Techniques in synaptic plasticity

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Synaptic plasticity Mapa Mental sobre Techniques in synaptic plasticity, creado por Cher Bachar el 02/05/2013.
Cher Bachar
Mapa Mental por Cher Bachar, actualizado hace más de 1 año
Cher Bachar
Creado por Cher Bachar hace más de 11 años
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Techniques in synaptic plasticity
  1. Electrophysiology

    Nota:

    • recording of changes in current OR voltage- in the postsynaptic cell usually in response to a stimulation protocol
    1. Extracellular
      1. How?
        1. populations of cells

          Nota:

          • used to record from populations of cells- filed EPDPs, compounds APs, synchronous oscillatory network activity
          1. single-cells

            Nota:

            • measures action potentials only
            1. (+) easier than intrace

              Nota:

              • easier than intracellular techniques or whole-cell patch clamping because there is less risk that the neurone will be damaged or lost because of mechanical movement
            2. in-vivo recordings
              1. As positive ions move into the cell, the extracellular space becomes more negative

                Nota:

                • graph show depolarisation as going down
                1. graph does down

                  Nota:

                  • for ion influx
              2. Considerations
              3. Intracellular
                1. Mainly in-vitro

                  Nota:

                  • acutely dissocated cells, neuronal cultures, brain slices
                  1. in-vivo

                    Nota:

                    • usually in anaesthetised animals (difficult)- also in head-fixed awake animals (very difficult)
                    1. Single-cell recordings
                      1. intracellular sharp microelectrode recordings
                        1. graph goes up

                          Nota:

                          • for influx
                        2. Current clamp
                          1. How?
                            1. records membrane potential-voltage
                              1. can inject current

                                Nota:

                                • to depolarise or hyperpolarise a cell
                                1. graph goes down

                                  Nota:

                                  • goes down for an ion influx- when plotting current- down will be an influx of ions when plotting voltage- up will be an influx of ions
                                2. Considerations
                                  1. Patch clamp
                                    1. configurations
                                      1. whole-cell

                                        Nota:

                                        • can be voltage-clamp or current-clamp mode
                                        1. cell attached
                                          1. inside-out/ outside-out
                                          2. Considerations
                                            1. Reductionist

                                              Nota:

                                              • studies shouldnt make grand claims of functional significance without corroboration from other experimental approaches that are more integrative
                                              1. Drugs

                                                Nota:

                                                • if drugs are used to block the action of ion channels that are not the focus of the study- it should be made sure that they dont alter the function of ion channels that ARE the focus of the study
                                                1. control- two drugs

                                                  Nota:

                                                  • with different selectivities- if the results are the same then it's not likely that they affect the target of interest
                                                2. Whole-cell
                                                  1. intracellular properties

                                                    Nota:

                                                    • using patch pippetes changes the properties
                                                    1. induction of LTP

                                                      Nota:

                                                      • sensitive to washout of intracellular components
                                                      1. alternatives
                                                        1. perforated patch
                                                          1. sharp electrode
                                                      2. Voltage clamp
                                                        1. Temperature

                                                          Nota:

                                                          • most are done at room temperature which can alter the function
                                                          1. Electrode resistance

                                                            Nota:

                                                            • Voltage error- when there are large currents, the voltage also increases (ohm's law) which means that the membrane potential might differ to the command potential applied to the cell
                                                            1. voltage error
                                                              1. monitor resistance

                                                                Nota:

                                                                • the higher the resistance the slower and smaller the recorded current becomes-  Don't record rapid membrane events because the membrane currents are being recorded
                                                              2. space clamp errors

                                                                Nota:

                                                                • when recording from complex shaped cells- e.g. pyramidal cells, not all ion channels are optimally clamped so the current measured will not be completely representative of the true current
                                                              3. Isolated patches
                                                                1. sampling errors

                                                                  Nota:

                                                                  • e.g. ion channel distributions aren't even; ion channel activity not uniform; temporal considerations- during the time of recordings the ion channel activity may vary; 
                                                                  1. Recording (amplifier) errors

                                                                    Nota:

                                                                    • Temporal resolution- channel currents might be very short (good papers will indicate the minimum open time, and estimate the proportion of events that have been excluded) Amplitude resolution- not all currents might be identifiable Such currents can be studied in the whole-cell configuration
                                                            2. Voltage clamp
                                                              1. Why?

