neurons 2 ms represented by their extracellular tempo potentials) -70 Membrane -75 INTRACELLULAR -80 Sharp electrodes (mV) IN 0 -85 Membrane potential -90 -95 51400 tempo 51500 Time (ms) Single channel current (pA) 400 current(pA) Membrane 200 closed PATCH CLAMP 0 open Membrane current -200 Imemb (pA) /single channel current -400 tempo -600 495 Time (s) 500 505 Instruments Microscope PC Stimulation Unit Amplifier Rat coronal brain slice Electrodes sharp Recording electrodes PULLER (vertical type) 1. Intracellular recordings: very thin tip (sharp) electrode with high resistance Heating (30-100 MΩ) wire Sequence 1. Fasten 2. Heat 2. Extracellular recordings: low resistance 3. Pull / separate electrode (10-20 M Ω) patch 3. Patch-clamp recordings: low resistance electrode (2-5 M Ω) Pulling force Stimulating electrode Metallic current conductor https://youtu.be/2vI4fCsVdXc Post-synaptic “sharp” recording electrode excitatory potential Stimulating electrode To record Vm variations to evoke a post- synaptic event Field potential Extracellular recording electrode for field potential The patch clamp technique is used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells the voltage across the cell the current passing across the membrane is controlled by the membrane is controlled by the experimenter and the resulting experimenter and the resulting currents are recorded changes in voltage are recorded, generally in the form of action potentials The patch clamp technique is used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells provides information on how specific manipulations may alter specific neuronal functions or channels in real-time. significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. approach Seal formation micropipette resistance ≈ 4MΩ Cell attached 1GΩ Whole-cell allows to record the ionic currents that cross the entire membrane, carrying out a "voltage clamp“ Inside-out achieved by retracting the electrode. A fragment of membrane will come off together with the electrode, with the cytoplasmic face facing towards the solution present in the tray; this solution can be rapidly and repeatedly changed to assess the role of intracellular messengers in regulating the properties of ion channels Outside-out obtained by retracting the electrode. A fragment of plasma membrane is attached to the electrode with the external face facing the contents of the tray, and the cytoplasmic face towards the inside of the electrode. Advantage to easily change the composition of the medium present on the external face of the membrane, and study the effects of substances (agonists, blockers) that can influence the properties of the channels present in that patch. https://youtu.be/mVbkSD5FHOw Intra-pipette solution The whole cell configuration is the most used. Patch pipette It allows to record the ionic currents that cross the entire membrane, carrying out a "voltage Ion channels clamp" with a single electrode. Similar to the intracellular derivation technique. A low access resistance produces low noise controls. Cell membrane Many channels contribute to the signal. cytoplasm A certain type of channel is isolated with voltage and drug protocols. the intra-pipette solution will flow into the cell and will balance the cytosolic solution The solution that fills the electrode must be such as to perturb the intracellular ionic composition as little as possible. It is possible to apply compounds or drugs directly into the cell Voltage clamp Intra-pipette 400 Patch pipette current(pA) solution 200 Membrane 0 Ion channels -200 Imemb (pA) -400 -600 495 Time (s) 500 505 time Cell current clamp membrane -70 cytoplasm potential (mV) -75 -80 Membrane (mV) IN 0 -85 -90 -95 51400 51500 Time (ms) time Voltage clamp: the membrane is "clamped" at a desired constant voltage and the signal recorded is the current flowing through all the open channels at that membrane potential Current clamp: the current injected is “clamped” at a desired constant value and the signal recorded is the change in the membrane potential +20 pA outward Voltage clamp 400 current(pA) 200 Membrane 0 pA 0 0 pA -200 Imemb (pA) -400 -600 495 Time (s) 500 505 inward time -20 pA -70 current clamp potential (mV) -75 -80 Membrane (mV) IN 0 -85 -90 -95 51400 51500 Time (ms) time Voltage clamp: the membrane is "clamped" at a desired constant voltage and the signal recorded is the current flowing through all the open channels at that membrane potential Current clamp: the current injected is “clamped” at a desired constant value and the signal recorded is the change in the membrane potential Voltage clamp 400 current(pA) 200 Membrane 0 -200 Imemb (pA) -400 -600 495 Time (s) 500 505 time -70 current clamp potential (mV) -75 -80 Membrane (mV) IN 0 -85 -90 -95 51400 51500 Time (ms) time Voltage clamp: the membrane is "clamped" at a desired constant voltage and the signal recorded is the current flowing through all the open channels at that membrane potential Current clamp: the current injected is “clamped” at a desired