Techniques in synaptic plasticity

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
      2. 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
      3. in-vivo recordings
      4. 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
2. Intracellular
   1. Mainly in-vitro

      Nota:

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

      Nota:

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

         Nota:

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

            Nota:

            • to depolarise or hyperpolarise a cell
         3. 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
      3. Patch clamp
         1. configurations
            1. whole-cell

               Nota:

               • can be voltage-clamp or current-clamp mode
            2. cell attached
            3. 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
            2. 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
            3. Whole-cell
               1. intracellular properties

                  Nota:

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

                  Nota:

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

                  Nota:

                  • most are done at room temperature which can alter the function
               2. 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
                  2. 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
               3. 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
            5. 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; 
               2. 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
   5. 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
      2. graph goes up

         Nota:

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

         Nota:

         • variation of voltage/current clamp that allows many types of cells to be voltage-clamped with one electrode
      4. 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
      5. 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
               2. (+) 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
               2. (-) 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
                  2. experimenter should monitor the voltage at the headset of the amplifier using an oscilloscope
               3. 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
                  2. need long recovery time (>15min)

                     Nota:

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

                     Nota:

                     • check for a clean trace- no more than 1mV variation in resting membrane potential
                  4. artefacts- due to electrode movement
               4. (-) 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)
3. Stimulate/ record from?

   Nota:

   • usually stimulate presynaptic, record postsynaptic neuron
4. 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
      2. Acute brain slices

         Nota:

         • Any age most local circuits intact, long range targets severed
      3. 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
      4. 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
5. plotting voltage
   1. up

      Nota:

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

      Nota:

      • down=influx of ions

1. Genetically modified mice
   1. transgenic
   2. knockouts/ knockins
   3. Cre-lox
2. immunocytochemistry
3. fluorescence imaging of ion activity
4. microinjection of cDNA

   Nota:

   • to see the effect of a specif protein/ subunit?
5. western bolt
6. 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

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
   2. Schaffer-commissural synapses

      Nota:

      • Similar effects are seen at Schaffer-commissural synapses in area CA1 (Andersen, 1960).
   3. 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
      2. (+) Other tests
         1. fluorescence imaging
         2. exocytosis detection
         3. Drugs
         4. Genetically modified
         5. 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
3. 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
4. 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
      2. Current clamp
      3. 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
      4. 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
      3. 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
   3. whole-cell voltage clamp
5. 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
         2. number of pulses determine long-term LTP
6. primed-burst stimulation (PBS)

   Nota:

   • a brief burst of stimuli 
7. Low frequency stimulation (LFS)
   1. 1-3Hz

1. Agonists
2. Antagonists
   1. that activate upon activation of a receptor
   2. APV/ DGG
   3. ligand-activated non-competitive antagonists

      Nota:

      • for example an antagonist that 
3. Ca-chelating agents
4. uncaging

Adjunto:

• Techniques for STP and LTP

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

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
   2. EPSCs amplitude- up=depol
3. dots=APs
4. % change in amplitude