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Ribbon Synapses<ul><li>In cone photoreceptors</li><li>Strong, electron dense</li><li>Also in auditory hair cells</li><ul><li>More circular in these</li><li>Structure attached to active zone that holds vesicles</li><li>Vesicles docked to the presynaptic membrane</li></ul><li>Specialised docking mechanism that is faster than a classical synapse</li></ul><div><br></div>
Ribbons in cone synapses<ul><li>Pedicle of cone - end of axon</li><li>Full of vesicles</li><li>Many vesicles specifically attached to the ribbons</li><li>Ribbons are actually plates, studded in vesicles</li><li>Specific filaments attaching vesicles to the ribbon and to eachother, alongside the presynaptic membrane</li><li>We don't know the identity of these specific filaments - it isn't actin</li><li>People have speculated they could be a motor protein that allow funneling to docking sites at the bottom of the ribbon</li></ul>
Comparing and contrasting ribbon structure to classical synapse<ul><li>Using high-quality EMGs, it seems like at the active zone, more vesicles at the ribbon synapse are attached to eachother and other structures by the filament</li><li>One way to mobilise a vesicle is to remove it from actin</li><li>Synapsin alleviates this bond through phosphorylation of tails, the vesicles become mobile</li><li>In ribbons, vesicles are not attached to actin, making them much more mobile</li><ul><li>KEY molecular difference - Ribbon synapses do not contain synapsin, as vesicles are much more mobile</li></ul></ul>
Ribbons in hair cells<ul><li>Same specific filaments connecting vesicles to eachother and ribbon</li><li>Circular structure in hair cells as opposed to plate-like structure in photoreceptors</li><li>We don't know why such different structures, but probably to do with function</li><li>Also found in the second order neuron involved in vision</li><li>In polar cells - also circular</li></ul>
The retinal circuit<ul><li>Signal in the retinal circuit flow either forward or laterally</li><li>All ribbon synapses are excitatory</li><li>Horizontal cells are inhibitory - modulating excitation</li></ul>
The voltage response in cones is just a few mV's<ul><li>Cone cells can pick up signals as small fraction of millivolt</li><li>Respons much faster than rod cells</li><li>EG: motion detection is better in bright light than dim light</li><li>Voltage response is just a few mV</li><ul><li>compared to depolarisation spike of 100mV, this is small</li></ul><li>Can transmit signals as small as a fraction of a millivolt</li><li>We know they're being transmitted as we can see them</li></ul>
ON and OFF responses originate in bipolar cells"<ul><li>The visual signal gets split into 2 pathways</li><ul><li>One is excited when the light goes on (on-centre bipolar cell)</li><li>The other is hyperpolarised when the light goes on (Off-centre bipolar cell)</li></ul><li>The glutamate receptor is metabotropic, acting through an amplifying biochemical cascade to control the ion cascade</li><li>Visual system breaks up visual scenes into areas where light intensity increases and areas where light intensity decreases</li><li>Splitting starts postsynaptic to the ribbon synapse - in receptors in off and on bipolar cells</li><ul><li>In off bipolar cells, the postsynaptic glutamate receptor is an AMPA receptor</li><li>So in the dark, glutamate is being released from the cone</li><li>This glutamate activates the AMPA receptor, which depolarises the boipolar cell in the dark</li><li>In the light, less glutamate is released, thereforethe off bipolar cell hyperpolarises as it receives less glutamate</li></ul><li>The effect of glutamate in reversing synapse is the opposite.</li><ul><li>Glutamate released in the dark closes cation channel</li><li>So when light comes on and the cone hyperpolarises, causing less glutamate to be released, the cationc ahnnels open in the on bipolar cell to then be depolarised.</li><li>GPCR which acts through amplifying bichemical cascade to control the opening and closing of ion channels</li></ul></ul><div><img src=""paste-bcc67afd7c00401219486e4c1a6048cae8292904.jpg""><br></div><div><br></div><div><img src=""paste-8a842681112f8b41aed5ea60e028135cca4ce254.jpg""><br></div>"
Separation fo rod signal and noise by thresholding<ul><li>Thresholding is thought to be essential from separating dim signals from noise</li></ul>
Rod cells can detect single photons"<ul><li>A single rod photoreceptor sucked into a glass electrode and a light flashed across it</li><li>The electrode reporting current across the rod cell</li><li>Light BLOCKS inward current, hyperpolarising the inside of the cell</li><li>light response HYPERPOLARISES</li><li>the amount of hyperpolarisation depends on light intensity</li><li>Application of very dim flashes shows probabilistic nature of transduction.</li><ul><li>From stimulus to stimulus, variable numebrs of photons are being absorbed</li></ul><li>This shows that photoreceptors are capable of responsing to individual photons</li><li>Extremely sensitive</li></ul><img src=""paste-a6393dca180da0ad4ae4472b934dc394473402ec.jpg""><br>"
Separation of rod signal and noise by thresholding"The fact that transmitted signals can be really small leads to the question - how are these signals differentiated from noise?<br><br><ul><li>Threshoding - the notion that the real signal is differentiated by applying a threshold</li><li>Only being transmitted once amplitude passes a threshold</li></ul><div><img src=""paste-60165572e47ea45c471f6b1d37fbcd1dfda23d49.jpg""><br></div><div><ul><li>Even in the dark, there is continuous glutamate release</li><li>What in the brain does thresholding?</li><ul><li>the threshold of postsynaptic neurons - voltage sensitive calcium channels.</li><li>This is thought to be an important factor in noise differentiation</li></ul></ul></div>"
ON rod-driven bipolar cells have very large synaptic terminalsbipolar cell extracted from retina of fish<br><ul><li>Can be large - up to 10 microns in diameter</li><li>This allows use in imaging / electrophysiology to understand ribbon function and see how the synapses operate</li></ul>
Continuous vesicle cycling at a ribbon synapse"<img src=""paste-001c6a3f1202b9d29d7c73eddccd1bd55b02fffb.jpg""><br><ul><li>FM-dyes</li><li>FM1-43</li><li>In the image:</li><ul><li>On the left the membrane is at rest</li><li>One to the right after depolarisation shows incr. in fluorescence within the terminal - vesicles which fuse with the membrane, taking up dye and becoming fluorescent</li><li>Following through the experiment over many minutes shows large incr. in fluorescence throughout the terminal</li></ul><li>Graph shows incr. in fluorescence against time</li><li>A-&gt;B fluorescence in absence of calcium</li><li>C-&gt;D continuous stimulation, incr. in fluorescence</li><li>Saturation is reached, meaning all vesicles have become loaded with the dye - no net change in fluorescence (D - steady state where endo- and exo-cytosis rates are balanced and every vesicle has got the label. The number of vesicles is equivalent to many times the terminal's surface membrane area)</li><li>We can see that this terminal is capable of maintaining high rates of release for long periods (continuous vesicle cycle)</li><li>This is a particular property of ribbon synapses</li><li>Important, as it reflects the key function of ribbon synapses - they have to be continually operating</li></ul>"
Three pools of vesicles"FM-dye technique short time-scale<br><ul><li>Patch pipette put onto synaptic terminal and membraen was depolarised for 5 seconeds</li><li>Before depolarisaiton, all calcium channels closed</li><li>For 5 seconds, channels were maximally open</li><li>Can see 3 distinct phases of fluorescence icnrease, reflecting three phases of exocytosis.</li></ul><div><br></div><div>1st phase - red - rapid (RRP)</div><div><ul><li>We think this reflects the fusion of the vesicles which are docked and primed ast the active zone under the ribbon</li><li>After release, we see a second slower phase</li></ul><div><br></div></div><div>2nd phase - green - slower (Reserve pool)</div><div><ul><li>Equivalent to 4 or 5 times as many vesicles</li><li>We believe that this reflects the reserve pool of vesicles which are attached to the ribbon behind the active zone</li><li>After continuous stimulation, the rate of exocytosis slows down and you see a third phase</li></ul><div><br></div></div><div>3rd phase - grey (Cytoplasmic)</div><div><ul><li>Limiting factor is the supply of new vesicles from the cytoplasmic reservopir to the ribbon</li><li>The pciture we form here is during continuous exocytosis, vesicles from the cytoplasm attach to the ribbon, travel down it and fuse at the surface membrane</li><li>This is called CONVEYOR BELT HYPOTHESIS</li></ul><div>This hypohtesis states that the ribbon is used as a conveyor belt to rapidly resupply the release sites at the plasma membrane</div></div><div><br></div><div><img src=""paste-4978d0214496fcc4d2e4ee29bffc83ac21faa571.jpg""><br></div>"
Monitoring exocytosis and endocytosis: the capacitance technique"<img src=""paste-b70d698f186d8516bd8b0fcdbca48cc3d3742e57.jpg""><br>Time resolution cannot be measured in electrophysiology<br><ul><li>to monitor exocytosis on shrot timescale, use capacitance technique:</li><ul><li>Patch pipette attached to synaptic terminal</li><li>Capacitance technique measures surface area of terminal</li><li>This is directly proportional to capacitance of the membrane</li><li>So when vesicles fuse, it incr. surafee area and therefore capacitance too</li></ul><li>The right graphs show capacitance recordings</li><li>A) responses to 2 pulses</li><ul><li>the forst is a weak activation of calcium current</li><li>Small incr. in surface area -&gt; small incr. capacitance</li><li>A second, longer pulse depolarises the membrane further, resulting i larger calcium current and larger capacitance incr. mirroring a larger number of vesicles fusing</li><li>The capacitance then falls, showing that it is not steady</li><li>This reflects endocytosis, as vesicles are retrieved from the surface, the surface area decreases.</li><li>This technique can monitor the fusion and retrieval of vesicles</li><li>Using this technique you can carry out a number of different experiments to investigate how rapidly vesicles can fuse and the kinetics</li></ul><li>B) black points represent incr. in fluorescence as a function of duration of depolarising pulse (time)</li><ul><li>First phase - rapid</li><li>2nd slower phase</li><li>See - Three pools of vesicles</li></ul></ul><div><br></div>"
Measuring the rapidly releaseable pool of vesicles"<div><br></div><div>L-type Ca channels open very rapidly in response to depolarisation<br></div><div><br></div><div>Exocytosis can be very fast</div><div><br></div><div>The ribbon synapse can maintain contnuous cycling at low rates and also very fast transient mode of exocytosis</div><div><br></div><div><img src=""paste-38c5985f260cdbe2c860ca18809984f06cfaaccb.jpg""><br></div><div><br></div><div>If the first pulse is longer (B), we see a large jump in capacitance, with the second pulse only having a small incr. as all Rapidly releasable vesicles have already been released by the initial stimulus</div><div><br></div><div>The graph below - black shows the response to the first pulse as a function of its duration</div><div><br></div><div>Show the whole pool is released in 10ms or so</div><div>release shows an exponential fit</div><div><br></div><div>the graph in the middle shows the rate of release when we maximally activate the calcium current - depolarising the membrane to -10mV and maximally activate inward calcium channel</div><div><br></div><div>We can ask how does this rate of exocytosis vary with different amplitude depolarisations?</div>"
The voltage-dependence of rapid exocytosis"Graph plots inward calcium current (continuous line) as a function of membrane potential - shown on x axis)<br><br>Activates at around 40mV or so<br><br>dots show rate of exocytosis as a function of membrane potential - showing that the rate of exocytosis is a very steep fucntion of memrbane potential - more than doubles within about 5 mV<br><br><img src=""paste-8c8dab8f9b51b08c63f53e27550acf80992b824a.jpg""><br><br>We can measure directly the rate constant of vesicle fusion and release as a function of membrane potential<br><br>- You can get measurable that changes the membrane potential at justa&nbsp; few millivolts<br><br>Key property of ribbon synapses - can respond to very small changes in membrane potential - smaller than sodium spikes that trigger exocytosis at normal synapses"
