LTP

Question 1

Hebb (1949)

Answer 1

The cellular basis of learning involves the strengthening of synapse that is repeatedly active when the post-synaptic neurons fires. Increase in synaptic efficiency arises from postsynaptic cells repeated and persistent stimulation of a post synaptic cell.

Question 2

• Bliss and Lomo (1973):

Answer 2

In vivo extracellular field recording of an anaesthetised rabbit. Repetitive stimulation of the perforant path to the DG.
o Found that with a short burst of high Hz pre-synaptic activity it is possible to produce a change in EPSP amplitude that can be maintained for a significant time period ‘
o Potentiated for periods ranging from 30 minutes to 10 hours after one of more conditioning trains at 10-20/s for 10-15 sec or 100/sec for 3-4sc
o Reduction in latency of population spike and increase amplitude → indicates 2 independent pathways

Question 3

• Bliss and Gardner-Medwin (1973):

Answer 3

Stimulation of the perforant path been recorded in the DG of the hippocampus. Extracellular recordings. Non-anaesthetised rabbits
o Able to generate LTP for 1h to 3 days. → showed even longer term changes

Question 4

• Anderson et al (1980):

Answer 4

Input specificity LTP studied in CA1 region of guinea-pig hippocampal slices in vitro. Stimulating pulses were delivered alternatively in 2 independent afferent pathways, stratum radiatum and stratum oriens. Presynaptic volleys and field EPSPs were recorded from the same 2 layers. Electrode in the pyramidal cell body layer recorded the population spike or intra or extracellular potentials
o Short tetanus of the two input pathways produced a long-lasting enhancement of the field EPSP
o No corresponding changes were seen in the field potential responses to stimulation of the untetanised input path
o Concluded that the LTP is specific to the pathway which received tetanisation
o Following tetanisation there is a short lasting depression most often seen for the control pathway but sometimes visible on the tetanised side as well→ conclude that STD is not confined to any particular pathway but generalised phenomenon

Question 5

• Barrionuevo and Brown (1983):

Answer 5

Interactions between 2 excitatory monosynaptic inputs to hippocampal neurons of the CA1 region were examined in the in vitro slice. One weak and one strong synaptic response. Rat hippocampal slices in vitro. Extracellular and intracellular electrophysiological recordings. Extracellular recording microelectrode in the stratum radiatum and intracellular recording microelectrode in the stratum pyramidale of the CA1.
o Simultaneous tetanic stimulation of both input pathways resulted in long-term enhanced synaptic efficacy in weak input under conditions in which the same tetanic stimulation of either input alone failed to have this effect
o Associative LTP
o Passive membrane properties (e.g. rmp, input resistance and membrane time constant) remained fairly constant so could not explain

Question 6

• Gustafsson et al (1987)

Answer 6

Recoding from cell and injecting current into it. 2 distinct inputs. Ca1 region of the hippocampus slice → guinea pig. PA1 pyramidal cells were synaptically activated by electrical stimulation of afferent fibres in striatum radiatum or orien. Intracellular recorded EPSPs resulting from single volley stimulation at low frequency in the stratum radiatum or oriens were pared with depolarising current pulses injected through recording microelectrode
o Single stimulation of either pathway fails to induce LTP and the EPSP stays the same
o Conjunction – S1 stimulus is now followed 7ms later by a 400ms depolarising pulse.
o Depolarization of the post-synaptic cell → allows induction of LTP (associativity/cooperativity)
o It was specific to paired input, blocked by AP5 and was largely blocked by prior homeostatic tetanisation
o Also supports the inportance of NMDAR as show the importance of post-synaptic depolarisation for LTP induction
o Results suggest that the co-operativity aspect of LTP is related to a need for sufficient post-synaptic depolarisation

Question 7

• Malenka et al (1989):

