Techniques for STP and LTP

1. Paired pulse stimulation
   1. highly used
   2. changes in PPR
2. Drugs
   1. open-channel blockers to estimate the probability of release, Pr.
      1. noncompetitive- ligand activates inhibitors of NMDARs
      2. highly used
3. Optical quantal analysis
   1. changes in Pr
4. Ca- indicators

   Annotations:

   • PCa= probability of evoking a Ca2 transient in the visualised spine Changes in Pr at single synapses following induction of LTP in pyramidal cells of areas CA1 and CA3 has been assessed using Ca2 indicators to monitor synaptic events at single visualised spines in organotypic hippocampal cultures
   • Emptage et al. 2003 argued that the probability of evoking a Ca2 transient in the visualised spine, PCa, provides an accurate measure of the probability of transmitter release, Pr, at that synapse, since manipulations that reflect an increase in Pr, such as paired pulse facilitation, also result in an increase in PCa, whereas manipulations that decrease Pr, such as bath application of adenosine, lead to a decrease in PCa.
   1. (-)

      Annotations:

      • The indirect nature of the Ca2 transient raises the possibility that the observed increase in PCa following LTP may reflect an increase in the coupling between the initial trigger Ca2 and store release, rather than an increase in Pr.
5. fluorescnece tagged proteins

   Annotations:

   • transfect neurons with a glutamate-binding protein tagged with a pair of fluorophores that yield a fluorescence resonance energy transfer (FRET) signal when L-glutamate is released from the presynaptic terminal (Okumoto et al., 2005).

1. presynaptic/ postsynaptic Ca2+

1. glial currents as a monitor of neurotransmitter release
   1. highly used
2. measuring L-glutamate

   Annotations:

   • 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. push-pull perfusion technique

      Annotations:

      • Errington et al., 1987, but see Aniksztejn et al., 1989
   2. glutamate-sensitive electrode

      Annotations:

      • Errington et al., 2003
   3. (-) doesn't show how glutamate increases
3. biotinylation of vesicular transporters

   Annotations:

   • measure presynaptic activity through the biotinylation (attaching biotin to a protein, nucleic acid or other molecule) of vesicular transporters in vesicles fused with presynaptic membranes during neurotransmitter release.  This method allowed us for the first time to selectively quantify the spontaneous or evoked release of glutamate or GABA at their respective synapses- Labeling of vesicular transporters occurs immediately when synaptic vesicles are released and exposed to the extracellular medium
   1. Xu et al (2013)
   2. How?

      Annotations:

      • Vesicular transporters are transmembrane proteins specifically located in vesicles within the presynaptic terminals and are therefore separated from the extracellular matrix by the presynaptic membrane (Fig. 1A). However, during either spontaneous or evoked transmitter release, synaptic vesicles fuse with the presynaptic plasma membrane and hence the lumenal domains of these transporter proteins are exposed to the extracellular medium, making them accessible to membrane-impermeable biotinylating reagents added to the extracellular medium. The vesicular transporters are specific for the type of synapses using the corresponding neurotransmitter (McIntire et al., 1997; Takamori et al., 2000), and therefore their biotinylation allows selective monitoring of the release of specific neurotransmitters
   3. Considerations
      1. -

         Annotations:

         • Despite these shortcomings, this method (when combined with currently available techniques) is a valuable approach to provide new insights into modifications and underlying mechanisms of presynaptic release.
         1. all synapses

            Annotations:

            • This technique measures the overall activity at all synapses in the culture without providing information on alterations in presynaptic activity at the single-synapse level
         2. indirect measure

            Annotations:

            • this technique measures synaptic vesicular turnover instead of the actual amount of released neurotransmitters, and as such, it can only be used as a semiquantitative method to indicate upregulation or downregulation but cannot be used to detect quantitative changes in transmitter release
         3. reduce the accuracy of using vesicular protein labeling

            Annotations:

            • surface membrane expression of vesicular proteins may reduce the accuracy of using vesicular protein labeling to estimate presynaptic release
      2. +
         1. simultaneous detection of presynaptic modifications and postsynaptic alterations

            Annotations:

            • allow the simultaneous detection of presynaptic modifications and postsynaptic alterations, such as the internalization of AMPARs.  This unprecedented advantage allows the analysis of potential relationships between presynaptic and postsynaptic changes under different experimental conditions and, in particular, after the induction of various forms of synaptic plasticity.
         2. differentiate various types of synapses

            Annotations:

            • can be used to selectively monitor vesicular turnover at glutamatergic and GABAergic synapses, thereby providing a selective, direct, and semiquantitative measurement of presynaptic glutamate or GABA release
4. antibody-fluoro lablelling

1. fuorescnece markers
2. antibodies- synaptic vesicle protein synaptotagmin

   Annotations:

   • Direct evidence for a presynaptic mechanism in cultured hippocampal neurons was obtained by measuring exocytoticendocytotic cycling with antibodies against the synaptic vesicle protein synaptotagmin (Malgaroli et al., 1995)
3. rate of destaining

   Annotations:

   • Another direct way of estimating the probability of glutamate release is to measure the rate of destaining of synaptic vesicles loaded with the steryl dye FM1-43- when vesicles are released then there would be a decrease in the staining seen

1. LTD
   1. spike timing dependent stimulation

      Annotations:

      • post before pre
      1. Anti hebbian
   2. low frequency stimulation
   3. PP-LFS
   4. agonist-DHPG- induced LTD
2. LTP
   1. Paired-pulse
   2. HFS/ tetanus
   3. paired
      1. spike timing dependent

         Annotations:

         • pre before post
         1. Hebbian
   4. priming/ theta burst stimualtion

1. 1. Prepared brain
2. 2. stimulating and recording electrodes located
3. 3. baseline measurements
4. 4. stimulation protocol
5. 5. return to low-frequency stimulation
6. 6. Pharmacological manipulations

1. changes in receptor expression
2. immiuno
3. fluoro

1. expression of LTP
   1. increase in no. receptors

1. knockouts/ knockdowns
2. overexpression/ underexpression of proteins/signalling molecules
   1. Cre-lox

1. subunits tagged with GFP

   Annotations:

   • recombinant AMPA receptor subunits are constructed containing a reporter, such as green fluorescent protein (GFP).
   1. mutant receptors
2. antibody labelling

   Annotations:

   • antibodies that recognize extracellular epitopes of AMPA receptor subunits are used, in conjunction with protocols that induce NMDAR-dependent LTP in dissociated cultured hippocampal neurons
   1. stimulation protocols
   2. fluorescently conjugated secondary antibodies

1. downregulation of receptors
   1. photolytic uncaging of caged glutamate

      Annotations:

      • Kandler et al., 1998; Rammes et al., 2003
   2. iontophoresis of NMDA

      Annotations:

      • Montgomery and Madison, 2002
   3. Immunocytochemistry

      Annotations:

      • immunocytochemical estimates of both AMPA and NMDA populations in tissue from animals in which LTD was induced in vivo (Heynen et al., 2000).

Annotations:

•    Describe the methodology that you would use, but don’t go into unnecessary detail. For instance, it will rarely be good enough to only state that you are doing electrophysiological recording. If it is in vivo you should state whether you will be recording ECG, EEG, EMG or extracellular field or unit activity (intracellular neuronal recordings are possible in vivo but technically difficult). If suggesting imaging, then what type, calcium fluorescence, voltage sensitive dye, intrinsic imaging, fluorescence microscopy, fMRI etc… If suggesting a genetic approach then outline how it would work even if you can’t describe the genetic modifications in detail. If you’re suggesting in vitro electrophysiology you need to state whether the recordings are sharp electrode, patch electrode etc.   

1. 1. receptor expression studies OR genetic manipulation;
2. 2. electrophysiology;
3. 3. behavioural testing.
4. strengths and weaknesses
5. Justify your use of preparation

   Annotations:

   • If experimenting on cells in vitro, then what type of cell? From which species? Cultured or freshly dissociated etc… If you are using animals then justify the species, and if appropriate the use of inbred vs outbred strains and the sex of the animals. Note that you may want to design an experiment using more than one strain or sex to increase the generality of the conclusions. If suggesting genetic modification then note that this is predominantly performed on lab mice and has only recently been extended to rats. You should not be suggesting germline genetic manipulation in any other species – use a viral vector etc… approach instead.
6. experimental groups and controls

   Annotations:

   •    Make sure that you discuss what experimental groups and controls that you would use. Most experiments either use methodological controls to ensure that the method is working correctly, or there are control groups, e.g. saline injections as controls for drug injection that is treated similarly to the experimental group so that the only thing that differs is the factor of interest.   
7. recording and anlysis

   Annotations:

   •    State what measurements would be recorded and how they would be analysed and how the results would be used to test the hypothesis.