constant value and the signal recorded is the change in the membrane potential Voltage-clamp recordings: holding potential and holding current The membrane potential Vm at rest (eg Vm = -65 mV) can be moved in the negative (Vm = -70 mV) or positive (Vm = -55mV) direction. The amplifier sends negative or positive current to the cell through the electrode to maintain a blocked Vm (voltage-clamp) at the chosen value. This value represents the holding potential (Vhold) and the current supplied is called the holding current (Ihold). With the fixed Vm (eg Vhold = -70mV) the amplifier measures and displays over time the current to be sent to maintain the established Vm. The transient opening of ion channels generates inward or outward ionic currents which add to the holding current causing a transient variation of the current recorded over time. Time (sec) -150 -160 -170 Vhold= -70 mV -180 I hold variation I (pA) The opening of the ion channels determines an inward (or outward) ionic current measured as a variation of the Ihold. The current is free to vary but the membrane potential is fixed. 4 400 Opening and closing of ion channels and passage of a cationic current (Na+ or Ca2+) 200 16.458 s 0 3.7 pA -200 holding current (Vhold = -60 mV) Imemb (pA) -400 -600 The holding current sent by the amplifier becomes more -800 negative to balance the incoming positive charges and keep the membrane potential constant (Vhold = -60 mV) -1000 495 500 505 Time (s) Effect of agonists and antagonists of membrane receptors administered in the extracellular environment Membrane receptors activation is associated with the opening of ion channels. It is possible to apply selective blockers in the extracellular medium or directly in the cell, through the recording electrode, to block receptors or intracellular enzymes. Receptor blocker agonist Short and repeated I(pA) 0 applications of a Glutamate receptor agonist evoke repeated transient currents -250 The response is reduced in the presence of a blocker of this receptor -500 1 min 20 patch-clamp recordings and intracellular calcium measurement Intracellular Ca2+ variations can be measured by injecting a fluorescent indicator (Fura-2) into the cell via a pipette and following the trend of fluorescence as well as the variations of the membrane current over time Visualization of intracellular Ca2+ in cells containing fura-2 agonist Calcium concentration https://youtu.be/0xJ7D8QGUrM Calcium concentration https://youtu.be/OcNrykR-AUs https://youtu.be/d27-YSVOneo https://youtu.be/9fTARVi7YhQ https://youtu.be/yy994HpFudc Fura-2 Ex at 340 nm excitation at 340 (high Ca2+) and 380 nm (low Ca2+). emission at 510 nm Ex at 380 nm [Ca2+] depends on Ex340/Ex380 ratio, regardless of the concentration of fura-2 and other external parameters The Pf is the percentage of current carried by Ca2+. Method developed by Neher to quantify the calcium current passing through ligand-gated cation channels (permeable also to Na+ and K+) such as glutamate and nicotinic receptors. Cells are loaded with cell-impermeant Fura-2 through the patch pipette used to measure NMDA-evoked currents Recordings of fluorescence signals and whole-cell membrane currents were synchronized DF≈ D[Ca2+]i INMDA NMDA Qtot=∫INMDA lex=380nm The F/Q ratio value was then measured as the slope of the Ca2+ permeability: Pf=QCa/Qtot linear regression best fitting the F–Q plot Voltage dependence of the Ca2+ flux through NMDA receptor channels from Garaschuk et al., 1996 A. Fluorescent traces and corresponding whole-cell currents after applications of NMDA at different holding potentials as indicated. Each trace is an average of n = 4 consecutive responses. B. QNMDA, calculated as the time integral of the NMDA-evoked currents, and the corresponding decrements in F380 (AF380), plotted as a function of holding voltage (Vh). Note that there is significant Ca2+ entry even at positive holding voltages (+10 to +30 mV) during NMDA-mediated net outward currents. Measurement of Ca2+ fluxes through dendritic NMDA receptor channels from Garaschuk et al., 1996 A. pseudocolour fluorescence images illustrating NMDA receptor-mediated Ca2+ changes in the dendritic region of a CA1 pyramidal neuron. Images taken before (a) and during (b) the NMDA application B. NMDA-evoked membrane current (bottom) and dendritic F380 trace (top) corresponding to the images shown in A. The shaded area corresponds to the total charge that entered through NMDA-activated channels (QNMDA) Stimulation Recording Synaptic plasticity refers to the activity-dependent modification of the strength or efficacy of synaptic transmission at preexisting synapses. In other words, synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity. Synaptic plasticity is considered the most important neurochemical foundations of learning and memory Long-term potentiation (LTP) is a process involving persistent strengthening of synapses that leads to a long-lasting increase in signal transmission between neurons. It is an important process in the context of synaptic plasticity. LTP recording is widely recognized as a cellular model for the study of memory. Long-term depression (LTD) is an activity-dependent reduction in the efficacy of neuronal synapses lasting hours or longer following a long patterned stimulus. Long-term synaptic activity High frequency stimulation protocol (HFS): 3 pulse trains at 100 Hz Mg2+-free LTP HFS HFS 1.2Mg2+ LTD Tozzi et al. J. Neurosci. 