Vesicles at ribbon synapses are more mobile than conventional synapses"<img src=""paste-929d5dd7c29d9e2e732331e87bb742a08968a7ca.jpg""><br><br><ul><li>Total internal reflection fluorescence microscopy</li><ul><li>Visualise vesicles by loading with FM143 - dye washed off membrane</li><li>Bipolar cells isolated on oversliop</li><li>Image is footprint</li><li>We can look about 100nm into the terminal</li><ul><li>In real time, we can see dots (vesicles) getting brighter as they move closer to the surface membrane</li><li>Shows random motion of vesicles as they collide with the membrane</li></ul><li>But on the right, shows a FRAP-type experiment where the vesicles were bleached</li><li>After bleach, we see fluorescence return as new vesicles appear</li><li>There are areas where lots of vesicles appear, making a very bright dot - This is a RIBBON at a specific site on the presynaptic membrane</li></ul><li>Using the diffusion coefficient, we can estimate how rapidly or often they bump into the ribbon<br></li><li>Is it feasible that diffusion alone is sufficient to resupply the vesicles to maintain continuous vesicle cycling?</li><li>the calculations say YES IT IS</li><li>The diffusion coefficient seen at the ribbon is much higher than at normal synapses</li><li>We belive their diffusion is much faster as the vesicles do not attach to the actin cytoskeleton</li><li>This is as there is no synapsin at ribbon synapses</li><li>The rate of supply of the ribbon is able to account for the third, slowest rate of release</li><li>This allows quantification of processing using isolated neurons in a dish</li></ul>"
Imaging synaptic activity in vivo"<ul><li>Using larval zebrafish allows us to observe ribbon synapses operate normally in the retina</li><li><img src=""paste-16324a04a0dc3a5e59a5b11d22d95aca67fdb200.jpg""><br></li><li>Zebrafish have very large eyes - strongly visually driven</li><li>8 days after fertilisation, half the neurons of the zebrafish are in the retina</li><li>Multiphoton imaging allows imaging into the retina whilst delivering visual stimuli</li><li>What we see in the image is the synaptic terminals of bipolar cells</li><ul><li>Lines up in multiple Layers</li></ul></ul>"
Synapses transmitting the visual signal to the innner retina"To image terminals, we take Gcap (fluorescent calcium-sensitive protein)<br><ul><li>We tether Gcap to synaptophysin in the membrane</li><li>by localising it to synaptophysin, we are localising to synaptic terminals</li><li>This allows us to monitor calcium signal that activates the synapses</li><li><img src=""paste-98e7c42de8af5d5b21feff4ea05307ad84cc2128.jpg""><br></li><li>Looking at the movie, you can as a bar of light moves across the retina, it is mirrored by populations of bipolar cell synapses activating along the same direction</li><li>We can now ask how a population of synapses is encoding the direction of motion of the bar</li></ul>"
Imaging synaptic activity in vivo: glutamate release"<img src=""paste-3da6927f536c59b09498ed97f55a8df8559834fb.jpg""><br><ul><li>Fluorescent proteins have been ngineered to respond to glutamate - INTENSE GLU SNIFFER</li><li>It sits on the surface membrane and binds calcium - incr. in fluorescence</li><li>In the moving bar movie, the pattern of synapse activation mirrors the motion of the bar across the screen</li><li>On a small dendritic tree - ypu can see synapses flickering on and off as they release glutamate</li><li>Very direct way of observing synaptic transmission</li></ul>"
Optical detection of multivesicular release in vivo"<img src=""paste-c560d36f717ba8741c4d7df77fb8c326e8b0dd3f.jpg""><br>B) Homing in on one synaptic terminal<br><ul><li>Scanning it in a line across the terminal</li><li>We can see in the movie that, after delivery of a periodic visual stimulus (sinusoidal modulation of light intensity) bursts of fluorescence that reflect the fusion of vesicles releasing their glutamate</li><li>Now we can ask how individual synapses encode a visual stimulus</li></ul><div><br></div><div>On top right image, we can see two regions of glutamate release</div><ul><li>Along x axis - time</li><li>along y -axis - distance along the line through the terminal</li><li>Fluorescence intensity greyscaled</li><li>The 2 active zones shown in black and red</li><li>You can see that at the output of bipolar cell are jumps of glu sniffer fluorescence reflecting fusion of vesicles.</li><li>This active zone doesn't respond to every cycle of stimulus</li><li>Sometimes there's a failure</li><li>When it does respond, amplitude of signal varies</li><li>This shows the stochastic probablistic nature of synaptic function</li><li>Also shows the variation of vesicles released between cycles</li><li>Signal caused by vesicles (D) shows quantisation of synaptic transmission</li><li>We can see how the probability of vesicle release varies in time in a fixed stimulus</li><li>We can thenvary the stimulus and see how the number of vesicles vary as a function of contrast</li><li>We can see how visual stimulus in encoded by individual synapses</li></ul><div><br></div><div>Larger jumps in glu sniffer signal reflect the release of multipe vesicles</div><div><br></div><div>It seems that ribbon synapses support the process of multivesicular release</div><div>This means diffusion of multiple vesicles almost at exactly the same time - synchronised to within a fraction of a millisecond. We don't know how this works.</div>"
What are the cellular mechnisms underlying multivesicular release"The mechanisms are still unclear, but there are hypotheses.<br><br><img src=""paste-88dce000212a926306e27b91be1b2dd55ac9353b.jpg""><br><br>1. Reflects the synchronised release of vesicles that are docked and primed under the ribbon (blue in diagram)&nbsp;<br><br>Somehow the release of these vesicles is coordinated so they fuse basically at the same time<br><br>2. Another hypothesis is that first vesicles fuse to eachother whilst attached to the ribbon at and behind the mambrane.<br><br>This multivesicular compartment then fuses with the membrane to release everal vesicles worth of glutamate (in lower diagram)<br><br>We don't know which of these it is<br><br>Arrows on right diagram show anisternie? just at end of active zone<br>These support fast endocytosis"
Ribbon synapses in the retina<ul><li>NOT activated by spikes</li><li>Are modulated by graded changes in Vm as small as a fraction of a mV</li><li>Hyperpolarise to light</li><li>Are also capable of transient and very rapid exocytosis</li><li>Can also encode light through a process of multivesicular release (MVR)</li></ul>
Transmission of the generator potential through ribbon synapses"<img src=""paste-1f55a7baa3214a492112dfc18d530b60f9bf1e09.jpg""><br><ul><li>Ribbon synapses of hair cells</li><li>Similar structure to retinal ribbon cells - spherical</li><li>Cahneg in membrane potential is graded as sound vibration change the hairs at the top</li><li>Depolarisation adn hyperpolarisation around the average membrane potention</li><li>Depolarisation -&gt; vesicle fusion</li><li>Hyperpolarisation -&gt; reduces vesicle fusion</li><li>Each ribbon in the auditory hair cell connects to just one psot-synaptic auditory nerve fibre</li><li>1:1&nbsp; relationship</li><li>The importance of this is to realise that the triggering of spikes is therefore done by the activation of ONE synaptic input</li><li>Very different picture to most areas of the brain - where a spike is determined by 100's of inputs</li><li>Properties of ribbon synapses are directly reflected by spiking activity in the nerve fibre</li></ul>"
Phase locking"<img src=""paste-681b4241b07cf01312958a55f00fdce12ed4509e.jpg""><br><ul><li>Again, spikes don't occur for every cycle of the stimulus</li><li>but we also see that timing of the spikes is phase-lcoked</li><li>Occuring at exactly the same time relative to the stimulus</li><li>Interval between 2 consecutive spikes tells us accurately the period of the sound-wave</li><li>1/300 Hz in this example</li><li>Being coded very directly by the spikes in the auditory nerve fibre</li><li>But because the fibre doesn't spike for every cycle, how can we accurately hear the sound?</li></ul>"
Encoded frequency: phase locking"even though fibres don't fire for every cycle of the stimulus, we can hear sound accurately as there are several fibres attached to a single hair cell<br><br><ul><li>In mammalian cochlear, may have 3 fibres connected to one ahir cell</li><li>There are 1000s of hair cells</li><li>This means avg firing of many fibres shows accurate periodic modulation in avg spike rate - reroducing the trime-cource of the input stimulus</li><li>Encoding dpends very strongly on teh ability of the ribbon synapse to very accurately in time respond to the auditory stimulus</li><li>This ties in with the ability to release vesicles very rapidly</li><li>This is important to hear sounds over a few hundres Hz</li><li>No single nerve fibre can spike more than 500Hz</li><li>So whole populations can have an avg firing rate of kilohertz</li><li>This shows that timing of exocytosis from ribbons is also very accurate</li><li>time accuracy of a fraction of a millisecond</li></ul><div><img src=""paste-36f3b0a0f8acb861a0cf48823eca1202e6051ccc.jpg""><br></div>"
Is the ribbon a conveyor belt?"<img src=""paste-d67cd9779e54a0791b354e64335b5c590c5b258d.jpg""><br>Conveyor belt hypothesis<br><br><ul><li>Cross-sections show that vesicles might be able to move across the structure, as they're already attached.</li><li>It would be good to know what filaments are - if they're motor proteins or kinesins</li><li>But we don't know the identity of these filaments</li></ul>"
Depletion of vesicles that are docked without depletion of ribbon"<img src=""paste-b8a845e1cf604b5d3a0815c4b7a2e394be85468e.jpg""><br><ul><li>Ribbon synapses in photoreceptors in lizards</li><li>Left - single electron micrograph - vesicles shown in yellow</li><li>B - accumulation of many micrographed aligned to the ribbon - superimposed average pciture of where vesicles are on ribbon relative to plasma membrane</li><li>We see lots of vesicles attached to the ribbons some distance away from the membrane</li><li>Not a lot of docked vesicles or vesicles behind the ribbon in the dark</li><li>In the dark they continually release vesicles</li></ul><div><br></div><div><ul><li>In the light, we see that there are more vesicles near the active zone - as we have slowed down their release in the active zone - allowing an accumulation of vesicles just behind the active zone</li><li>This fits the conveyor hypothesis</li></ul><div><br></div></div><div><ul><li>If you remove calcium, shutting down all vesicle fusion, we can see that not only is there accumulation of vesicles on the lower part of the ribbon, but also many docked vesicles</li><li>This allows us to build a picture of the function of the ribbon being to supply vesicles to release sites at the active zone</li></ul><div>Very compatible with conveyor belt hypothesis</div></div><div><br></div><div>We don't know how, but we have evidence they move along the ribbon. But we don't know how fast</div>"
Damaging the ribbon: effect on synaptic transmission"Damaged the ribbon (Farley Fluorophor assisted liht activation)<br><br>Made a peptide that binds to the ribbon which is labelled with a fluorophor&nbsp;<br>We can now use this to cause photodamage by applying very bright lights which damages the ribbon through the energy absorbed by the peptide.<br><br>How does this affect exocytosis?<br><img src=""paste-e3b2416fc99a26a7ac67da31b48381b319291926.jpg""><br>Graphs show post-synaptic response of bipolar cell<br>Inward current caused by glutamate release<br><br><ul><li>In grey - normal stimulus in control - glutamate activated current&nbsp;</li><li>In red - first resposne after ribbon damage - reduction in amount of rapid release</li><li>In black - subsequent responses after damage, get even smaller after each activation, shwoing ability to resupply vesicles is reduced</li></ul><div>This is good evidence that the ribbon is involved in resupplying</div><div><br></div><div>A suboptimal aspect of this experiment is that this technique isn't sufficient to completely oblitorate the ribbon.</div><div><br></div><div>the electron micrographs shwo ribbons in control and after bleaching</div><div><br></div><div>damage of the ribbon - not complete removal</div><div><br></div><div>Another way to measure it would be complete removal of the ribbon, which has been done. A KO of the protein ribeye (major protein component of the ribbon)</div>"
The phenotype of a RIBEYE KO"<img src=""paste-ac54024786ed63bd45da967fde155f975e3127da.jpg""><br><ul><li>In mice</li><li>Consequences of this KO are we can't see ribbons attached to the membrane</li><li>Can see lots of floating vesicles, but not at active zones</li><li>At top: voltage clamp stimulus</li><ul><li>Goes from hyperpolarised at -70mV to depoalrised at -10mV</li><li>Black trace is calcium current</li><li>EPSC is black line bellow</li></ul><li>In control, strong activation of postsynaptic current is seen</li><li>In the KO, we see that the calcium current is similar to control, meaning calcium is still getting in</li><li>However, we see fusion has been almost completely removed - lack of EPSC.</li></ul><div>This shows that the ribbon has a key role in maintaining fast exocytosis at ribbon synapses&nbsp;</div>"