                                                                Nota:

                                                                • ion channels have different properties at different membrane potentails in current camp voltage varies so the properties of the channel change during the event
                                                                1. graph goes up

                                                                  Nota:

                                                                  • goes up for an ion influx
                                                                  1. Patch-clamp

                                                                    Nota:

                                                                    • variation of voltage/current clamp that allows many types of cells to be voltage-clamped with one electrode
                                                                    1. two-electrode

                                                                      Nota:

                                                                      • for larger cells
                                                                      1. control membrane voltage- record current

                                                                        Nota:

                                                                        • 2 electrodes- one measures membrane potential, one injects current to control the voltage
                                                                      2. single-electode (SEVC)

                                                                        Nota:

                                                                        • was developed before patch-clamp technique was available
                                                                        1. considerations
                                                                          1. +
                                                                            1. (+) recording from deep layers of the brain

                                                                              Nota:

                                                                              • can enable voltage clamping ofcells that cannot be accessed by patch clamping
                                                                              1. (+) Minimal disruption to intracellular messenger systems

                                                                                Nota:

                                                                                • that may be critical for regulation of ionic mechanisms in cells
                                                                              2. -
                                                                                1. (-) can't clamp large currents

                                                                                  Nota:

                                                                                  • theres a limit to the amount of current that ca be passed by the electroge because of the high resistance of microelectrodes
                                                                                  1. (-) clamping the microelectrode, but the preparation /cell

                                                                                    Nota:

                                                                                    • can result from a failure to recognize a necessary compromise between the speed of the clamp (maxim- switching faster is better) and the requirement for the electrical potential difference of the electrode to decay fully before the value of remaining potential (i.e. the potential of the impaled cell)  is sampled and the switch is made to current passing (maxim= switching too fast is bad))
                                                                                    1. errors from very large very fast currents
                                                                                      1. experimenter should monitor the voltage at the headset of the amplifier using an oscilloscope
                                                                                      2. indicators of poor clamping
                                                                                        1. Na/ Ca channels- negative slope region of current-voltage (I/V) relationship

                                                                                          Nota:

                                                                                          • poor clamping- there will be a sudden all or nothing increase in the amplitude of the inward current- which will show up as a very steep negative slope in the I/V relationship
                                                                                          1. need long recovery time (>15min)

                                                                                            Nota:

                                                                                            • Some evidence of damage upon microelectrode penetration- SEVC >>need longer recovery time (over 15 minutes)
                                                                                            1. noise- no more than 1mV

                                                                                              Nota:

                                                                                              • check for a clean trace- no more than 1mV variation in resting membrane potential
                                                                                              1. artefacts- due to electrode movement
                                                                                              2. (-) APs are attenuated because of capacitance
                                                                                            2. SEVC vs patch-clamo

                                                                                              Nota:

                                                                                              • APs more attenuated in SEVC- due to greater capacitance  Some evidence of damage upon microelectrode penetration- SEVC >>need longer recovery time (over 15 minutes)
                                                                                        2. Stimulate/ record from?

                                                                                          Nota:

                                                                                          • usually stimulate presynaptic, record postsynaptic neuron
                                                                                          1. Preparations of brain slices
                                                                                            1. in-vitro
                                                                                              1. Dissociated/ organotypic neuronal cultures

                                                                                                Nota:

                                                                                                • perinatal cultures, inappropriate hyperwired connected (may be hard to generalize), no anatomical correlate with CNS wiring in vivo
                                                                                                1. Acute brain slices

                                                                                                  Nota:

                                                                                                  • Any age most local circuits intact, long range targets severed
                                                                                                  1. Living brain slice

                                                                                                    Nota:

                                                                                                    • This preparation allows ready access to all pathways of the dentate gyrus and hippocampus, provides mechanical stability for intracellular recording, and makes possible rapid pharmacological manipulation of the extracellular environment
                                                                                                    1. synaptosome

                                                                                                      Nota:

                                                                                                      • A synaptosome is an isolated synaptic terminal from a neuron. Synaptosomes are obtained by mild homogenization of nervous tissue under isotonic conditions and subsequent fractionation using differential and density gradient centrifugation
                                                                                                    2. in-vivo
                                                                                                      1. Whole animal

                                                                                                        Nota:

                                                                                                        • Any age; all circuits intact, can correlate behaviour with electrophysiology, technically very demanding, especially for cellular recording and freely moving animals
                                                                                                    3. plotting voltage
                                                                                                      1. up

                                                                                                        Nota:

                                                                                                        • up= influx of ions
                                                                                                      2. plotting current
                                                                                                        1. down

                                                                                                          Nota:

                                                                                                          • down=influx of ions
                                                                                                      3. Molecular
                                                                                                        1. Genetically modified mice
                                                                                                          1. transgenic
                                                                                                            1. knockouts/ knockins
                                                                                                              1. Cre-lox
                                                                                                              2. immunocytochemistry
                                                                                                                1. fluorescence imaging of ion activity
                                                                                                                  1. microinjection of cDNA