Answer 7

Patched CA1 pyramidal cells and stimulating the Schaffer collaterals of CA3 cells. Hippocampal slices. Intracellular recordings onto pyramidal cells. Tetanic stimuli consisted by 2 1s 100Hz separated by 20 secs
o Need both pre and post synaptic activity to generate LTP. Providing a simple stimulus pattern and pairing it with a post-synaptic depolarisation of CA1 results in enhancement of EPSP. If simply provide post-synaptic depolarisation but no pre-synaptic stimulation then see no LTP
o Intracellular injection of into CA1 pyramidal cells of protein kinase inhibitor H-7, or calmodulin antagonist calmidazolium Blocks LTP
o CBP ( a potent inhibitor of CaM dependant activation of CaM-KII, → blocked LTP
o Appears that activation of post-synaptic CaM is critical for generation of LTP
o – Potential roles of other kinases can’t be rules out

Question 8

• Collingridge et al (1983):

Answer 8

fEPSP recordings of synapses in CA1 region of rats with administration AP5 (NMDA receptor antagonist). Applied to brain slice . Trying to induce LTP with 100Hz for 1 second
o AP5 blocks the induction of LTP (see an small change in EPSP magnitude but quickly returns back to baseline)
o Had no effect on baseline response o on LTP that has already been induced

Question 9

• Nowak et al (1984):

Answer 9

Analysing the response of mouse central neurons to glutamate using patch clamp recordings. Performed on Mescenphalic or striatal neurons . In vitro, whole cell recordings:
o NMDARs required both depolarisation and Glu binding to open. Important as showed why NMDAR are active in LTP. However something else must carry the current to generate EPSP during baseline so must be another Glu receptor
o Found a link between voltage sensitivity and Mg2+ sensitivity
o In Mg2+ solution, NMDA open cation channels, the properties of which are voltage independent
o In the presence of Mg2+ probability of the opening of channels is reduced, increase steeply with hyperpolarisation (thereby accounting for the negative slope of the I-V relationship of the glutamate response
o The voltage dependence of NMDA receptor-linked conductance appears to be consequence of the voltage dependence of the Mg2+ block

Question 10

• Lynch et al (1983):

Answer 10

rats. Stimulating electrode of the Schaffer collaterals and extracellular recording electrode in CA1 of the pyramidal cell body layer. Injection of EGTA (Ca2+ chelator) via intracellular electrode into the post-synaptic neurons. HSF
o Prevent subsequent induction of LTP in response to HFS

Question 11

• Malenka et al (1988):

Answer 11

Photolysis of Ca2+ loaded nitro-5 (makes it inactive, light sensitive, Ca2+ chelator) (mechanism predates optogenetics but still an important study). Nitr-5 injected into hippocampal CA1 pyramidal cells. Extracellular population EPSP recordings
o Ca2+ alone is sufficient to induce LTP
o Buffering intracellular Ca2+ at low concentrations blocked LTP . Examined the effect of nitr-5 on LTP- tetanic stimulation (before photolysis it is a chelator of Ca2+)→ suggests influx of Ca2+ is important
o But how does this increase in Ca2+ trigger ltp? May be activation of Ca2+ dependant protein kinase

Question 12

• Rehehr and Tank (1990):

Answer 12

Performed microfluorometric (a fluorescent spectrophotometer combined with microscope) measurements in individual CA1 pyramidal cells during HFS to induce LTP. Pyramidal cells filled with Fura-2. Guinea pig hippocampus slices. Stimulation electrode in stratum radiatum . 
o	HFS stimulation induced NMDAR dependant increase in intracellular Ca2+ 
o	Spatially localised to dendritic area near activated afferents → localised component blocked by NMDAR antagonists Calcium accumulations observed during synaptic activation can be divided into 2:
o	AP5 insensitive accumulations widely distributed over the apical and basal dendritic trees and produced in response or moderate and high freq stimulus strain→ could play part in second messenger modification E.G. LTD anti-hebbian reductions of synaptic strength

Question 13

• Malinow et al (1989):