Design principles of for a ribbon synapse"<ol><li>Capable of supporting both fast and slow modes of exocytosis - limiting step being transport of vesicles from ribbon down to release site at plasma membrane. We suspect this may involve the conveyor belt mechanism</li><li>Fast and slwo modes of vesicle retrieval (endocytosis) after release. Retrieval is important in maintaining the reservoir of rapidly mobile vesicles. Continuous cycle of fusion and retrieval</li><li>Couple these throught he calcium signal</li><li>Fast refilling of the RRP (also driven by calcium)</li><li>High mobility of vesicles in the cytoplasmic pool to refill ribbon</li><li>Move vesicles on ribbon down active zone?</li><li><img src=""paste-27c443775d2c18422b211df22aefade494a284a6.jpg""><br></li></ol><br>"
Processe involved in the sensation of pain, temperature and touch"<ol><li>Transduction - detection of stimulus by sensory neuron -&gt; spinal cord</li><li>Transmission - second order neuron from spinal cord -&gt; brain to be perceived</li><li>Modulation - curtailed or exacerbated by touch, stress, anticipation etc</li></ol><div><img src=""paste-ba9512d7dd1cc346687a99f06563d238297066f8.jpg""><br></div>"
Defining Pain and its benefitsUnpleasant sensory, emotional and cognitive experiences provoked by real or perceived tissue damage and manifested by certain autonomic, psychological and behavioural reactions<br><br>Our ability to recognise noxious stimuli functions as a defence mechanism to avoid futher hard by the adoption of nocifensive behaviours<br><br>Noxious stimuli generate acute pain and patients who lack the abiliuty to perceive pain because of some hereditary disease have injuries, self-mutilate and unrealised infections
Pathological pain"Switching from acute to persistent / chronic.<br><br>Our understanding of the mechanisms that cause this switch are incomplete, and this is one of the reasions why pain is an area of unmet medical need<br><br>Persisten pain includes:<br>Hyperalgesia: pain intensity to a stimulus ois enhances<br>Allodynia: stimuli that would not normally be perceived as painful, then become painful (e.g. light feather touch)<br><br>Persistent pain is often caused by inflammation and is triggered by inflammatory mediator such as histamine and prostaglandins (Inflammatory pain).<br><br>It can also be caused by injury of pain sensing neurons and this is known as Neuropathic pain<br><br><img src=""paste-8ce4ad01548bb132669f0becfebfb41cdb269961.jpg"">"
The ascending pathway for detection and perception of pain"Cell bodies of primary sensory neurons (nociceptors) lie outside of spinal cord in DRG. Those that detect pain are A-delta and C fibres<br><br>their axons enter the dorsal horn of the spinal cord via the dorsal root, where they synapse with secondary relay neurons in a region of the spinal cord known as the Substantia Gelitanosa. (Transmission at this synapse is subject to modulation by inhibitory interneurons and descending pathway).<br><br>Axons of secondary neurons corss over and travel via the Spinothalamic tract to the Thalamus. Here they synapse with tertiary neurons, which project to somatosensory cortex<br><br>Perception of pain occurs in somatosensory Cortex. Sensation is on opposite side of brain to the injury site<br><br><img src=""paste-80006ea79e58120f2af82ffa66f267556b63b54d.jpg"">"
different parts of somatosensory cortex are mapped to distinct areas of the body"third order neuron projects from thalamus to the area of the somatosensory cortex that relates to the area of injury.<br><br>So this allow both concious awareness and localisation of pain/touch/temperature<br><br><img src=""paste-ffdec422c2178cb7dea312b4ff7ec9ab5d186e05.jpg"">"
Pathway for sensation of touch/pressure (low threshold)"The primary neuron, the A-beta fibre, enters the spinal cord and travels on same side of spinal cord (Lemniscal tract) to the Medulla oblongata before forming a synapse with the 2nd order neuron.<br><br>It is the axon of the 2nd order neuron that crosses over ot the other side of the spinal cord and then projects to the Thalamus where it synapses onto the 3rd order neuron<br><br>In the dorsal horn of the spinal cord, the primary neuron branches and forms a synapse onto an inhibitory interneuron which modulate transmission in the pain pathway<br><br><img src=""paste-389e65905424d777408038eda63b6f554f60855c.jpg"">"
summary of differences in route for sensing pain and touch"these differences can have clinical significance. Injuries that affect only one side of the spinal cord can affect touch but not pain and vice versa<br><br><img src=""paste-70929b268ad71d1d7742b589566b6904ef324ca5.jpg"">"
Nociception"Detection of noxious stimuli, defined as stimuli that can cause tissue damage. Can either be mechanical, chemical, heat (above 40 degrees) or cole (less than 10 degrees)<br><br>Sensed by specialised peripheral sensory neurons called nociceptors<br><br><img src=""paste-d122a9b8906aebdc0112131956290f128b577bca.jpg"">"
Nociceptors: A-delta and C fibresDifferent types of sensory neurons respond to different stimuli, e.g. chemical, mechano or thermal<br><br>C-fibres are unmyelinated and respons to all 3 types of stmuli with a high threshold. They carry information related to slow, poorly localised pain<br><br>A-delta fibres are myelinated and respond to mechanical and thermal stimuli. they mediate fast, well-localised sharp pain
Properties of mammalian peripheral sensory fibres"<img src=""paste-8ff3c21008634e53b45be3aef7324f0d0d04f8b9.jpg"">"
How do nociceptors respond to noxious stimuli?Activation of nociceptors requires that the stimuli depolarise peripheral terminals with sufficient amplitude and duration (generator potentials) to reach threshold for generation of an AP
Ion channels located in membrane of freee nerve ending open in response to noxious stimuli"<img src=""paste-c46ce373674b553bbe6b0fcaf61f37df31e42d1b.jpg"">"
There are a great many inflammatory mediators which act on their receptors at nerve endings of nociceptors"<img src=""paste-7b3f5012ff3ec418a2207897d04978835a61b321.jpg"">"
Nociceptors themselves also release mediators which act upon receptors on the surface (autocrine signalling)"<img src=""paste-26e9fed7690dd3d37d71d5c380f58b7679e0aec9.jpg"">"
Multiple ion channels and receptors are expressed by nociceptors and are involved in transduction"<img src=""paste-7d39b0280f23f55a820d6684a52b0e4880116384.jpg""><br><br>Nociceptors are very heterogeneous, differing not only in the channels and receptors that they express but also transmitters that they release<br><br>Shown at the terminal are receptor families that are involved in generation and modulation of receptor potential<br><br>If this reaches threshold, teh voltage gated channels in the axon, generate and conduct an AP<br><br>All of these receptors and ion channels are potential therapeutic targets for reducing pain (i.e. for analegsics)"
Nociceptors differentially express a variety of anatomical and biochemical markers leading to functional heterogeneity"The identification of molecules that contribute to heat-, cold-, mechanical and chemical-induced generator potentials has been achieved by characterisation of currents in native cells and the identification of genes encoding channels and receptors.<br><br><img src=""paste-eccaa60271d05b9e1b801c9f59830e5d73e96a4c.jpg"">"
Structure of TRP cahnnels"<img src=""paste-c4e70d3cfb5c2d3864d2c4709ba498f7d936b743.jpg"">"
TRP channels are activated by multiple different modalities including temperature"Chemical stimuli affect the temperature threshold for activating the channel.<br><br>EG: TRPV1 with a typical heat activation threshold of 42 degrees becomes active at 37 degrees when there is a decrease in pH. Its activation threshold reduces even further (to around 32 degrees) in response to inflammatory mediators<br><br><img src=""paste-ed237da61bc322903d4776b4903525c506d8ff5a.jpg"">"
Transduction of noxious heatBoth A-delta and C fibres respond to noxious heat. A-delta fibres rapidly activate and adapt whereas c fibres respond slower<br><br>Genetic deletion of TRPV1 in mice only partially reduces noxious heat sensitivity and has no effect on heat responsiveness of C fibres. Another TRPV channel subtype involved?? TRPV2 becomes activates at more than 52 degrees<br><br>Are TRPV1 receptors useful targets for the development of analgesics?<br><br>Desensitisation to topical TRPV1 agonists (eg capsaicin creams and pacthes) has been in clinical use for decades to treat chronic painful conditions like diabetic neuropathy<br><br>Potent small molecule TRPV1 antagonists have been advanced into clinical trials for pain relief but there are side-effects, EG: hypothermia and impaired noxious heat sensation
Transduction of noxious coldBoth A andC-fibres respond to cooling skin to 4 degrees. Spontaneously active fibres show enhanced activity with decreasing temp<br><br>KO mice experiments suggest TRPM8 is responsible for detection of innocuous cooling. Alos, behavioural tests inficate a role for TRPM8 in sensing noxious cold in inflammation models<br><br>Analgesis effects of cold temperature were also lost in TRPM8 KO mice in context of formalin-induced inflammation<br><br>TRPA1 might contribute to detection of nocious cold under oxidative stress conditions. Human genetics liunk variation in TRPA1 to variations in cold pain sensitivity
Sensation of mechanical stimuliPerception of innocuous and noxious touch relies upon mechanosensitive sensory neurons<br><br>Family of mechanosensitive ion channels called PIEXO channels are involved in sensing touch/pressure both external and internal<br><br>A-beta fibres have low mechanical thresholds and respond to light touch, and a subset of A-delta and C fibres have high mechanical threshold so respond to harmful stimuli
Identifying mechanosensitive ion channels"Patapoutian and Coste started with a type of mouse cell that is capable of transforming a tiny poke from a pipette into a measurable electric current. KO candidate ion channel genes, a different one in each batch of cells, and look for a batch that has lost touch sensitivity. This this way identified mouse gene Piezo1, (from the Greek word for pressure) and soon afterwards identified Piezo 2<br><br><img src=""paste-67a9fbffc38bdb80668e7ae6ef85a89012b4c407.jpg"">"
the piezo family of ion channels"Gene encodes a polypeptide that contains around 2500 amino acids and spand membrane 38 times (300kDa).<br><br>Three piezo proteins come together in a trimer that crosses the plasma membrane.<br><br>From the centra pore, three arms spiral out like propeller blades. they curve uo and out creating a deep divot in the surface of the cell<br><br><img src=""paste-8faa8316b717da739a87f633c70ffbf270b22f63.jpg"">"
Piezo 2 is the major transducer of mechanical forces for touch sensation in mice - Ranade et al. 2014"Generated a mouse expressing GFP-Piezo2 they demonstrated that Piezo2 expression is found in a broad range of LTMRs that sense mechanical stimuli relevant to touch sensation<br><br>Piezo2 deletion in mice leads to perinatal death 9but interestingly not in humans). They generated mouse line in which Piezo2 expression selectively KO in adult mouse sensory neurons (DRG neurons). Loss of mechanically activation, rapidly adapting currents recorded from these cells<br><br><img src=""paste-d9dded6b29091c17959b29b1cabd12cb3b73a1ce.jpg""><br><br>50% decrease in percentage of A-beta neurons showing mechanosensitivity for conditional KO<br><br>A-delta mechanonociceptors showed elevated mechanical threshold for activation, suggesting some role for Piezo2 in these neurons.<br><br>C fibres showed no change in threshold for mechanical stimulation<br><br>Behavioural test: detection of Von Frey Filament of varying forces to hindpaw, Greater force for CKO mice indicates loss of response to forces below 4g"
What about the role of Piezo2 in humans? Chesler et al. 2016"2 indivisual identified with LOF utations in Piezo2. Presented with neuromuscular disorders. The following were tested:<br><br><ul><li>Punctate-touch detection and pinprick pain using calibrated Von Frey monofilaments</li><li>Proprioception by measuring the detection of the vector of a 10-degree vertical angular motion at the following joints.</li><li>thresholds of thermal detection and pain using Peltier-based thermode. The stimulus temperature was decreased or incr. (in incraments of 1 degree from a baseline temp of 32 degrees), and the participants verbally indicated when they perceived cooling or warming and the shift from hot to painfully hot</li></ul><div><br></div><div><img src=""paste-414029bd47e0328a0cf9357de7a313055780fdbb.jpg""><br></div><div><br></div><div>Loss in vibration detection and touch discrimination in patients with LOF mutation in Piezo2</div>"