                                                                                                                    Nota:

                                                                                                                    • to see the effect of a specif protein/ subunit?
                                                                                                                    1. western bolt
                                                                                                                      1. Detection of exocytosis
                                                                                                                        1. measuring L-glutamate- in synaptic cleft

                                                                                                                          Nota:

                                                                                                                          • The studies that have measured the gross amount of L-glutamate released cannot distinguish between the mechanisms by which there was glutamate release- increase in no. of vesicles, Pr, etc or other alterations that affect the extracellular concentration of L-glutamate.
                                                                                                                          1. (-) doesn't show how glutamate increases
                                                                                                                      2. Stimulation protocols
                                                                                                                        1. Paired-pulse stimulation

                                                                                                                          Nota:

                                                                                                                          • Protocol- Pairs of action potentials were elicited in the presynaptic cell with short depolarizing current pulses (each 10-20 ms duration, 0-2- 0 7 nA) separated by 20-3000 ms. Pairs of pulses were delivered at intervals of 7-10 s. Magnitude of responses are recorded in the postsynaptic neuron in response to both stimuli and are compared in the analysis
                                                                                                                          • The aftereffects on synaptic transmission of a single stimulus can be tested by delivering a second stimulus at a variable time after the first.
                                                                                                                          • two stimuli are delivered with an interval of, say, 50 ms, and the amplitude of the first and second synaptic responses are compared. The amplitude of the second response relative to the first (facilitation ratio) is then a reflection of the increase in the probability of transmitter release (Pr).
                                                                                                                          1. dentate gyrus in vivo

                                                                                                                            Nota:

                                                                                                                            • When pairs of stimuli are delivered to the perforant path, the amplitude of the second response recorded in the molecular layer of the dentate gyrus in vivo is typically facilitated at interstimulus intervals of less than 200 to 300 ms and depressed at longer intervals of up to a few seconds (McNaughton, 1980
                                                                                                                            1. Schaffer-commissural synapses

                                                                                                                              Nota:

                                                                                                                              • Similar effects are seen at Schaffer-commissural synapses in area CA1 (Andersen, 1960).
                                                                                                                              1. test for pre/ postsynaptic mechanism of LTP
                                                                                                                                1. (-) suggestive but not conclusive

                                                                                                                                  Nota:

                                                                                                                                  • For example, no change in facilitation would be observed if LTP resulted from recruitment of a population of silent boutons (that is, boutons with zero probability of release before the induction of LTP) that, following induction, assumed a distribution of release probabilities similar to that of the population activated before induction
                                                                                                                                  1. (+) Other tests
                                                                                                                                    1. fluorescence imaging
                                                                                                                                      1. exocytosis detection
                                                                                                                                        1. Drugs
                                                                                                                                          1. Genetically modified
                                                                                                                                            1. tests for LTP
                                                                                                                                              1. stimulation protocols
                                                                                                                                        2. high-frequency/ tetanus

                                                                                                                                          Nota:

                                                                                                                                          • stimulating the presynaptic neuron, and recording in the postsynaptic neuron- this will lead to neurotransmitter release and depolarisation in the postsynaptic neurons- Mg+ block removed because NMDAR activation is slow you need to depolarise for a long time
                                                                                                                                          1. frequency of 100Hz for 1 second

                                                                                                                                            Nota:

                                                                                                                                            • depolarising for a long time can induce LTP
                                                                                                                                          2. Theta burst stimulation (TBS)
                                                                                                                                            1. 5 bursts of 4 pulses at 100 Hz with 200ms break between the bursts
                                                                                                                                              1. Hyman et al (2003)

                                                                                                                                                Nota:

                                                                                                                                                • when TBS was applied at the trough of theta EEG- LTD at the peak- LTP
                                                                                                                                              2. Lever et al (2010)

                                                                                                                                                Nota:

                                                                                                                                                • investigated theta phase of firing in principal cells in subiculum and CA1 as rats foraged in familiar and novel environments. We found that the preferred theta phase of firing in CA1, but not subiculum, was shifted to a later phase of the theta cycle during environmental novelty. Furthermore, the amount of phase shift elicited by environmental change correlated with the extent of place cell remapping in CA1. Our results support a relationship between theta phase and novelty-induced plasticity in CA1
                                                                                                                                              3. Paired

                                                                                                                                                Nota:

                                                                                                                                                • pairing a stimulation of the presynaptic neuron with depolarisation of the postsynaptic neuron
                                                                                                                                                1. Spike timing-dependent

                                                                                                                                                  Nota:

                                                                                                                                                  • When did the stimulation arrive? You stimulate the presynaptic and depolarise postsynaptic neurons at different time e.g. Post before pre, or pre before post- This will lead to either LTP (pre before post) or LTD (post before pre)
                                                                                                                                                  1. Voltage clamp
                                                                                                                                                    1. Current clamp
                                                                                                                                                      1. Backpropagating

                                                                                                                                                        Nota:

                                                                                                                                                        • APs fires in the cell body also goes back up the dendrite towards the cell body, and interact with incoming inputs from presynaptic neurons
                                                                                                                                                        1. Bi and Poo (1998)
                                                                                                                                                        2. induces LTP/ LTD

                                                                                                                                                          Nota:

                                                                                                                                                          • depends on the depolarisation of the postsynaptic neuron- less Ca influx means that it won't remove the Mg block
                                                                                                                                                          1. insilences silent synapses

                                                                                                                                                            Nota:

                                                                                                                                                            • which would induce LTP
                                                                                                                                                            1. Isaac et al (1995)

                                                                                                                                                              Nota:

                                                                                                                                                              • paired stimulation lead to release of neurotransmitter coupled with depol, which leads to the insertion of receptors in the postsynaptic density
                                                                                                                                                            2. LTD- Nakamura et al (2011)

                                                                                                                                                              Nota:

                                                                                                                                                              • dependent on the Ca influx
                                                                                                                                                              1. Wigström et al., 1986

                                                                                                                                                                Nota:

                                                                                                                                                                • even low-frequency stimuli could induce LTP in single CA1 pyramidal cells if each stimulus was given in conjunction with a strong depolarizing pulse
                                                                                                                                                              2. whole-cell voltage clamp
                                                                                                                                                              3. Considerations
                                                                                                                                                                1. Theta vs high frequency

                                                                                                                                                                  Nota:

                                                                                                                                                                  • major factor controlling the magnitude of LTP is the number of stimuli in a train rather than the pattern of stimulation
                                                                                                                                                                  1. Hernandez et al. (2005)

                                                                                                                                                                    Nota:

                                                                                                                                                                    • major factor controlling the magnitude of LTP is the number of stimuli in a train rather than the pattern of stimulation
                                                                                                                                                                    1. difference in early phase, but not late phase LTP
                                                                                                                                                                      1. number of pulses determine long-term LTP
                                                                                                                                                                  2. primed-burst stimulation (PBS)

                                                                                                                                                                    Nota:

                                                                                                                                                                    • a brief burst of stimuli 
                                                                                                                                                                    1. Low frequency stimulation (LFS)
                                                                                                                                                                      1. 1-3Hz
                                                                                                                                                                    2. Pharmacological
                                                                                                                                                                      1. Agonists
                                                                                                                                                                        1. Antagonists
                                                                                                                                                                          1. that activate upon activation of a receptor
                                                                                                                                                                            1. APV/ DGG
                                                                                                                                                                              1. ligand-activated non-competitive antagonists

                                                                                                                                                                                Nota:

                                                                                                                                                                                • for example an antagonist that 
                                                                                                                                                                              2. Ca-chelating agents
                                                                                                                                                                                1. uncaging
                                                                                                                                                                                2. Techniques for testing STP/ LTP

                                                                                                                                                                                  Adjunto:

                                                                                                                                                                                  1. CONFOUNDING FACTORS
                                                                                                                                                                                    1. Temperature (Hedrick and Waters, 2011)
                                                                                                                                                                                      1. At 24-25C
                                                                                                                                                                                        1. Intrinsic membrane properties that increase
                                                                                                                                                                                          1. Resting input resistance
                                                                                                                                                                                            1. membrane time constant
                                                                                                                                                                                              1. , curvature of the I–V relationship
                                                                                                                                                                                            2. At 36-37C
                                                                                                                                                                                              1. no difference in resting membrane potential
                                                                                                                                                                                            3. Stimulation protocol
                                                                                                                                                                                              1. no. of pulses
                                                                                                                                                                                            4. Plotting data

                                                                                                                                                                                              Nota:

                                                                                                                                                                                              • usually they plot the data using EPSPs, or amplitude of EPSCs; depol=up- easy to understand that way
                                                                                                                                                                                              1. Voltage (mV)- EPSPs
                                                                                                                                                                                                1. up=depol
                                                                                                                                                                                                2. Current (pA)- EPSCs
                                                                                                                                                                                                  1. down=depol
                                                                                                                                                                                                    1. EPSCs amplitude- up=depol
                                                                                                                                                                                                    2. dots=APs
                                                                                                                                                                                                      1. % change in amplitude
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