Answer 13

dialysed into the post-synaptic neurons. Inhibitors of CAMKII. Intracellular microelectrode used to record synaptic potentials and deliver protein kinase inhibitors to Ca1 pyramidal cells of rat hippocampal slices. Intracellular recordings
o Blocks LTP (Also use a scrambled version of the peptide, fails to block LTP)
o Induction of LTP is blocked by intracellular delivery of H7, a general protein kinase inhibitor, or PKC(19-31), a elective protein kinase C inhibitor, or CAMKII(273-302), a selective inhibitor of CAMII. (modified PKC(19-31) which is less effective against PKC did not block LIP not did a modified CaMKII which does not inhibit CAMKII acitivt )
 After its establishment, LTP appears unresponsive to post-synaptic H7 although remains sensitive to externally applied H7
o both PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP
o CAMKII is not required for the maintenance of LTP
o - Surprising as they knew that phosphorylation is a short process yet LTP can be expressed for years. So is CAMKII is responsible for phosphorylating AMPA then presumably one needs to keep it activated to repeatedly phosphorylate AMPA
o Resulted in concerns with the CAMKII hypothesis model

Question 14

• Silva et al (1992):

Answer 14

-CAMKII KO mice. Whole cell recordings / Feid potential recordings and whole cell recordings. Hippocampal slices, stimulating Schaffer collaterals using whole cell recordings
o Deficient in ability to produce LTP→ No LTP (a couple of cells produced LTP and where is does occur appears indistinguishable from what is observed in normal animals)
o So CAMKII may be involved in regulatory pathway for the processes responsible for LTP
o - Deficit in LTP could be caused by decreased number of connections between CA3 and CA1 neurons→ though no anatomical evidence relating to number of connections, but electrophysiological data make it highly unlikely

Question 15

• Derkach et al (1999):

Answer 15

Identified a potential mechanism by which CaMKII potentiates AMPARs. Co-expression in HEK-293 cells of activated CaMKII with GLuR1 a mutation increased the contribution of the higher conductance states (phosphorylation site→ attempted to mimic the effect by introducing a negative charge through mutation). Patch pipettes and glutamate applied. Whole cell and outside out patches
o (CAMKII enhances phosphorylation of GluR1 )
o Did not effect the glutamate affinity of the receptor, kinetics of desensitisation, and recovery, channel rectification, open probability of gate
o Single channel recordings identified multiple conductance states for GluR1 and co-expression with CaMKII increased the contribution of higher conductance states → indicates that CaMKII can mediate plasticity at glutamatergic synapses by increasing single-channel conductance of existing functional AMPAR or by recruiting new High-conductance state AMPAR
o Only channel conductance increased significantly
o (unlikely that number of functional GluR1 receptors with same conductance properties also

Question 16

• Le Roux et al (2013):

Answer 16

PV-expressing interneurons (major source of inhibition and they influence both spike timing precision and network oscillations) of CA1. Afferent-specific stimulation was paired with either postsynaptic huperpolarisation or depolarisation
o Show that FF and FB synapses onto PV Ins are endowed with distinct postsynaptic glu receptors which set opposing long-term plasticity rules
o Inward rectifying CP-AMPARs expressed at both FF and FB inputs mediate a form of anti-Hebbian LTP, relying on coincident membrane hyperpolarisation and synaptic activation
o NMDArs are abundant at FB afferents (largely devoid in FF inputs) → confer an additional form of LTP with Hebbian properties
o The specific endowment of FF and FB inputs with distinct coincidence detectors allow them to be differentially tuned upon high frequency afferent activity
o Propose that the differential input specific learning rules may allow PV Ins to adapt to changes in hippocampal activity while reserving their recisely timed, clockwork operation
o Used Application of Naspm to block CPAMPAR prevented antihebbian LTP
o Application of AP5 block NMDARs and induction of Hebbian LTP
o Antihebbian plasticity may ramp up synaptic strength id network excitation fails to recruit inhibitory circuits → maintain E/I balance
o Hebbian plasticity might enhance lateral feedback inhibition for persistently active pyramidal neurons
o Results reveal the 2 major excitatory inputs onto CA1 PV+ interneurons undergo long term plasticity with different frequency regimes of afferent activity which is likely to influence their function under both normal and pathological conditions