Piezo1 mediated injury-induced tactile pain in mice and humans Szczot et al. 2018"Piezo2 KO in large subset of snsory neurons in mice. Compare with control mice.<br><br>Calcium imaging of the trigeminal ganglion (using fluorescent Ca 2+ reporter) as a means to record activity by in vivo imaging from hundreds of sensory neurons responding to a range of sensory stimuli<br><br>Loss of response to gentle mechanical stimulation but not to higher intensity stimulation<br><br><img src=""paste-343762c52d9136b643fb7a879f11064421df8996.jpg"">"
Piezo2 is required for Tactile allodynia in humans"Tactile allodynia is evaluated using heat in combination with capsaicin cream to induce short-term but intense inflammation in humans (via activation of TRPV1). Occurs in both control group and individuals with LOF mutation in Piezo2. Map primary and secondary areas using fine brush.<br><br>Control group report light touch surrounding the inflamed areas as unpleasant and painful compared with the same stimulus applied to a neighbouring uninflamed area of skin.<br><br>Those with LOF mutation could not discriminate between two areas.<br><br>Could blocking Piezo2 prevent tactile allodynia?<br><br><img src=""paste-096dac5d9580d051bff72cd106282fa0e738c288.jpg"">"
Piezo2 mediates sensitivity to mechanical pain in mice (Murthy et al. 2018)Activation of Piezo2-expressing neurons evokes painful behaviour in mice<br><br>Expressing the light activated channelrhodopsin in Piezo2 expressing sensory neurons, showed that in response to incr. activity of these neurons, mice displayed nocifensive behaviours. eg paw licking<br><br>the behavioural response to noxious mechanical stimuli (eg: pinprick) was compromised in KO mice<br><br>The no. of mechanically insensitive A-beta fibres was higher in KO mice and although the number of A-delta and C-fibres with mechanical responsed was not affected,. the threshold for response in A-delta fibres was higher. Also, at the highest mechanical force used, both A-delta and C-fibres in KO mice had reduced activity
Mechanical allodynia is mediated by Piezo2"Acute inflammation model:<br><br>Capsaicin was injected intradermally causing transient inflammation.<br><br>Mechanical threshold from both von Frey Filament stimultion and brush stroke reduced in wildtype mice<br><br>In the Piezo2 KO mice there was no change in mechanical threshold<br><br><img src=""paste-1d31e6fbea0db26c8ea58228bc5423a9eece1b31.jpg"">"
Piezo2 expressing nocieptors mediate mechanical sensitisation i experimental osteoarthritis - Obeidat et al. 2023"In this study they selectively KO Piezo2 in mouse nociceptors (using NaV1.8 as marker) to examine its role in the development of mechanical sensitization in experimental osteoarthritis. Used in vivo recordings of DRGs using fluorescent calcium indicator (GCaM6f) to examine response to mechanical force applied to knee.<br><br>(check slide for slide info)<br><br><img src=""paste-f293136d375238b72f2b2d01ddb9bba0cd5212c8.jpg"">"
Depletion of Piezo2 from nociceptors protects mice from mechanical sensitisation associated with acute joint inflammation"<img src=""paste-74056a9655fabfa9cadc01717ef412f3c70246fc.jpg"">"
Piezo2 conclusionsPiezo2 is expressed by nociceptors, however its primary function on nociceptors doees not appear to be the sensation of high intensity mechanical forces under hea;thy conditions; rather Piezo2 appears to become sensitised in settings of inflammation and tissue damage
NTs and their receptors at synapse between primary and secondary neurons in pain pathwayA-Delta fibres release glutamate (fast NT) which binds and activates AMPA and NMDA receptor ionc hannels in post-synaptic membrane of secondary neuron<br><br>C-fibres also release glutamate (fast acting) primarily Substance P (acting at neurokinin 1 receptor, GPCR), also excitatory
Gate control theory (Melzak + Wall, 1965)'a gate control system modulates sensory input from the skin before it evokes pain perception and response<br><br>Ascending pain suppression model and the basis of why rubbing can reduce pain signals<br><br>The role of A-beta fibres in inhibiting pain transmission<br><br>Circuits involved in the dorsal horn, the role of inhibitory interneurons and the transmitters involved
A-Beta fibre conveys sense of touch/pressure (low threshold)"In the dorsal horn of the spinal cord, the primary neuron branches adn forms a synapse onto an inhibitory interneuron which modulate <br>transmission in the pain pathway<br><br><img src=""paste-5953d9a539cf45750d44de1d28e827185731751d.jpg"">&nbsp;<br><img src=""paste-b8dd77f4bd1a0c66dbe68785e83c6c189e7fb998.jpg""><br><br>A-delta and C fibres responding to painful stimulus<br><br>Rub area to reduce pain sensation"
A-beta touch fibre acts via inhibitory interneuron to reduce transmission between primary and scondary nociceptive neurons"Stimulation of A-beta fibres stimulates inhibitory interneuron to release GABA and enkephalins<br><br>These transmitters act pre- and post-synaptically to reduce transmission in the pain patwhay<br><br>Enkephalins act at opioid receptors (u and delta, both GPCRs) and GABA acts at GABA(A) and GABA(B) receptors.<br><br><img src=""paste-f3a93f739d9a999a6bcb609322a32491043684cc.jpg"">"
More connections involved"Inhibitory interneurons have basal activity which acts to suppress transmission in the pain pathway.<br><br>C-fibres suppress the activity of these interneurons whereas A-beta fibres potentiate the activity<br><br><img src=""paste-3d70c8426e0579beb0f3587db32e41089cdcde05.jpg"">"
More on ascending tracts"Other pathways involved in the modulation of the synapse between primary and secondary neuron<br><br><img src=""paste-96d1c9358651cee7a5baf11c5ea6be2497ef8eac.jpg""><br><br>A-Delta/C fibres synapse onto the secondary neuron, teh neuron crosses over to the other side of the spnal cord. Thn up through spinothalamic tract, then onto tertiary neuropn in the brain.<br><br>There are 2 pathways/tracts via which nociceptive informaiton can be centrally transmitted<br><br>In addition to the spinothalamic tract there is the spinoreticular tract"
The Spinoreticular Tract"Secondary neurons that ascend in spinoreticular tract project to a variety of limbic structures that regulate emotion<br><br>Include: parabrachial nucleus, amygdala, hypothalamus and thalamus, and from there information sent to various regions of the cortex&nbsp;<br><br><img src=""paste-7aa019e166826f8f4504d9fc3355eeb4fe04a1cb.jpg""><br><br>Branch points at PAG and RVM<br><br>These activate the descendign pathway leading to modulation of pain transmission in the dorsal horn"
The descendign pathway"Regulates synaptic transmission within the ascending pathway<br><br>PAG is output centre from limbic system receiving inputs from hypothalamus and amygdala in addition to activation of spinoreticular pathway<br><br>Neurons project from PAG to RVM and synapses onto neurons that project to dorsal horn at same region as primary adn secondary neuron synapse<br><br><img src=""paste-559306ffee73f0a431bafdbd8fabb213d09a397e.jpg""><br><br>Activation of the ascending pathway can block transmission"
Modulation by descending serotonergic and noradrenergic RMV neuronsdifferent types of inhibitory interneuron<br><br>Inhibitory interneuron releases GABa and enkephalins<br><br>There's another input into interneuron from the descending pathway.<br><br>NA and serotonin (5-HT) released from RVM neurons acts on GPCRs on interneurons to excite<br><br>the interneurons release enkephalin and GABA which act both on A-Delta / C-fibres to inhibit NT release, and postsynaptically on secondary neurons, to reduce excitability<br><br>In these ways pain sensation is reduced
Descending pathways: a role in masking of pain by another painful stimulusWhen two pain stimuli are applied to two differeent areas of the body, perception of the weaker of the 2 stimuli is inhibited<br><br>EG: in ancient times, burning hot metal tips were applied to points on patients bodies to relieve certain kinda of pain elsewhere<br><br>Cattle ranchers apply clamp to animal's nose to relieve pain of castration
Identifying a role for descending GABAergic RVM neurons in modulation of transmission in spinal cord"<img src=""paste-52516e940f5a8d161676e0b35fa00a2c5075a00a.jpg""><br><br>To identify enkephalinergic dorsal horn neurons, manipulate their activity and define their inputs, knock-in mice were generated in which the promoter of the preproenkephalin gene (Penk) drives Cre recombinase<br><br>this enables selective expression of protein of interest (in this case YFP) in neurons that use enkephalins as transmitter, Penk+<br><br>Used markers to co-label to identify a population of YFP labelled neurons that are glutamatergic (TLX3) and population that are GABAergic (PAX2)<br><br><img src=""paste-5e13006b912535ed5e06555cb752addb6156e782.jpg"">"
Use of chemogenetics to manipulate activity of Penk+ neurons"Express an engineered inhibitory GPCR in Penk+ neurons (hM4R). Upon addition of agonist (clozapine CNO, IP) to mice this inhibits the activity of these neurons. Can observe effects on mice behaviour.<br><br>Incr. in mechanical hypersensitivity with no effect of heat and light touch (disinhibition)<br><br>went on to show GABAergic Penk+ neurons mediate this effect<br><br><img src=""paste-9bba375c34aa573bacc8954156ee5a4ba5df7a11.jpg"">"
Which brain descending neurons engage the enkephalinergic spinal neurons?"Use of rabies virus to mediate retrograde trans-synaptic tracing<br><br>Neurons strongly expressing GFP were only identified in the RVM<br><br>~90% of GFP expressing RVM neurons are GABAergic<br><br>The release of GABa from the RVM neurons acts to suppress the firing of the inhibitory interneurons<br><br>It is a disynaptic inhibitory circuit<br><br><img src=""paste-4233cf58266ecb0d46e95564c677f0c1d978ab5d.jpg"">"
GABAergic RVM neurons facilitate nociception"Inhibition of GABAergic RVM neurons using selective expression of inhibitory receptor (hM4) showed that this produced mechanical hyposensitivity<br><br>Tonic activity in the GABAergic RVM neurons drives mechanical hypersensitivity<br><br><img src=""paste-06039cd7e8fac3aca60b34b288c4197d275db16f.jpg"">"
Use of optogenetics to identify how GABAergic RVM neurons regulate mechanical pain sensitivity"Selectively express excitatory channelrhodopsin 2 and inhibitory Halorhodopsin<br><br>Implanted optical fibre in lumbar spine allows light stimulation of one or other of these channels whislt monitoring mouse behaviour<br><br>Activation caused hypersensitivity and inhibition caused hyposensitivity (incr. mechanical threshold)<br><br>Hyposensitivity dependent upon enkephalins, blocked by naloxone<br><br>GABA released from RVM neurons acting via GABA(A) receptors to generate iPSC in Penk+ neuron<br><br><img src=""paste-ad4547a9a845c1818f375e6c0597edd74d242642.jpg"">"
Pre- and post-synaptic inhibition of nociceptive signalling by GABA and enkephalins"Stimulating the dorsal horn and recording activity in the synapse through recording of EPSCs<br><br>Looking at transmission in the pain pathway by measuring an EPSC caused by stimulation of the presynaptic fibre.<br><br>They then expressed channel rhodopsin in inhibitory interneuron<br><br>showed that, stimulation of A-Delta fibre results in nice EPSC. If you activate inhibitory interneuron through shining a light into the mouse, the EPSC disappears in response to the same stimulus. It is blocked.<br><br><br>Pharmacology shwoed that:<br><br>Stimulation of interneuron inhibits transmission between primary and secondary neuron in nociceptive pathway in 2 phases<br><br>Short-lived phase is dependent upon GABA acting pre-synaptically on GABA(A) receptors<br><br>Longer lived phase is dependent upon enkephalin acting pre-synaptically upon opiod receptors. There is also a post-synaptic action<br><br><img src=""paste-0de17dd378e9d459d5db0e7f655ec4d5dd22d420.jpg"">"
Regulation of the activity of RVM neurons"RVM neurons receive inputs form hypothalamus. Acute stress induces pain hyposensitivity and chronic stress hypersensitivity. Evidence that number of GABAergic interneurons is up and down regulated respectively<br><br>RVM GABAergic neurons express opioid receptors which suppress their activity, thereby promoting disinhibition/increase activity of interneuron and analgesia<br><br><img src=""paste-b838e971065b818f274591e95d9cbfd7c790b8a0.jpg""><br><br>How do pre-and post- synaptic u and Delta-opioid receptors reduce transmission between primary and secondary neurons?"