2011;31:1850-1862 ©2011 by Society for Neuroscience General organization of the nervous system Peripheric nervous Central nervous system (PNS) system (CNS) Sensory component (afferent system) Sensitive input Integrative component Sends information of (Central) external/internal Integration Sensor Integrates peripheric environment information and produces a response Motor-autonomic component (efferent system) motor output Sends information to effectors Effector Central nervous system Spinal cord: receives sensitive information of skin, joints and muscles of limbs and body and controls their movements. Brainstem: bulb, pons, midbrain: receives sensitive information of skin, joints and muscles of the head and controls the movements. Cerebellum: implied in motor control. Diencephalon: Contains the thalamus (analyzes information directed to the cortex) e hypothalamus (regulates endocrine and visceral functions). Brain: Includes the cerebral cortices (frontal, parietal, occipital and temporal lobes) and subcortical structures: basal ganglia, hippocampus, amygdala Central nervous system 1. Afferent component 2. Integrative component peripheric nervous system 3. Efferent component skin receptors somatosensitive system 2 1 spinal cord 3 axon afferent and terminals efferent axons skeletal muscle Sensitive system It includes all the systems that transmit, analyze and interpret information coming from outside or inside the body in order to produce a conscious perception of the stimuli 1. transmission of the stimulus conscious perception 2. analysis of the stimulus of the stimulus 3. interpretation of the stimulus The sensory system includes: Receptors Primary sensory neurons Secondary sensory neurons Neurons of higher order Afferent pathway Receptor structures mean line transducers that encode stimuli Upper order cerebral cortex neurons coming from 1. external environment Third order (exteroceptors), neuron 2. internal organs (enteroceptors) Thalamus 3. joints and muscles brainstem (proprioceptors) Second order neuron Core of receptors transform stimuli into connection signals interpretable by the CNS motor activity spinal cord Graded potentials EPSP/IPSP Receptor and action potentials First order neuron Afferent pathway mean line Primary sensory neurons: Upper order cerebral cortex in relation to receptors, they neurons send information about the Third order characteristics of the stimulus to neuron the integration center through Thalamus the sensory afferent fibers. Second order brainstem Secondary sensory neurons neuron receive information from Core of connection primary neurons and send motor activity spinal cord them to third order neurons (thalamus) and then to the cerebral cortex Receptor area specific for that type of First order neuron sensitivity. Afferent pathway mean line The consciousness of a sensory Upper order cerebral cortex neurons stimulus is called sensation. Third order neuron Thalamus Sensory information from internal Second order brainstem neuron organs, joints and muscles does not reach the level of consciousness; Core of connection however, it produces responses for motor activity spinal cord maintaining homeostasis (visceral afferents) or for motor control (proprioceptive afferents). Receptor First order neuron Receptors based on their morphology can be divided into three classes: Type I receptors: formed by the bare terminals of a primary sensory neuron (tactile, muscular and articular receptors are of this type); Type II receptors: specialized cells in synaptic relationship with the termination of the primary sensory neuron (i.e. Ear receptors); Type III receptors: receptors that communicate with the primary sensory neuron by an interposed transmission cell (only in cones and rods retina). primary sensitive neuron Type I receptor stimulus class I free terminals primary sensitive synapse Type II receptor neuron stimulus class II specialized cells primary sensitive transmission cell neuron synapses Type III receptor stimulus rod class III Nature of environmental stimuli A stimulus is a specific form of energy that is able to produce the appropriate response of a receptor. Typical stimuli are: Electromagnetic: radiant heat or light Mechanic: pressure, sound waves, vibrations Chemical: acidity, molecules of different shapes and sizes Intensity and threshold All stimuli are characterized by intensity, the amount of energy able to interact with the receptor. For each type of stimulus there is a threshold intensity below which the receptor cannot be activated. Appropriate stimulation Each receptor is characterized by an adequate stimulus. An adequate