Question 17

• Lamaa et al (2007):

Answer 17

Looked at associative LTP in the interneurons in stratum oriens of hippocampus (CA1), (many neurons here show NMDAR-independent LTP) while recording with gramicidin perforated patch techniques to preserve intracellular polyamines.
o Report that LTP at synapses on hippocampal interneurons mediating feedback inhibition is ‘anti-hebbian’. Induced by presynaptic activity but prevented by postsynaptic depolarisation
o Anti-hebbian LTP may occur in interneurons that are silent during periods of intense pyramidal cell firing such as sharp wave and lead to their altered activation during theta activity
o So LTP in the excitatory synapses on interneurons is prevented by associative pairing (direct contrast to NMDAR dependant LTP)
o 8 out of 11 cells, pairing with hyperpolarisation but not depolarisation resulted in LTP
o And low frequency stimulation was found to trigger LTP if interneurons were hyperpolarised → anti-Hebbian
o Tested the same LTP induction protocols in Ins of the stratum radiatum
o Hebbian LTP could be elicited in about half of these cells ((many of which mediate feedforward inhibition) whereas mediating HFB or low freq stimuli with hyperpolarisation was uniformally unsuccessful
o Anti-hebbian LTP is thus characteristic of excitatory synapses made by local pyramidal cells on interneurons in the oriens/avleus but not schaffer collateral synapses on interneurons in the stratum radiatum (was found in 2 neurons)

Question 18

• Ling et al (2002):

Answer 18

PKMζ (introduction of new messenger). Staurosporine is a inhibitor of PKC family but doesn’t inhibit PKMζ so is a useful control in this regard. Whole cell recordings of CA1 pyramidal cells with PKMζ in electrode. Voltage clamped to -75mV to isolate MAPAR mediated events. Rats hippocampal slices
o First and only time in the literature where see that when the messenger is inhibited, once LTP is established the EPSP goes back to baseline
o Diffusion of PKMζ into cells enhanced EPSC amplitude within 6 mins and stabilised after 10min
o PKC inhibitor reverse potentiation. →Chelerythrine
o CNQX( a non-NMDAR glu receptor antagonist) suppressed PKMζ augmented EPSCs indicating enhancement of AMPAR currents.
o PMKζ mediated increases in EPSCs completely occluded LTP (initial PTP was still observed)
o (Staurosporine ( general kinase inhibitor but doesn’t block atypical PKCs) Chelerythrine at low concs selectively inhibits PKMζ relative to convention and novel PKCs and CaMKII)
o Staurosporine blocked induction but had no effect on maintenance after tetanisation

Question 19

• Volt et al (2013):

Answer 19

Knocked-out PKMζ in a model system. Hippocampal slices Generated transgenic mice lacking PKCζ and PKMζ. Auditory trace fear conditioning, spatial reference memory
o LTP was normal and in a whole bunch of behavioural tests so was the capacity to learn
o Show normal conventional and conditional KO mice show normal synaptic transmission and LTP at schaffer collateral- CA1 synapses and have no deficit in several hippocampal dependant learning or memory tasks
o ZIP still reverse LTP in PKCζ/PKMζ KO mice, indicating that effects of ZIP are independent of PMKζ

Question 20

• Zakarenko et al (2001):

Answer 20

Load neurons with FM1-43 (marker of presynaptic activity) before and after the induction of LTP. (Logic if vesicles are released in greater number/higher probability then should see a change in the fluorescence profile of the labelled terminals that would decay more quickly as a function of there being more release and therefore more FM1-43 being released) Mice hippocampal slices. Directly image changes in presynaptic function during both short-term and long term forms of plasticity in presynaptic boutons of CA3-CA1 excitatory synapses in acute hippocampal slices. Combine with 2 photon laser scanning microscopy
o FM1-43 unloading is accelerated after induction of LTP (interpretation that there has been a change in NT release in erms of number or probability after LTP and either of these things are true then suggests strongly that LTP is accompanied with a change in the presynaptic terminal )
o Enhanced presynaptic function during LTP induced wither chemically (CH3(4)NH4) or by HFS
o -Possible released for increased unloading: increase probability of synaptic vesicle fusing, increase rate in which a vesicle releases its cargo following fusion, increase number of active sites
o Induction of LTP with 200Hz tetanic stimulation but no significant effect for FM1-43 unlading following induction of LTP with lower frequency (50 or 100Hz)