u and Delta opioid receptors and signalling pathways"Presynaptically:<br>Opiod receptor-induced inhibition of Ca 2+ channel is mediated by binding of the dissociated G-beta-gamma subunit directly to voltage-gated Ca 2+ channel<br><br>Postsynaptically:<br>the direct binding of dissociated G-beta-gamma subunit to Kir3 (potassium) channels activates the channel and hyperpolarises the neuron<br><br>Both receptors couple to G-alpha i/o proteins which inhibit activity of adenylyl cyclase<br><br><img src=""paste-d9350ccdbc59020b25963e559c94579bf6755d73.jpg"">"
Development of tolerance to analgesic effects of opiod receptor agonsistsu opioid receptor activation strongly inhibits severe acute pain and is a major target for post-operative and cancer pain management. Norphine most commonly used agonist<br><br>u opiod receptor agonists have side effects of producing respiratory depression, sedation, constipation and abuse liability, related to tolerance<br><br>Opiod tolerance is defined typically in the clinic as the need to increase a dose to maintain the analgesic effects<br><br>Originally thoguht to be down-regulation of opiod recptors after chronic agonist exposure induces tolerance. However, more recent evidence suggests that opioid receptor expression is not down-regulated but instead may be desensitised and uncoupled from downstream signalling pathways.<br><br>Tolerance also caused by adaptive changes to the cell. Chronic morphine exposure produces elecated cAMP signalling, including the up-regulation of adenylyl cyclase, pkA<br><br>Agonsists at delta-opiod receptors are less effective in treatment of severe acute pain, but may have a role to play in treatment of chronic pain
Central Sensitisation"due to high level of activity in primary C fibres leading to incr. production and release of substance P<br><br>Substance P acts on secondary neuron to strengthen synaptic transmission. lowers the threshold for activation of an AP<br><br>Incr. in cell membraen glutamate receptor number leads to enhanced glutamatergic transmission<br><br>Changes in axonal ion channels to increase inward current and reduce outward current<br><br>Reduction in GABA production by interneuron<br><br>Sprouting of fibres of pre-synaptic neuron<br><br><img src=""paste-7d664afe9b19eae3b7e769a0b31c656bf79eb185.jpg"">"
Hypersensitivity summary"Pain hypersensitivity can be caused by changes in the periphery (as a result of inflammation of nerve damage), or centrally (level of spinal cord), or involve remodelling of the brain and altered connectivity<br><br><img src=""paste-9def760a6949e60d38414f3044eecf1fc769661f.jpg"">"
Chemical synapses vs electrical synapses"Chemical:<br><ul><li>Require conversion of chemical into electrical signal</li><li>Direct transmitter action - NT binding to ionotropic receptor/ indirect transmitter action - metabotropic receptor -&gt; indirect second messenger -&gt; channel opens</li></ul><div><br></div><div>Current flow at electrical vs chemical synapse:</div><div><img src=""paste-32df51764287e72a418edc0db4cfdec4ffe1dd23.jpg""><br></div>"
Discovery of the electrical synapse - Furshpan and Potter (1959)"The first E-synapse was discovered in the crayfish CNS<br><br>In each abdominal segment between the lateral giant axon of an interneuron and segmental giant motor neurons. A single AP in the intrneuron can trigger abdominal flexion which results in escape behavior<br><br>The synapse is electrically rectifying, ie current flows from the interneuron to the motor neuron but not in the reverse direction.&nbsp;<i>This prevents a single motor neuron from activating the escape response</i><br><br>Rectification indicates that there must be an asymmetry at a molecular level but until as late as 2008 little was known about the molecular mechanism of rectification. Most e-synapses do not rectify<br><br><img src=""paste-14929a7eae2feacb5f5fd29e176dc39a5f527b6d.jpg""><br><br>Lateral giant axon:<br><br><br><img src=""paste-e4e6f85c96a34afeaa1e359e0e516dfc8aa66037.jpg""><br>Picture shows cytoplasmic bridge connecting 2 neurons at an e-synapse"
How to test for the rectification of an electrical synapse"<img src=""paste-79f2d4c043f8cfc0433518ceaeafe343e5892d95.jpg""><br><br>2 neurons direct contact<br>Current / voltage detecting electrode in each.<br><br>Top left:<br>Injecting current causing a voltage step neuron A shows a voltage step occur in neuron B - non-rectifying, A to B rectification of depoalrising response<br><br>Top right:<br>Injecting current into neuron B causes a depolarising voltage in B, but not in A - rectifying as it cannot travel backwards, rectification of depolarising response<br><br>Bottom left:<br>Inject hyperpolarising current into A results in hyperpolarising response in A, but not B - B to A rectification of hyperpolarising response<br><br>Bottom right:<br>Injecting hyperpolarising current into B results in hyperpolarising response in both A and B- Rectification of hyperpolarising response"
The first Electrical synapses: Mediate Escape behaviour in Crayfish"<ul><li>The giant fibres (MG and LG) excite the flexor motor-neurons of the abdomen via electrical synapses</li><li>MG produces escape to the rear and LG produces forward directed escape</li><li>The different behaviours can be explained by different connectivity patterns</li><li>Rectification functions to allow motor neurons to be used for other behaviours without activating MG or LG</li></ul><div><img src=""paste-74186976ecb9cbf8f9cbaeace3cd952b470d65c9.jpg""><br></div>"
Molecular machinery I. Connexins form Gap junctions in Mammals"<ul><li>1980s: first members of the connexin family of proteins discovered (in cardiac muscle where they fuinction to synchronise msucle contraction)</li><li>21 genes in the human genome encode different connexins</li><li>SIX connexin proteins form&nbsp;<u>Hemichannels&nbsp;</u>called connexons</li><li>Connexons from adjacent cells link with one another to form intercellular tunnels, effectlely joining the cytoplasm of adjacent cells</li><li>Linked connexons cluster together forming rafts of intracellular channels called&nbsp;<u>Gap Junctions</u></li><li>Connexins are restricted to the Deuterostomes (Chordates, Echinoderms, Hemichordata)<br></li><li>There are NO connexin encoding genes among the Protostomes (arthropods, nematodes, molluscs, annelids, flatworms)</li></ul><div><img src=""paste-521e300d7a5dc2c602024e48430dcbce772079fa.jpg""><br></div>"
Connexin topology and structure of a gap junctional channel"<img src=""paste-d6053b6ba2885c88cc5d51779a9be41e0d03e5ec.jpg""><br><br>Basic structure of 4 membrane spanning domains, extracellular domains E1 and E2 + intracellular terminals Nt, CL and CT<br><br>We know this from techniques such as X-ray diffusion<br><br>C) shows how six connexins can forma connexon<br><br>B) Hemichannels are rotated by 30 degrees"
Connexin channel structure"<img src=""paste-a9cd989889fa5678b688b02a606f18fba148a0ea.jpg""><br><br>A: Three different types of channel using connexin units: <br><br>1) Homomeric / homotypic<br>2) Heteromeric / homotypic<br>3) Homomeric / heterotypic<br><br>B: E1 and E2 have 3 conserved extracellular cysteine residues - critical for docking and a variable cytoplasmic loop and c-terminal region containing various targets for kinases (serine, threonine, tyrosine)"
Functional consequences of variation in subunit composition"Connexin-specific selectivity among second messengers - connexon hemichannels have unique molecular permeability<br><br><img src=""paste-3aa5861528742f1fad448d1148bf038a86265367.jpg""><br><br>If you construct a homomeric connexon from connexin 32, you will see that the channel lets through cAMP and cGMP<br><br>But a heteromeric connexon with connexin 32 and 26, cAMP will not let through but cGMP will be"
Mechanism for variable conductance and permeability?"connexons exist in 2 states: open and closed<br><br><img src=""paste-289aa55ced11eeb2537be68f9e4a0d0225474f92.jpg"">"
Atomic force microscopy"Sample with uneven surface - EG: a raft of connexins<br><br>Like a record table cantilevel, a silicon cantilever moves along the peaks and troughs of the sample surface. A photodiode detecter then transmits the heigh information to a computer that processes the information into an image.<br><br>Through this, we can see connexins in 2 states<br><br><img src=""paste-24c9a9ce23b2637d527aa33481d9f9dd361a5f28.jpg"">"
Molecular Machiner II. Connexins are regulated by <u>kinases, voltage, calcium and pH</u>"They can be regulated from:<br><br>Outside - the extracellular domain - mediation of extracellular loops E1 and E2<br><ul><li>These allow voltage gating and variation of heterotypic channel formation</li></ul><div><br></div><div>Inside - intracellular - mediation of intracellualr loops</div><div><ul><li>These deal with voltage gating, polarity and pH gating</li><li>Very important for regulation of the whole function of the connexon and hemichannel</li><li>Indirectly linked to the fuinctioning of the two neurons that it is connected to</li></ul><div><br></div></div><div><img src=""paste-d6348a473ccd073450c7c319893295e66cbaa4b9.jpg""><br></div>"
Molecular MAchinery. III Innexins"If connexins are absent from protostomes (exclusive to the deuterostomes) what is the molecular basis of the first-discovered E-synapsee in the crayfish??<br><br>This has remained a puzzle until research at the Univeersity fo Sussex leading (directly or indirectly) to:-<br><br><ul><li>Discover of a new gap junction forming protein family - the innexins</li><li>Extension of the innexin family to the deuterostomes</li><li>Connexin, Innexin and Pannexin</li><li>Two types of e-synapse in the mammalian brain - Cx and Px based</li><li>New roles for hemichannel-forming Cx and Px proteins#</li></ul><div>the following experiments show that sometimes to discover something new about a subject, you should study something apparently completely unrelated and trust in serendipity!</div><div><br></div><div><img src=""paste-c50a45301b2120c2f47124f06669e7f4efbd8732.jpg""><br></div>"
Examples of escapes"Long duration escape: Wing-raised, coordinated, takes a minute for realisation, flies away in a single direction<br><br>Short duration escape: Un-coordinated, turns in flight<br><br><img src=""paste-d2d20328c35f50ec4cd4a6379ef999ff6391e0f8.jpg"">"
The Drosophila giant fibre system"Similar to crayfish giant fibre system<br><br>Activates mtoor neurons, making them jump leading to the escape<br><br><img src=""paste-3beb372631d6b74ea4147c6cacc1f5a9b416570f.jpg"">"
Recording Giant fibre&nbsp; activity"A single spike is responsible for the escape response<br><br><img src=""paste-31c8bac8c060f20327d06163ad4e225ccf3509da.jpg""><br><br>showing the effectiveness of this synapse<br>"
Road to discovery of another family of gap junction protein - innexin and pannexin - via neural circuit for fly escape behaviourexperiments on mutant shaky-B^2 mutants show there is not electrical coupling between the giant fibre systems and motor neurons<br><br>Also no dye coupling - dye injected to interneuron stains connected neurons<br><br>Absence of dye coupling and electrical coupling