stimulus is a type of stimulation that is able to activate the receptor with the lowest possible intensity, and therefore to produce a sensation. Example: A mechanical stimulus, as a strong compression of the eye, can evoke a visual sensation but that stimulus is not an adequate: the appropriate stimulus for the retinal receptors is light. In fact, light provides a much lower quantity of electromagnetic energy than the mechanical one, sufficient to activate eyes receptors. Based on the adequacy of the stimulus, the receptors are classified as: Photoceptors: Activated by light, involved in the visual sensation. Chemocetors: Activated by chemical stimuli, involved in the sense of taste and smell and are stimulated by specific compounds in the blood and body fluids (O2, CO2). Mechanoceptors: Activated by physical deformations, involved in the senses of touch and hearing and can measure the stretching of a muscle or a tendon. Thermoceptors: Activated by the temperature, they detect heat. Nociceptors: Activated by harmful stimuli that are associated with the sensation of pain. Specificity of sensation The sensation is region-specific Any sensation (visual, acoustic, tactile, etc.) is region specific in the brain and it is determined by the connection of the activated receptor with the cerebral areas (code of the active line). A specific sensation is generated by neurons that are part of a certain cortical area. An expert of the sensory system stated: "If it were possible connecting the optic nerve with the auditory cortex and the acoustic nerve with the visual cortex, we would be able to see thunders and hear lightnings during a thunderstorm”. Stimulus transduction The stimulus applied to the receptor mechanisms in a nerve signal causes a modification of membrane permeability, due to the opening of ion channels (a graded potential). 1. The stretching of the membrane associated with mechanical stimuli causes opening of ion channels. 2. The application of heat (thermal stimulus) to the membrane causes opening of ion channels. 3. The interaction of chemical molecules (chemical stimulus) with specific membrane receptors causes direct ion channel opening or mediated by the production of second messengers. Receptor potential Change of membrane permeability in the receptor causes a variation of its electric membrane potential Vm (depolarization, in almost all receptors), which is named receptor potential. The receptor potential is a graded phenomenon (its amplitude depends on the intensity of the stimulus) whose amplitude decreases with the distance as it propagates on the membrane surface. The receptor potential triggers the action potential in sensitive neurons (sensory nerve fibers). Amplitude and duration of the stimulus determine the amplitude and duration of the receptor potential. The receptor potential depolarizes the trigger zone of the primary sensory neuron generating the AP (reaching the threshold potential). The frequency and duration of AP discharge is proportional to the intensity and duration of the receptor potential (and therefore of the stimulus). The APs, propagating along the axon, determine the amount of neurotransmitter released from the terminal. Through this mechanism the peripheric nervous system sends information to the CNS about the characteristics of the stimuli (intensity and duration) and consequently the CNS can grade the responses associated with these stimuli. Receptor adaptation Adaptation is the reduction of the response of a receptor as the stimulus persists on that receptor. The adaptation can be: Fast: the response is present only at the beginning and at the end of the stimulation. Slow: the response is present for all the duration of the stimulation but tends to be reduced in amplitude. Rapidly adapting receptors (phasic) are used to signal conditions that change over time, because they are deactivated when the stimulus reaches constant intensity and are reactivated if the intensity changes. Slow-adapting receptors (tonic) are used to signal conditions that are maintained over time. Tonic receptor Phasic receptor tonic firing firing at the beginning and the end Receptive field of a sensory neuron is the peripheral area that if stimulated is able to activate that neuron. Primary sensitive neuron A primary sensory neuron is connected with many receptors. The specific area from which the receptors detect information is the receptive field of that neuron. Peripheric terminals of the primary sensitive neuron Receptive field: Is the peripheric area innervated by that specific sensitive neuron Receptors Receptor field of a secondary sensory neuron Many primary sensory neurons project to the same secondary neuron (convergence), thus the receptive field of the secondary neuron is the sum of the receptive fields of all the primary neurons that converge on it. Receptor field of a tertiary sensory neuron Receptor field of a Primary sensitive neuron primary sensitive neuron Secondary sensory neurons Third order sensitive neuron of the sensitive