Question 21

• Emptage et al (2003):

Answer 21

Optical quantal analysis. Used confocal microscopy and Ca2+ sensitive dyes, BAPTA-1 to study LTP at individual visualised synapses. Synaptically evoked Ca2+ transients were imaged in distal dendritic spines of pyramidal cells in cultured hippocampal slices (rats). Monitor transmission at individual synapses made by associational to Schaffer collateral fibres on distal apical dendrites of CA3 and CA1 pyramidal neurons. Optical methods have been used to monitor quantal parameters of transmission at individual synapses
o Show that with induction of LTP the probability of NT release is increased
o Strongly suggests that there is an increase in probability of Ca2+ release and so LTP does induce changes in presynaptic locus
o Most synapses sustain an increase in amplitude of evoked Ca2+ transients reflecting an increased release of Ca2+ from internal stores in the spine
o Increase in PCa2+ seen in most spines after LTP induction indicates increase in probability of NT release

Question 22

• Schuman and Madison (1991):

Answer 22

EPSP were evoked in the striatum radiatum of hippocampal slices by test stimuli delivered to Schaffer collaterals. Tetanic stimulation
o Extracellular application or post-synaptic injection of 2 inhibitors of NOS block LTP.
o Extracellular application of Haemoglobin which bind NO also attenuates LTP → suggests that NO liberated from post-synaptic neurons may travel back to the presynaptic terminal to cause LTP expression
o Data provide evidence that the post-synaptic pyramidal neuron is site of NO production.
o – Does not directly show that it is a retrograde simgnaller

Question 23

• Padamsey et al (2017):

Answer 23

Using photolytic Glu uncaging technique So take a synapse where optical quantal analysis approach has been used to measure synaptic transmission and show that it’s a relatively low NT release probability and then artificially present Glu at the synapse in a way of synaptic transmission whilst not stimulating presynaptic terminal. So post-synaptic receptors believe that NT transmission is occurring, but the presynaptic terminal is not being activated. Looked at CA3-CA1 hippocampal synapses. Use both Ca2+ imaging and PPR(paired pulse ratio) in cultured and acute hippocampal slices using a number of pharmacological and genetic manipulations. (PPR: reflects aggregate changes in PR over large number of stimulated synapses.. Indirect measure of Pr)
o If create the ‘perfect’ synapse, where Glu is presented at each and every stimulus. If do this then don’t get LTP, so despite that you’ve provided all this Glu you don’t get a change in NT release probability (Pr)
o If however you don’t provide artificially a perfect synaptic transmission, you do get an increase in Pr. Seems to suggest when the system is already optimised (Where NT release is occurring out of proportion with its impact on the post-synaptic neurons) there is no LTP, but where NT release is occurring at a time where there is insufficient transmitter release to account for the amount of post-synaptic depolarisation then the presynaptic terminal adjusts its efficiency its. Weight, up to the level one may predict for the amount of post synaptic depolarisation so it’s a sort of trial and error
o Glutamate is required for depression but not potentiation of long-term presynaptic function
o - Paper was very controversial for the field and produced a lot of upset as was saying in the paper that Glu release is actually required to generate depression and glu release per se does not potentiate the presynaptic terminal.
o Find that Hebbian activity, via LVGCC triggered NO signalling, is sufficient to induce LTPPRE but Glu released it inhibits LTPPRE and promotes LTD pre by activating NMDARs. So for presynaptic potentiation to occur, a presynaptic neuron must not only fire together with its presynaptic partner byut also fail to release Glu
o When glu release occurs it decreases Pr by activating presynaptic NMDARS and promotes presynaptic LTD
o Net changes in Pr depending on opposing factos: 1 Hebbian activity which increase Pr and 2 glutamate release which decrease Pr
o Potentially operate to tune synaptic function. May explain why sometimes the same pairing protocol that produces LTPpre at low Pr synapses, produce LTDpre at higher Pr synpses
o In this new model the levels of Gu release at synapse will not only depend on basal Pr but also on the pattern of presynaptic activity on the state of the synapse.