Another Sussex discovery"Differential voltage&nbsp; gating of structurally asymmetric (heterotypic) gap junctions underlies rectification at arthropod electrical synapses<br><br><img src=""paste-386dc29a4598d17cff4d3be1cb94d9b5bd78291a.jpg"">"
Connexins vs innexins"Differences:<br><ul><li>No DNA or protein sequence homology (consider evolutionary implications)</li><li>Connecins: 3 extracellular cysteines. Innexins: 2 extracellular cysteines</li><li>Innexins: longer extracellular loops, correlating with a wider gap between membranes</li></ul><div>Similarities:</div><div><ul><li>Membrane topology</li><li>Function</li><li>Similar types of channel formed: homotypic, heterotypic and heteromeric</li><li>Gating</li></ul><div><br></div></div><div>Innexins: in invertebrates only.</div><div><br></div><div>Connexins: in vertebrates only</div><div><br></div><div>Pannexins both in invertebrates and vertebrates</div><div><br></div><div>Pannexins have more similarity to innexins than connexins</div><div><br></div><div><img src=""paste-eed8625f22d5178bfdd4c9f188baefefa60c5a39.jpg""><br></div>"
Electrical synapses between motor neurons and Central pattern generator (CPG) neurons"These endow the motorneurons with the ability to modulate the CPG neurons<br><br><img src=""paste-38b0ec0b89a5423f28ab036806c52402e8f002b9.jpg""><br><br><img src=""paste-808b08962003053891d6e89b619f4077c7584380.jpg"">"
Main points of first electrical synapses lecture"<img src=""paste-8d190b2c5fdbd971e5a07dfb0c9834373d1e5830.jpg"">"
E-synapses lecture 1 summary"<img src=""paste-63388e6630985bbb7f553bd55a9c7361cdfc566f.jpg"">"
Electrical synapses in the mammalian brain"Physiological coupling is not simply 1-to-1 but involves low-pass filtering<br><br>Functional consequence: spikes are attenuated and slower potential changes are favoured<br><br>This can even result in preferential transmission of slow hyperpolarising after-potentials!<br><br><img src=""paste-8034783276ee07aeef56f542ae0cfa4bed68c788.jpg""><br><br>Electrical synapses form between similar neurons throughoout the mammalian brain. Therefore they cansunchronise activity among distinct classes of neuron<br><br>Note: evidence of low-pass filtering in the inferior olive (c) - part of the medulla and associated with the cerebellum. These neurons exhibit synchronised oscillations - functionally related to the cerebellum and movement control.<br><br><img src=""paste-1962cb902a54d67217fc421bb058b2a7d192c432.jpg"">"
Synchronous rhythmic activity in Electrical synapses"E-synapses formed from the Cx36 connexons promote synchronous rhythmic activity among inferior olive neurons in wild-type mouse brain<br><br>In Cx36 KO, there is synchrony, thought not oscillation<br><br>The functional consequence of loss of synchrony in the Cx36 KO mouse<br><br><img src=""paste-010e1f680de91cafc65ecf8e4b1802a11a1b73da.jpg"">"
Beyond Synchrony - Low-pass filtering at inter-neuronal electrical synapses: Spike-driven generation of postsynaptic inhibition between coupled neurons"<img src=""paste-781d6a2270e4c9bb797b11495708070bf8ce7f18.jpg""><br>A) presynaptic and postsynaptic neurons - electrically coupled<br>Upon application of large depolarising current in the presynaptic neuron, no EPSP in the post-synaptic neuron is seen as the spike freq. is v high and due to low-pass filtering properties, the spikes don't show up at all in the post-synaptic cell. Only the summed slow after hyperpolarisations are causing the postsynaptic cell to hyperpolarise in response to large presynaptic activity. This is unexpected.<br><br>D) A single spike - we see the post-synaptic effect of the spike is attenuated, whereas the long hyperpolarisaiton is expressed on the post-synaptic neuron.<br><br>E) When you depolarise the cell, you hold it in depolarised state - causes bursting activity instead of steady firing. Even then, initial response is slow post-synaptic depolarisation, but when the bursts come in, it tends to dip down after hyperpolarisation<br><br>F is gradual build up of progressive hyperpolarising effect<br><br>Electrical synapses are sophisticated machines that can operate in many interesting ways"
Extensive electrical coupling between neurons and receptor cells of the retina has functional consequences in light-dark adaptation"<img src=""paste-fa59ed99cea67f791164e341083efa5faeda6142.jpg""><br><br>Little red-blue synapses are gap junctions (E-synapses)<br>They are everywhere in the retina, showing their importance<br><br><ul><li>all 50 cell types in the retina express Cx genes, mostly Cx36, Cx45 and Cx57</li><li>Three important electrical couplings are functionally regulated by light. Generally coupling is incr. by low light and cells are uncoupled by bright light</li></ul><div><ol><li>Photoreceptor (rods and cones) coupling: incr. coupling enhances luminance discrimination but reduces colour discrimination</li><li>Horizontal cell coupling: regulates the coupling of the rod and cone pathways - couples rod pathway to cone pathway in low light. This enhances the fidelity of rod signalling at the expense of colour discrimination</li></ol><div><img src=""paste-41ca0140a20c48157b59a1aa7fea4fa6556fd69a.jpg""><br></div></div><div><br></div><div>Light-activated neuromodulators (DA and NO) released by amacrine cells alter or modulate the conductance (g) of gap junctions. Light can increase and/or decrease conductance (g) depending on brightness level</div><div><br></div><div>Other sources of modulation include calcium, pH and voltage.</div><div><br></div><div><img src=""paste-e95b3219e459b072af8b64de49257b4442f554cf.jpg""><br></div><div><br></div><div>Like horizontal cells, Amacrine cell coupling is also light regulated in a triphasic manner. Extensive coupling in starlight conditions would dissipate the very weak signals, whereas extensive coupling in daylihgt would lead to blurring of the image. However, incr. coupling in twilight conditions helps preserve signal fidelity</div><div><br></div><div><img src=""paste-bf00a6bafdfed5fb594ac7a3aec00690c8176f24.jpg""><br></div>"
Electrical synapses and circadian rhythms - Coupling in the Suprachiasmatic nucleus (SCN) is mediated in part by Cx36"Cx36 KO mice show disrupted circadian behavioural rhythms and reduced synchronous firing of SCN neurons<br><br><img src=""paste-5e02e6af9c5f976597e46cbeb48de5de1027f612.jpg"">"
Role of gap junctions in circadian rhythm"Biocytin spreads between cells in the SCN - blocked by halothane which uncouples electrical synapses<br><br><img src=""paste-0363869c0d0cf41a040be688ecb4d0b1681971e7.jpg"">"
Coupling in the SCN exhibits a circadian rhythm"<img src=""paste-143a2b039d3250118f18345399f72581531a1821.jpg"">"
Plasticity of electrical synapses - the Mauthner cell system: E-synapse and synaptic plasticity"<img src=""paste-8eaf211f252a9ec076cf27588b06b1def352b429.jpg""><br>Fish show fast escape reactions<br><br>This response in mediated by the MAuthner cell - a giant cell that forms electrical synapses with theimput pathways that alert the fish to danger<br><br>Organisation is simialr to giant axons of drosophila and crayfish<br><br>But there are some differences<br><br>What ahppens is, upon injection of AP, it will activate the right nerve cell, with an immediate result of an effect in left cranical nerve but inhibition of the left mauthner cells, as activation of both cells would stop the fish from turning.<br><br>"
A mixed electrical and chemical synapse"At the axon of the 8th cranial nerve, there are both glutamatergic chemical and electrical synapses onto the Mauthner cell<br><br><img src=""paste-d4174493381e9f56d72e79e1c695adb3730b8532.jpg""><br><br>"
Activity-dependent synaptic enhancement at the mixed e-c synapse on the Mauthner cell dendrite"<img src=""paste-21d39a4f1229e4ffbf08d3f455c6a5848205a3c6.jpg""><br>Can evoke both electrical and chemical response through nerve stimulation<br><br>Then an LTP protocol - high freq stimulation<br><br>If you then measure the size of electrical post-synaptic potential and chemical, they both undergo LTP.&nbsp;<br><br>This shows they work together to enhance synaptic transmission<br><br>inter-dendritic calcium also incr. similarly iin both types of synapse<br><br>Summary:<br><br>Tetanic stimulation:<br><ol><li>Activates NMDA receptors to</li><li>Initiatiate a calcium dependent process that</li><li>Modulates BOTH non-NMDA receptors AND gap junction channels</li><li>Resulting in LTP</li></ol><div><br></div><div><img src=""paste-5a7a9b94c6451ea4f5ccace76e51827814820fb7.jpg""><br></div>"
Three genetic Diseases resulting from mutations in Cx genes"Human Peripheral Neuropathy<br><br>Congenital Cataracts<br><br>Genetic Deafness<br><br><img src=""paste-0d6d3ef4be0c68817678221fb57158db3ab60134.jpg""><br><br>Example:<br><img src=""paste-f650d4ff8aa89920a4ae5e66a52f5b1a9c5413dc.jpg"">"
E-synapse conclusions"<img src=""paste-13dab04459ac1f3a70a1792488f7cc3e37df70d3.jpg"">"
Plasticity"Plasticity manifested at the level of individual neurons ('neuronal plasticity' or 'synaptic plasticity') and brain circuits ('brain plasticity' or 'neural plasticity') are the physiological substrates for learning-induced changes in behavioural performance ('memory')<br><br>Examples of links between synaptic plasticity and memory:<br><ul><li>Heterosynaptic facilitation and implicit learning in invertebrates</li><li>Long-term potentiation (LTP) and fear conditioning in rodents</li><li>Long-term depression (LTD) and eye-blink conditioning in rabbits</li></ul><div><img src=""paste-ab65c1ffa40bd94eb298964c092791bb0b7ed542.jpg""><br></div><div><br></div><div><b>Common function: They all chaneg the efficacy of synaptic transmission</b><br></div>"
A conceptual framework for investigating how memory is formed, encoded, maintained and retrieved I. The reductionist (simple systems, bottom-up) approach<ul><li>Physiological level analysis: In vitro analogues of learning paradigms leading to the formaiton of in vitro analogues of memory (eg: long-term potentiation). Also, manipulation of neuronal activity leading to plastic changes in vitro</li><li>Molecular level analysis: Activation or inactivation of intracellular signalling cascades and genes underlying in vitro analogues of behavioural plasticity</li><li>Behavioural level analysis: Testing the hypothesis that the discovered in vitro mechanisms underlie behavioural learning</li></ul>
A conceptual framework for investigating how memory is encoded and retrieved II. The top-down approach<ul><li>Behavioural level analysis: Learning paradigms leading to the formation of memory</li><li>Physiological level analysis: Neural (systems level) and single neuronal expression of the learned response (the electro-physiological or optical 'readout' of the memory trace). Also, manipulation of neuronal activity leading to the behavioural or electrophysiological expression of memory</li><li>Molecular level analysis: Learning-induced activation or inactivation of intracellular signalling cascades and genes underlying physiological and behavioural level plasticity</li></ul>
Kandel, Spencer, Thompson, Berger (starting from the 1960's) - reductionist approach to non-declarative (implicity) memory storage"Implicit memory: encoded within the same network of enurons that mediates learning. This makes studying the mechanisms of memory formation easier<br><br><img src=""paste-30996fdf64756b8b93b571b781c414d75853321c.jpg"">"