cortex Receptor field of a cortical sensitive neuron Receptor field of a secondary sensitive neuron Many secondary neurons project to a tertiary neuron whose receptive field is the sum of the receptive fields of all secondary neurons that activate it. If this neuron is a neuron of the sensory cortex, the magnitude of its receptive field determines the sensitivity of that area to a stimulus. Sensory information Primary somesthetic cortex Vision Taste Hearing Smell Sensitive Homunculus In the primary somatosensory cortex, the extent of peripheral areas is inversely proportional to the size of the receptive fields of the neurons. The smaller and more in number are the receptive fields, the larger will be the cortical areas involved. Face: large areas related to lots of small receptive fields (lots of neurons receive inputs from that area. Foot: small areas because the receptive fields are large and involve a low n. of neurons. SOMATO-SENSORY SYSTEM Allow the transmission of information of the following sensory modalities: Cerebral Touch-Pressure by mechanoceptors with rapid or cortex slow adaptation, located below the skin, respond to mechanical stimuli, light (caressing) or intense Thalamus (deformation). Proprioceptions mediated by proprioceptors Brain localized in the muscles, tendons or joints stem (neuromuscular spindle and Golgi muscle-tendon organ). Motor output Spinal Temperature mediated by thermoreceptors, which cord signal the temperature of the skin to the CNS. Receptor Nociception mediated by nociceptors, which respond to mechanical, thermic or chemical stimuli able to produce real or potential tissue damage. TOUCH-PRESSURE Fast- and slow-adapting mechanoceptors: bare or encapsulated nerve endings: Meissner corpuscles (rapid), Pacini corpuscles (rapid), Ruffini corpuscles (slow), Merkel receptors (slow). rapid The nerve endings of sensitive tact C and pressure receptors use rapid myelinated type Aβ fibers E slow B slow D B, Merkel's disks: slow adaptating, allow the continuous contact of objects on the skin, pressure on the skin and spatial characteristics of the object. C, Meissner corpuscles: rapid phasic, sensitive to the movement of light objects on the skin and to low frequency vibrations. D, Ruffini’s corpuscles: poorly adapting, signal prolonged stimuli and intense tactile pressure (also present in the joint capsules). E, Pacini's corpuscle: rapid and very rapidly adapting, they detect high frequency vibrating stimuli, on-off response with stationary stimuli (also in muscles and joints). THERMOCEPTORS: receptors activated by the skin temperature. Sensitive to rapid variations of T (rapid adaptation). Classified in receptors for: Cold, active for T 30 °C maximum T activation approx. 43 ° C. T = 28-35 ° C zone of indifference there is no heat or cold sensation. AP frequency (n./sec) Skin temperature (°C) NOCICEPTION: process by which it is possible to perceive pain. From the periphery to cerebral cortex (where the perception of pain occurs) the nociceptive message crosses three main areas: the spinal cord the brain stem the thalamus NOCICEPTION: Nociceptors consist of free (bare) terminal endings of C type (not myelinic) and Ad type (myelinic) nerve fibers. Nociception is mediated by slowly adapting receptors (tonic) activated by stimuli capable of producing tissue damage. The damaged tissue can release chemicals that activate the receptor. NOCICEPTION Pathogenic steps: Descendant paths of 1. Transduction and control and gate control inflammation 2. Conduction Neurotransmitters (Glutamate, P-substance, 3. Transmission norepinephrine) 4. Modulation 5. Perception Aδ - myelinated fibers C - amyelin fibers PGE, bradykinin, P- substance, serotonin, histamine MECHANICAL, PHYSICAL, CHEMICAL.. Two main classes of nociceptors: Mechanical nociceptors part of small diameter, myelinated Aδ fibers, mediate the stinging and highly localized pain. Respond to intense mechanical stimuli (skin puncture, crushing). Respond to thermic stimuli but with high activation threshold. Polymodal nociceptors part of amyelinic C fibers, mediate delayed, more widespread and long-lasting pain. Respond to all types of harmful stimuli: mechanical, thermic (T> 45 ° C) and chemical. Nociceptors Type of fiber Conduction Type and diameter speed Mechano-thermic receptors Polymodal receptors The anatomical differences between the two types of nociceptors (presence or absence of myelin) influence the speed and frequency of action potentials. Along the C type fibers, the velocity of APs is particularly low. Perceived pain intensity A nerve normally contains both Aδ and C fibers, so nociceptive stimulation induces a first (initial) pain and a initial pain delayed pain Time second (delayed) pain. Perceived pain intensity When Aδ fibers are damaged, only delayed pain appears. Time When C fibers are damaged, only Perceived pain intensity initial pain appears. Time Periferic tissue Spinal cord Transmission of nociceptive inputs from Spinal cord the peripheral neuron to the spinal cord (spinal synapse) requires excitatory mediators (ex. Glutamate, substance P, CGRP and neurokinin A). In the spinal cord inhibitory interneurons release opiates or GABA and inhibit spinal transmission both at the post- synaptic and pre-synaptic levels. GABA oppiacei R OP- A- R Secondary GA B CGRP CGRP-R Nociceptoràterminal projection NMDA-R of primary sensitive Glu neuron AMPA-R neuron SP SP-R NK1/2 NKA CENTRAL CONTROL OF PAIN The activation of descending inhibitory systems on nociceptive pathways leads to reduction of the intensity of painful sensations (strong emotions or stress). The main pathway originates in the periaqueductal gray matter (PAG) (midbrain), projects to the nucleus raphe magnum (bulb) and locus coeruleus which projects to the spinal cord. These projections activate the inhibitory interneurons in the spinal cord releasing endogenous opioids, inhibiting pain transmission. (endorphins: enkephalin and dinorphine, similar to morphine) Gate control hypothesis (spinal cord inhibition) https://kin450-neurophysiology.wikispaces.com/Pain+Modulation Stimulation of a tactile pathway (mechanoceptor-Aβ fiber) activates an interneuron that inhibits the neuron of pain sensitivity. Nociception is the result of an algebraic sum The intensity of the nociceptive information to the cerebral cortex is the modulation transmission result of all the excitatory phenomena (transmission through the ascending pathways) and inhibitors (segmental and descending modulation) that take place in the CNS. Physiological pain Physiological pain can easily be experienced in daily life by exposure to brief stimuli (for example, contact with a very hot or pungent surface). mechanic pressure It represents an alarm signal and it is aimed at the defending the body heat integrity by activating reflexes able to avoid harmful stimuli. inflammatory molecules A low intensity stimulus (S) applied to a healthy tissue does not reach the threshold necessary to evoke a response (R); A stimulus of greater intensity causes a response (physiological pain), which ends when the stimulus ends; A further increase of the stimulus intensity leads to a proportional increase of the response. The response ends with the ending of the stimulus. Under normal, physiological conditions, the sensory system is not sensitized. A low intensity stimulus does not cause pain while a higher intensity stimulus causes pain. Low intensity stimulation High intensity stimulation Activation of fibers A β Activation of C and Aδ fibers Non-sensitized system Non-sensitized system NO PAIN PHYSIOLOGICAL PAIN In pathological conditions (damaged tissue or with inflammation), the sensory system is sensitized. Low intensity stimuli cause pain perception. Low intensity stimulation High intensity stimulation Activation of fibers A β Activation of C and A d fibers Sensitized system Sensitized system PAIN (ALLODYNIA) PAIN (HYPERALGESIA) 1. A low intensity stimulus (S) applied to a damaged tissue (for example, inflamed) evokes an answer (R). The response remains for longer, beyond the end of the stimulus; 2. As the stimulus intensity increases, the response increase is exponential (both in intensity and duration); 3. As the stimulus intensity increases, the response increase is exponential. The response is even more prolonged than the previous situation. The two phenomena that characterize pathological pain are: HYPERALGESIA Accentuated perception of painful stimuli ALLODYNIA Perception of pain in response to non-painful stimuli They both depend on sensitization (or facilitation) of peripheric nociceptors and/or central neurons HYPERALGESIA and ALLODYNIA If the nociceptive fibers are repeatedly stimulated by production of local chemicals (damage/inflammation) they lead to sensitization (lowering of the response threshold to the stimulus). In these situations hyperalgesia appears: reduction of pain threshold and enhancement of painful sensations. Primary hyperalgesia affects the area of damaged tissue and depends on the sensitization of the primary sensitive neuron. Secondary hyperalgesia affects larger areas outside areas of damaged tissue and depends on the sensitization of the secondary sensitive neuron. In these areas appears allodynia, pain sensation resulting from harmless tactile stimulation. In hyperalgesia (primary) the main mediators of sensitization (lowering of the activation threshold) of peripheral nociceptors are: Prostaglandine Istamina TNFα Interleuchina-1 (IL-1) Sostanza P (SP) CGRP (Calcitonin gene- related peptide) Primary sensitive n. is sensitized in primary hyperalgesia Central sensitization represents the secondary hyperalgesia In these areas allodynia appears, Emergence of pain following harmless tactile stimulation. primary hyperalgesia Allodynia Secondary hyperalgesia appears when the secondary sensitive neuron is also sensitized REFERRED PAIN It is felt in superficial areas of Cuore the body, but generated by the Fegato stimulation of nociceptors located at the level of the internal organs. Appendice Examples: - pain in the left arm during a heart Stomaco Intestino attack tenue - pain of renal colic referred to the Colon Ureteri pubic area. Cutaneous stimulus (frequent) Primary sensory neurons Visceral stimulation (rare) Sensitive path ascending to the Secondary sensory somatosensory cortex neurone The “referred pain” is due to convergence of nociceptive stimuli, coming from the viscera, on the same neurons that receive nociceptive afferents from the body surface. This allows the cortex to detect only one location of the stimulus, which is normally the superficial one. Animal research is not a game…!!! All research projects that involve the use of vertebrate animals must be authorized by the Ministry of Health and carried out within authorized user estabilishments Rules aimed at the maximum protection of animals. The use of animals is allowed only when the person in charge of the research project is able to demonstrate the impossibility of achieving the desired results using scientific experimentation methods that do not involve the use of living animals Novelty: development, validation, acceptance and application of ‘’alternative methods’’ that make possible to avoid the use of animals in scientific experimentation KEEP IN MIND!!!!! Favored procedures: Require the least number of animals Use animals with the least ability to experience pain, suffering, distress, prolonged damage Offer the greatest probability of satisfactory results Have the most favorable relationship between harm and benefit studies are performed with microorganisms, cells, biological molecules or tissues outside their normal biological context. In vitro experiments fail to replicate the precise cellular conditions of an organism. Because of this, in vitro studies may lead to results that do not correspond to what occurs in a living organism. In vivo refers to experimentation using a whole living organism. Animal testing and clinical trials are major elements of in vivo research. Which animals are used for animal research? mouse rat rabbit gerbil monkey living organisms whose genetic material has been artificially manipulated in laboratory through genetic engineering food production scientific research industrial protein purification (including drugs) Vectors agriculture vaccines scientific research food production scientific research industry and pharmaceuticals new colors in plants scientific research. enhanced crops (herbicide tolerance or insect resistance) Genetic modification of an animal involves altering its genetic material by adding, changing or removing certain DNA sequences in a way that does not occur naturally Mammals are the best model organism for humans Transgenic animals Knock-in animals Knock-out animals animals bearing a foreign gene deliberately inserted into their genome animal models in which a gene sequence of interest is altered by on-for-one substitution with a transgene, or by adding gene sequences that are not found within the locus. The insertion of a transgene is typically done in specific loci. Knockin mice are used for the study of regulatory elements or causal gene of diseases. Animal models in which one or more genes have been turned off or "knocked out." Disruption of a gene of interest by deleting portions of the gene's DNA sequence or by replacing the gene with an altered sequence. Knockout mice are used to study what happens in an organism when a particular gene is absent. Neurological diseases Parkinson’s disease Epilepsy Ischemia Autism Multiple slerosis Alzheimer’s disease Parkinson’s disease massive degeneration of the dopaminergic neurons of the nigrostriatal pathway presence of Lewy bodies containing α-synuclein dramatic alterations of motor activity associated cognitive functions The identification of several genes whose mutations are causative of rare familial forms of PD has lead to the creation of genetic models of PD. However, none of these models turned out to be a perfect replica of PD. Parkinson’s disease ideal model of PD 1) A normal complement of dopamine neurons at birth with selective and gradual loss of commencing in adulthood; 2) Easily detectable motor deficits, including bradykinesia, rigidity and resting tremor; 3) Development of Lewy bodies; 4) If the model is genetic, it should be based on a single mutation to allow for the robust propagation of the mutation; 5) relatively short disease course of a few months, allowing rapid screening of therapeutic agents; Parkinson’s disease model mechanism advantages disadvantages 6-hydroxydopamine injection kills dopamine neurons presence of a quantifiable not characterized by the gradual loss of neurons nor by through generation of free motor deficit the formation of Lewy bodies radicals rotenone injection kills neurons by oxidative - progressive degeneration of large variability damage nigrostriatal neurons - presence cytoplasmic inclusions - appearance of motor deficits MPTP Generation of free radicals replicates all the clinical signs - acute or subacute process of PD - applicable only in primates - absence of Lewy-bodies α-synuclein expression Toxicity by α-synuclein - Dopamine neurons - applied in Drosophila melanogaster overexpression degeneration -absence of correlation between motor deficits and - Development of locomotor dopamine dysfunction disfunction with age - no differences in toxicity observed between wild-type - Inclusions that resembled and mutant α-synuclein expression Lewy bodies Transgenic mice α-synuclein overexopression - progressive loss of motor - absence of Lewy-bodies in substantia nigra overexpressing α-syn under toxicity function - it does not faithfully replicate PD the control of Thy-1 - Lewy-bodies accumulation promoter - dementia with Lewy bodies Parkinson’s disease Toxin-induced model: 6-Hydroxydopamine (6-OHDA) is a neurotoxic synthetic organic compound used to selectively destroy dopaminergic and noradrenergic neurons The classical method of intracerebral infusion of 6-OHDA involving a massive destruction of nigrostriatal dopaminergic neurons, is largely used to investigate motor and biochemical dysfunctions in Parkinson's disease. HOW to identify the correct brain’s region? Stereotactic surgery : a minimally invasive form of surgical intervention based on three main components: stereotactic planning system, including atlas stereotactic device or apparatus stereotactic localization and placement procedure STEREOTACTIC APPARATUS SKULL SUTURES The stereotactic apparatus uses a set of three coordinates (x, y and z). The mechanical device has head-holding coronal suture clamps and bars which puts the head in a fixed position (the so-called zero sagittal suture or origin). lamboid suture All the procedures must be performed under anaesthesia!!!!!! Fix the animal on the sterotactic apparatus Cut the skin, in order to see the skull Starting from the bregma move the Hamilton siringe in the right position Inject the substance Conditioned apomorphine-iduced turning test Apomorphine is a non-selective dopamine agonist which activates both D2 and D1 receptors. It also acts as an antagonist of 5-HT and α-adrenergicreceptors. Apomorphine-induced turning has been used to evaluate the extent of unilateral nigrostriatal denervation after 6-OHDA lesions LEFT lesioned RIGHT healty Over 200 turns: FULL! Apomorfine Behavioral test rotation test Pharmacological treatments injection sacrifice Electrophysiology Molecular analysis weeks Morphological assays days Is a small pre synaptic protein that in particular conditions, shows the tendency to aggregatein Lewy bodies. Lewy bodies represent the neuropatological hallmarks of the patology. In vitro model of PD a-syn oligo Incubation with α-syn oligomers cause an impairment of the LTP of striatal neurons In vivo a-syn-injected model of PD Protofibrils of α-syn are injected in the dorso-lateral striatum. Injection of α-syn impairs LTP 6 weeks post-injection 12 weeks post-injection Epilepsy is a neurological disorder in which brain activity becomes abnormal, causing seizures or periods of unusual behavior, sensations, and sometimes loss of awareness. Genetic model: Bassoon mutant mice Bassoon is an integral component of the presynaptic cytoskeleton that interacts with other proteins to organize the cytomatrix at active zones of regulated neurotransmitter release. Mice lacking the central domain of the Bassoon gene, display alterations in neuronal activity associated with the occurrance of spontaneous cortical seizures. Acquired (syntomatic) epilepsy Epilepsy develops after an Kindling Electrical induction electrically-induced status epilepticus Pilocarpine Epilepsy develops after an Kainate Chemical induction chemically-induced status epilepticus In vitro model: 0 Mg++ and bicuculline The activation of the NMDA receptors (Mg++ free ACFS) and the antagonism of the GABA receptors (Bicuculline) cause a major exitability of the synaptic transmission that results in the induction of epileptic-like activity. R S Is this true epilectic-like activity? R S Lafora Disease is a rare form of autosomic recessive myoclonic epilepsy caused by mutations in the genes EPM2A and EPM2B encoding for Laforin and Malin respectively Nitschke et al., Nat Rev Neurol, 2019 Lafora Disease is a rare form of autosomic recessive myoclonic epilepsy caused by mutations in the genes EPM2A and EPM2B encoding for Laforin and Malin respectively Nitschke et al., Nat Rev Neurol, 2019 Structural, functional and behavioral abnormalities similar to LD patients Development of Lafora bodies in different organs and brain regions (hippocampus, cortex, thalamus, cerebellum) 3 months 6 months 1 month 12 months WT VS Epm2aR240X Electrophysiological characterization of LD mice to identify time- and region-specific alterations that underlie the disease and drive its progression. Epileptic-like activity in Dentate Gyrus 12-month-old vs 3-month-old Epm2aR240X mice 0 Mg2+ + 0.1 μM bicuculline A B 8 mean number of PSs 6 *** *** 4 CTRL 2 R240X 12m R240X 3m 0 0 10 20 30 40 50 time interval (min) * p

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