Question 24

• Isaac et al (1995):

Answer 24

): Electrophysical quantal analysis. So in an attempt to reduce the number of synapses that could be recording from, performing minimal stimulation, potential that would be adequate to relieve the Mg2+ block of post-synaptic NMDA receptors. Use NMDA antagonist AP5, CA1 cells
o One issue with this technique (electrophysical quantal release) is that not really sure how many synapses are contributing to EPSP that you record in the cell soma. Still assuming that just recording from single synapse, may or may not be true, may just be recording a few very weak synapses.
o Held at -60mV then no evidence of EPSP. However when moved the clamp recording to +30mV see evidence of EPSP being generated, reflect that a current being detected. It cant be AMPAR otherwise would see a current at -60mV
o At +30mV, AP5 abolished synaptic events→ looking at a signal similar to that at -60mV. So appear there is a situation where we have a response being carried by NMDAR alone. That at rmp is silent but at depolarised potential becomes active
o When functionally silent synapses are subjected to LTP induction protocol, EPSCs mediated by AMPAR appear and remain for the duration of the experiment → conversion of silent synapse to functional synapse is blocked by AP5
o Suggest that LTP may involve modifications of AMPAR that prior to LTP were either not present in the postsynaptic membrane or electrophysiologically silent → may account for several experimental results previously attributed to presynaptic changes in quantal content

Question 25

• Liao et al (1999):

Answer 25

Immunohistochemical localisation for NR1 and Glu1. Cultured hippocampal neurons (Rats)
o Show that there are synapses that only express NMDAR and there are synapses that show both
o But none of just AMPAR. Initially most synapses contained NMDAR but only a few contained detectable AMPAR → synapses progressively acquired APMAPR as cultures matured
o AMPAR blockade increased the number size and fluorescent intensity of AMPAR receptor clusters and rapidly induced the appearance of AMPAR at silent synapses
o Whereas NMDAR blockade increased size, intensity and number of NMDAR clusters and decreased the number of AMPAR receptor clusters, resulting in an increase in proportion of silent synapses
o Suggest that the number of silent synapses is regulated during development and by changes in synaptic activity
o Only looked at blockade not looked at changes in synaptic activity

Question 26

• Shi et al (1999):

Answer 26

To monitor changes in AMPAR distribution in living neurons. GluR1 tagged with GFP.2 photon laser scanning microscopy or electron microscopy. Hippocampal slice cultures CA1 region
o Used GFP attached to GluR1 → trafficked into the dendritic spines, presumably to the surface membrane of dendritic spines, in response to induction of LTP.
o Tetanic stimulation induced rapid delivery of tagged receptors to the dendritic spines as well clusters in dendrites
o Required NMDAR activation (AP5 )
o May contribute to the enhanced AMPAR receptor mediated transmission observed during LTP and activity dependant synaptic maturation

Question 27

• Granger et al (2013):

Answer 27

Artificially express Knate receptors (receptors that wouldn’t usually be found at the particular synapse they studied) and demonstrated that you can maintain LTP through the insertion of these receptors in the membrane. To find minimum necessary requirement of CluA1 C-tail for LTP in mouse CA1 hippocampal pyramidal neurons→ used single cell molecular replacement strategy to replace all endogenous AMPAR receptors with transfected subunits . Whole cell and outside out patch recordings Slice hippocampal recordings
o Found no requirement of GluA1 C-tail for LTP
o Replacement with GluA2 subunit showed normal LTP as did an artificially expressed kainate receptor