Cellular / molecular mechanisms underlying short-term non-associative leanring and memory storage -&nbsp;<i>Aplysia&nbsp;</i>learning studies: the original boldly reductionist experiments"<img src=""paste-c10a98859b220e0ea872f9b7c9801337247c46ff.jpg"">"
Simple systems: Invertebrate models of learning"<div>Experimental advantages in using invertebrate nervous systems:</div><div><ul><li>Small nervous systems</li><li>Large neurons</li><li>Identifiable neurons</li><li>Identifiable circuits</li><li>Simple Genetics</li></ul><div><br></div></div><div>Non-associative learning in&nbsp;<i>Aplysia&nbsp;</i>- gill withdrawal reflex</div><div><img src=""paste-ed57f15b7538baa493d526daa5587fc0629b4004.jpg""><br></div>"
Non-associative Learning"Habituation:<br><ul><li>Learning to ignore stimulus that lacks meaning</li><li>A decrement of behavioural response that occurs when an initially novel stimulus is repeatedly presented - usually harmless / neutral stimulus</li></ul><div><br></div><div>Sensitisation:</div><div><ul><li>Learning to itensify response to stimuli</li><li>Facilitation of enhancement of response by presentation of different, usually strong / dangerous stimulus</li></ul><div><br></div><div><br></div><div><img src=""paste-08a6e802cc2fcdf3fc207697c6102eac57d0b559.jpg""><br></div></div><div><br></div><div><img src=""paste-2baa886713a34a877ecd78ad5fd7a50a12f7bd84.jpg""><br></div><div><br></div><div>Need to demonstrate the existence of these reflexes and pin down the identity of the circuits involved</div>"
Habituation of the Gill-withdrawal reflex"<img src=""paste-670d16b0a01f0d5eed0edb1592a936ef3edbafce.jpg""><br><br>homosynaptic plasticity - only affects a single synapse between sensory neuron and motor neuron between syphon skin and gill muscle<br><br><b>Sensitisation of the Gill-withdrawal reflex</b><br><br><img src=""paste-6c1b6ce771ea9bf1ebbd1dac0ffb4fd8689aa87b.jpg""><br>The sensory and motor neurons are the same<br><br>Facilitatory interneuron is now synapsing onto the motor neuron - now heterosynaptic plasticity<br><br>the plastic change between the sensory neuron and the motor nuron arises as an effect of the thirs facilitatory interneuron<br><br>"
Basic mechanisms of non-associative learning"Locus of the plastical changes underlying habituation and sensitisation of the&nbsp;<i>Aplysia&nbsp;</i>gill-withdrawal reflex: the sensory neuron (SN) terminal, presynaptic to the motorneuron (MN).&nbsp;<br><br>This can undergo homosynaptic depression (leading to habituation), and the heterosynaptic facilitation (leading to sensitisation), the latter due to inputs from facilitator neurons (FN)<br><br><img src=""paste-fd394c73b1d30a95c4e7422bbf525124035d2504.jpg"">"
Biophysical analysis of short-term habituation and sensitisation<ul><li>Duration of sensory neuron AP is reduced during homosynaptic depression (underlying habituation and prolonged during heterosynaptic facilitation (underlying sensitisation)</li><li>Homosynaptic depression results from a progressive decrease in a voltage-sensitive inward Ca 2+ current and in a decrease of NT quanta released, due to an impairment in vesicle mobilisation.</li><li>Heterosynaptic facilitation results from a decrease in an outward K+ current that prolongs the depolarisation during action potentials and thus enahnces the voltage-sensitive inward Ca 2+ current, which in turn incr. transmitter mobilisation and enhances synaptic release</li></ul>
Molecular analysis of short-term sensitisation<ul><li>Serotonin (5-HT) or peptide transmitters, released from facilitatory interneurones, activate G-protein couples receptors.</li><li>This increases adenylate cyclase activity in the sensory neuron terminals, thereby incr. intracellular cAMP levels</li><li>cAMP in turn stimulates the activity of protein kinase A (PKA), responsible for phosphorylating substrate proteins, one of which is the S-type (serotonin sensitive) K+ channel.</li><li>Phosphorylation closes this channel, giving rise to increased spike duration, more Ca 2+ influx, and thus more transmitter release</li><li>5-HT also activates protein kinase C, which is involved in increased transmitter mobilisation</li></ul>
Summary figure of facilitation"<img src=""paste-fbe96d7a0766aa063ab695585ba9a295ac734ea6.jpg""><br>Sertononin binds to GPCR activation 2 cascades:<br><ul><li>adenylyl cyclase PKA cascade</li><li>PKC cascade</li></ul><div>This will have the effect of broadening the presynaptic AP, resulting in release of more NT per spike</div><div><br></div><div>A broader spike maintains depolarisation for a bit longer -&gt; more calcium enters presynaptic terminal -&gt; more NT release</div><div><br></div><div>PKC contributes to mobilisation of the reserve vesicles -&gt; contributing to enhacnement of NT release from presynaptic terminal</div><div><br></div><div>Leads to incr. activation of the motor neuron</div>"
A detailed diagram of the signalling events underlying short-term sensitisation in&nbsp;<i>Aplysia</i>"<img src=""paste-2199740e317867d2849cfe4a5325e6046159986f.jpg"">"
A summary of teh sequence of biochemical, electrophysiological and behavioural events underlying short-term sensitisation in&nbsp;<i>Aplysia</i><ol><li>Spike triggered in facilitatory interneuron (FIN) by sensitising stimulus</li><li>5-HT (serotonin) released from terminal of FIN onto terminal of sensory neuron (SN)</li><li>Activation of adenylate cyclase (SN)</li><li>Incr. levels of cAMP (SN)</li><li>Activation of PKA and PKC (SN)</li><li>Phosphorylation of K+ channel protein (SN)</li><li>Decreased conductance in K+ channels (SN)</li><li>Broader AP evoked by non-sensitising stimulus (SN)</li><li>Incr. Ca 2+ influx (SN)</li><li>Incr. presynaptic release of transmitter (SN)</li><li>Incr. postsynaptic response (MN)</li><li>Incr. behavioural response</li></ol>
Cellular / molecular mechanisms underlying long-term non-associative memory storageAfter only one sensitising stimulus, the sequence of molecular events underlying short-term sensitisation will only lead to changes that last from minutes to hours, after which elevated cAMP levels return to normal<br><br>Repeated applications of sensitising stimuli lead to long-term sensitisation but not to maintained elevation of cAMP levels<br><br>So, how does long-term memory form, if not by keeping cAMP levels persistently high?
Cellular and molecular changes underlying long-term, non-associative memory storage"Cajal: learning does not result in the proliferation of new nerve cells (mature nerve cells have lost their capacity to divide)<br><br>Instead, existing nerve cells grow more branches to strengthen their connections with other nerve cells so as to be able to communicate with them more effectively<br><br><img src=""paste-dca7cd551c63dd219aeec06e05e7904b0544cd94.jpg""><br><br>Two sets of questions raised by this diea:<br><ul><li>Does the formation of long-term memory involve persistent changes in synaptic strength? If so, what are the molecular underpinnings of these changes?</li><li>How do short-term synaptic changes differ from those that support lng-term storage? Do they occur in differnt neurons, or can teh same neuron store information for both short- and long-term memroy?</li></ul><div>Models: Long-term habituation and sensitisation in&nbsp;<i>Aplysia</i></div>"
Important observations from work on long-term habituation and sensitisation"<ul><li>The same set of connections that undergo short-term plastic changes (homosynaptic depression, underlying habituation and heterosynaptic facilitation, underlying sensitisation), can also undergo long-term changes&nbsp;</li><li>Long-term habituation and sensitisation are accompanied by morphological changes</li></ul><div><br></div><div>the nmber, size and vesicle complement of sensory neuron active zones are smaller in animals showing long-term habituation and larger in animals showing long-term sensitisation. Time course of changes is similar to duration of memory</div><div><br></div><div><img src=""paste-f1d9cfbd233fadf3a061beec506852479bc17dfc.jpg""><br></div>"
A key observation concerning long-term sensitisation / heterosynaptic facilitation:"Inhibitors of protein and RNA synthesis block long-term sensitisatino (semi-intact preparations) and the long-lasting heterosynaptic facilitation and morphological changes associated with it (cell cultures)<br><br><img src=""paste-59b15c9d2492f4422ca1b9df829b6d8825143bb6.jpg""><br><br>Important consequence of initial observations concerning the transcription- and translation-dependence of long-term sensitisation: focus shifted from the presynaptic terminal, where the short-term changes occur, to the cell body of the neuron, where RNA is transcribed and new protein molecules are syntehsised.<br><br><img src=""paste-34fd43a2daec052cedca2aeaa5859eb64ad07757.jpg""><br><br>This opened the way for a detailed molecular analysis of lon-term memory formation resulting from in vitro correlates of sensitisation"
Use of co-cultured sensory and motorneurons to study synaptic facilitation"<ul><li>Spike activity in the sensory cell substitutes for weak touch to the siphon</li><li>Serotonin (5-HT) applications substitute for noxious stimuli (5HT)is one of the transmitters used by facilitatory interneurons activated by noxious stimuli)</li><li>A large variety of biophysical, biochemical and molecular changes measured in the sensory neuron</li></ul><div><img src=""paste-15daf5960a0c8d8e339461569746be0acfd139f5.jpg""><br></div>"
Long-term heterosynaptic facilitation is dependent on the transcriptional activator protein CREB1 binding to specific gene regions containing the CRE oligonucleotide sequence TGACGTCA"<img src=""paste-a70289faea57d4731bdb5204d33c208028fa16e8.jpg"">"
&nbsp;The activatino of transcriptional activators, such as CREB, represents the most important step in the process of switching from short-term to long-term synaptic plasticity"<img src=""paste-4b83968a1088e696c588ab227d58052412dd6cc0.jpg"">"
the molecular processes of long-term synaptic plasticity have been highly conserved during evolution"<img src=""paste-11bd9f0009eccbc162c0f4d675e776481cf993fe.jpg"">"
A direct comparison of the changes underlying short-term and long-term sensitisation in the sensory neurons of the&nbsp;<i>Aplysia&nbsp;</i>gill-withdrawal system"<img src=""paste-8f6a2a3bc94fe0ee38202802302f6c8eb8a0545c.jpg"">"
Summaries of short-term and long-term facilitation"Short-term:<br><img src=""paste-9839231342040fefd785e4b967c3065d26004c57.jpg""><br><br>Long-term:<br><img src=""paste-1662fe3c2db70f1ec77965366785ebf1774595f5.jpg"">"
Associative learning: based on pairing either two stimuli (classical or Pavlovian conditioning) or a behavioural act of the animal with a stimulus (operant or instrumental conditioning)"<ul><li>Conditioning can be either aversive or non-aversive, depending on the anture of the unconditioned stimulus (US)</li><li>Aversive US: produces a defensive unconditioned response (UR), such as reflexive withdrawal of a body part</li><li>Non-aversive US: usually evokes a consumatory UR, such as feeding, so also known as appetitive US</li><li>The neutral or weak stimulus that is reinforced by the US in classical conditioning is called the conditioned stimulus (CS)</li><li>The response that forms as a result of pairing the CS with the US is called the conditioned response (CR). After conditioning, it can be evoked by the CS alone, and resembles the UR which could origginally be evoked by the US</li></ul><div><img src=""paste-257212caa45eea446973d11f046997020af744b1.jpg""><br></div>"
Simple systems: Invertebrate models of learning (again)"<ul><li>Associative learning in&nbsp;<i>Aplysia</i></li><ul><li>Classical conditioning<br></li><li>CS-US pairing</li><ul><li>Cellular level</li><li>Molecular level</li></ul></ul></ul><div><img src=""paste-f6b8d0e319aae8524013e149c6caddb08c201e70.jpg""><br></div><div><br></div><div>Application of mild tactile stimulus to siphon, same when testing for habituation or sensitisation</div><div><br></div><div>But for associative learning, the touch is paired with an electric shock to the tail. It's important that these occur almost simultaneously.</div><div><br></div>"
The molecular basis for classical conditioning in&nbsp;<i>Aplysia</i>"<ul><li>The molecular basis for classical conditioning in&nbsp;<i>Aplysia</i></li><ul><li>Pairing CS and US causes greater activation of adenylyl cyclase because CS admits Ca 2+ into the presynaptic terminal<br></li></ul></ul><div><img src=""paste-f4c16af981bc2fc6d385de145a7fae7f0e9f3f85.jpg""><br></div><div><br></div><div>Molecular co-incidence detector, sitting in membrane.</div><div><br></div><div>Detects coincidence by Calcium and G-protein interacting with it at the same time</div>"
The first proposed mechanism for associative conditioning was based exclusively on the pre-modulatory coincidence model and was called 'activity-dependent enhancement of presynaptic facilitation'"Interactions involved in the activity-dependent enhancement of presynaptic facilitation in Aplysia sensory neurones. Ca 2+ influx during the sensory neuron spike potentiates the activatino of the adenylate cyclase by the facilitatory transmitter<br><br><img src=""paste-94d5a3ea890e6a20e4012c8c0f94cabaa79bc856.jpg"">"
Summary of the molecular mechanisms underlying short-term associative synaptic plasticity based on activity-dependent enhancement of presynaptic facilitation"A1. CS alone - Low amount of cAMP<br>A2. US alone - Higher amount of cAMP as no calcium channel activated<br>A3. Both simultaneously - very high amount of cAMP<br><img src=""paste-1230900580e35e167fe744d757f596154ea7d61b.jpg"">"
Concise summary of the molecular mechanisms of the formation of short-term associative memory in the aplysia gill withdrawal network based on both the non-Hebbia presynaptic dual adenylate cyclase activation mechanism (co-incidence detector 1) and the more recently discovered Hebbian pre-post coincidence detection mechanism (co-incidence detector 2)"Detector 1 - presynaptic<br>Detector 2 - post-synaptic<br><br><img src=""paste-e7cff30153bb7f11db15dde7a90eaa6938e5082a.jpg"">"
There are 2 key co-incidence detection mechanisms involved in classical conditioning of the Aplysia gill-withdrawal response:"<ol><li>Dual activation of adenylate cyclase by Ca 2+/calmodulin and derotonin (5-HT) within the presynaptic terminal of the sensory neuron. Detects co-incidence between the presynaptic spike activity (CS) and input from the facilitatory interneuron (US) in the same sensory neuron</li><li>An NMDa receptor mediated Hebbian pre-post coincidence detection mechanism. Detects coincidence of pre-synaptic spike activity in teh sensory neuron (CS) and psot-synaptic depolarisation in the motor neuron caused by the US</li></ol><div><img src=""paste-0a86cec3393a1cb745236d513703d90f591ad692.jpg""><br></div>"
Formation of long-term associative memory (LTM) after classical conditioning and its in vitro analogues involves the same molecular steps as those underlying long-term non-associative memory (see Plasticity II)"<img src=""paste-57f209b9158a324a33f568d7b84f408ba62fb2ee.jpg"">"
The key steps of learning-induced transcriptional activation leading to long-term associative plasticity in both invertebrates and vertebrates"<img src=""paste-0cec2cf7f74a7425b6bf6209f1a8774374a1f3ec.jpg""><br><br>a) Baseline condition - No transcription, Hox promotor region just sits there suppressing transcription<br><br>b) When CREB-1 is upregulated, CREB-2 is knocked off of its position<br><br>c) CREB-1 sits here and is phosphorylated by PKA and MAP kinase mainly. This activates transcription."
LTP and LTD: first described as mechanisms of persistent regulation of synaptic strength in the mammalian neocortexLTP - long-lasting enhancement of synaptic strength<br><ul><li>Occurs at a variety of sites within the nervous system, incl. hippocampus</li><li>The hippocampus is important in the formaiton of new, long-term, declarative memories. If the hippocampus is removed, the ability to form short-term memories remains unimpaired. Likewise, long-term memory of events that occurred before the destruction of teh hippocampus remains unaffected.</li></ul><div>However, the ability to place new information into long-term memory is severely impaired</div>
Vertebrate models of learning"<ul><li>Synaptic plasticity in the hippocampus (continued)</li><ul><li>Anatomy of the hippocampus</li></ul></ul><div><img src=""paste-f805bf4ef06865ccee90ecb8372fce0e37eab8b7.jpg""><br></div>"
How to induce LTP experimentally?"Long-term potentiation of Schaffer collateral Ca1 synapses<br><br><img src=""paste-a45cdf3f947b7b2b5b562e6b2d8902c48e3058a5.jpg""><br><ul><li>2 CA3 pyramidal cells<br></li><li>Can stimulate and record post-synaptic activity</li><li>record baseline activity - will show spontaneous activity</li><li>Pathway 1 - stimulated with strong stimulus - buildup of LTP shown through enhanced size of EPSP</li><li>Pathway 2 - not stimulated by tetanic stimulus - remains unpotentiated</li></ul><div><br></div><div>LTP of excitatory synapses shows associativity</div><div><br></div><div>A: Experimental arrangement. Axons 1, 2 and 3 can be separately stimulated</div><div><br></div><div>B: prior to potentiation, each input synapse produces a similar EPSP</div><div><br></div><div>C: A rapid burst of AP is triggered in axon 1, while axon 2 fires at a low rate and axon 3 remains inactive</div><div><br></div><div>D: One hour after the potentiating burst, the EPSPs elicited by axon 1 and axon 2 are larger, while the response to input from axon 3 remains the same as before potentiation</div><div><br></div><div><img src=""paste-ba86b2061c5db22be684e0c578dd2d05c95249e0.jpg""><br></div>"
What aspect of the strong stimulation triggers synaptic strengthening in&nbsp;<b><u>both&nbsp;</u></b>&nbsp;the strongly stimulated synapse <u><b>and</b></u>&nbsp;the coactive, but much more weakly stimulated synapse?Strong synaptic stimulation produces substantial depolarisation of the psotsynaptic neuron, and this initieated LTP if it coincides with presynaptic activity
Direct test of the idea that depolarisation of the postsynaptic activity causes LTP"Single stimuli applied to a Schaffer collateral synaptic input evokes EPSPs in the postsynaptic CA1 neuron. These stimuli alone do not elicit any change in synaptic strength<br><br>However, when the CA1 neuron's membrane potential is briefly depolarised (by applying current pulses through the recording electrode) in conjunction with the Schaffer collateral stimuli, there is a persistent increase in the EPSPs<br><br><img src=""paste-2cf873d8ac3ab6b2fbbe03428b72acd127c43f66.jpg"">"
Pre- and post-synaptic molecular mechanisms of LTP"Dependence of LTP on coincidence of activity in the pre- and postsynaptic cell makes LTP a Hebbian type synaptic plasticity<br><br><img src=""paste-645ce576ab389430db4a3f5ce857ee4f33b58387.jpg"">"
Invertebrates also show LTP"Pieces of evidence pointing to the possibility that mammalian-type LTP amy play a role in classical conditioning in Aplysia<br><br><ul><li>Mechanosensory neurons use glutamate as a transmitter</li><li>Gill motoneurons possess an NMDA-type receptor</li><li>NMDA receptor blockers block conditioning</li><li>Tail shock not only activates facilitatory neurons but also depolarises motorneurons</li></ul><div><br></div><div>Indeed the Aplysia model has provided the first direct link between LTP and learning at the level of individually identificable neurons and synapses!</div><div><br></div><div><img src=""paste-bfbcd2bb1284e6728fee6c8170d5e3be4404d1d4.jpg""><br></div>"
Aplysia classical conditioning: both non-Hebbian and Hebbian synaptic mechanisms"feedback from motor neuron to sensory neuron<br><br><img src=""paste-b77f2b4bc5dfe2af326bcf795317c76db10c5ea1.jpg"">"
Compare and contrast: the molecular mechanisms of early and late LTP"<img src=""paste-fa8ac060c9d770bd2502532dad2233f7f3db2d51.jpg"">"
General model for learning-related enhancement of excitatory glutamatergic synapses. This model is based on data from studies of synaptic plasticity in both invertebrates and vertebrates"<img src=""paste-e1732afdc78668439ce7e4112939ab83cb555d06.jpg"">"
Induction of early and late LTP in the hippocampus"a single train of high-frequency stimuli elicits early LTP; four trains at 10-minute intervals elicit late LTP<br><br>Early LTP lasts about two hours; late LTP lasts more than 24 hours<br><br><img src=""paste-5c7e134186acb7d89c153b9219de0fd77832336d.jpg"">"
Structural changes associated with late LTP in the hippocampus"A) The dendrites of a CA1 pyramidal neuron were visualised by filling the cell with a fluorescent dye<br><br>B) New dendritic spines (white arrows) can be observed to appear approximately 1 hour after LTP-inducing stimulus<br><br>The presence of novel spines raises the possibility that LTP may arise, in part, from formation of new synapses.<br><br><img src=""paste-acad03e5519ef7535f62a6eda2e17e3a875f3469.jpg"">"
LTD: A long-lasting decrease of synaptic strength"Results from low-frequency (~ once per second) stimulation of the presynaptic pathway, repeated for a prolonged period of time (~15 mins)<br><br><img src=""paste-45b57e4756eadd43a0d88d9ef10920ce8a507e62.jpg"">"
Side by side compaison of mechnisms of LTP and LTD"<img src=""paste-f51fed114ef48711866f830e54f250b36af30699.jpg""><br><br>Short-duration, high-freq stimulation -&gt; high ic. Ca 2+ cc kinase activation -&gt; induction of LTP<br><br>Long-duration, low frequency stimulation -&gt; low ic. Ca 2+ cc phosphatase activation -&gt; induction of LTD<br><br><img src=""paste-318213c66788f3b78dcd8e1087685557aa0f89e6.jpg""><br><br><ul><li>Both LTP and LTD are dependend on Ca 2+ influx in the postsynaptic cell. Little calcium is thought to give LTD, a lot of calcium is thought to give LTP</li><li>'Silent synapse'hypothesis: most synapses have only one NMDA receptors and LTP can only occur after the addition of non-NMDA receptors</li><li>LTD was proposed to remove non-NMDA receptors from the postsynaptic membrane</li><li>Cerebellar LTD elevates Ca 2+ levels through voltage-gated Ca 2+ channels and a metabotropic receptor-mediated second messenger cascade. LTD leads to sustained de-sensitisation of the ionotropic, non-NMD-type AMPA receptor.</li></ul><div>Thus, the final common pathway for the induction and expression of both LTP and LTD is an elevation of intracellular Ca 2+, an activation of enzymatic cascades, and a modification of postsynaptic AMPA receptors</div>"