Excitation Flashcards
(15 cards)
• Lante et al (2011):
o investigate the impact of sleep on excitatory synaptic transmission, we obtained whole-cell patch-clamp recordings from layer V pyramidal neurons in acute slices of somatosensory cortex of juvenile rats Aim of the study was to investigate the influence of the sleep wake cycle on synaptic AMPAR composition and then to assess whether these modifications could result from NREM associated firing patterns
o Study shows time of day fluctuations in AMPAR composition at excitatory cortical synapses with CP-AMPARs being present after the dark episode and being detectable after the light phase
o The CP-AMPAR removal occurred gradually over time and was significant after 4 hours.
o Reasonable to conclude that the selective loss of CP AMPARs from cortical synapses is associated with sleep
o – could be a role of clock related parameters
o In animals after the dark period, (PhTx)-sensitive calcium-permeable AMPA receptors (CP-AMPARs) accounted for 25% of total EPSP size, and current–voltage (I–V) relationships of the underlying EPSCs showed inward rectification. After the light period, EPSPs were PhTx insensitive with linear I–V characteristics, indicating that CP-AMPARs were less abundant. C
o Subsequently, we show that burst firing during slow-wave sleep has a dual impact on synaptic AMPA receptor composition: repetitive burst firing without synaptic stimulation eliminated CP-AMPARs by activating serine/threonine phosphatases. Additionally, repetitive burst-firing paired with EPSPs led to input-specific (LTD), affecting CP- AMPARs via protein kinase C signalling. In agreement with two parallel mechanisms, simple bursts were ineffective after the light period but paired bursts induced robust LTD. In contrast, incremental LTD was generated by both conditioning protocols after the dark cycle.
• Nowak et al (1984):
Mesencephalic or striatal neurons in mouse. Patch clamp, whole cell recordings
o In Mg2+ free solution, glu elicited and inward current at neg potentials→ varied linearly with membrane potential, reversal potential was 0mV. → voltage independent
o Mg2+ single channel currents observed at positive potentials but not at negative potentials→ fast channel block (transient burst of activity) → link between voltage sensitivity and Mg2+ sensitivity.
o Assumes that NMDA as NMDA elicited similar responses to L-glut
o Mg2+ a voltage dependant ‘gate
• Stern et al (1992):
Looked at shape of fast EPSP at different holding potentials in the neurons Voltage and current recordings were made from visually identified nonpyramidal neurones in slices of layer IV of rat primary visual cortex using the whole-cell patch clamp technique. Excitatory postsynaptic currents (EPSCs) were recorded from these neurones during focal stimulation of neighbouring cells by a second patch pipette.
o >-70 mV mp, the decay of stimulus evoked EPSCs showed two components with very different time courses. In standard extracellular solution the (I-V) relation for the fast component was almost linear whereas the slow component showed a J -shaped I-V relation with a region of negative slope conductance between -30 and -70 mV.
o The fast EPSC component was blocked by CNQX-AMPAR antagonist), the slow component by APV – NMDAR antagonist. This suggests that EPSCs are mediated by both the AMPA/KA and the NMDA subtypes of the GluR channel.
o The I-V relationship of the slow component of EPSCs changed when Mg2+ was omitted from the solution. In Mg2”-free solution the slope conductance of the slow component showed a linear voltage dependence suggesting that the region of negative slope conductance arises from the voltage-dependent Mg2’ block of NMDA receptor channels.
• Berretta and Jones (1996):
Layer II neurons if entorhinal cortex. Control and DAP5 treated.
o Electrophysiological evidence for the existence of presynaptic NMDA receptors which can tonically facilitate glutamate release in the CNS.
o AP5 reduced the frequency, but not amplitude, of glutamate-mediated spontaneous excitatory postsynaptic currents in layer II neurons of the rat entorhinal cortex.
o This effect was also observed in the presence of tetrodotoxin and when postsynaptic NMDAR receptors were blocked by dialysis with dizocilpine maleate.
o Tendency for paired-pulse facilitation of excitatory postsynaptic currents evoked at short (50 ms) intervals with postsynaptic NMDARs blocked) to be reduced by AP5, although this did not reach significance.
o These data strongly support the presence of presynaptic NMDA autoreceptors which may facilitate glutamate release in layer II of the entorhinal cortex.
o Paired pulse facilitation is considered to be a presynaptic phenomenon, resulting from a transient increase of presynaptic calcium, caused by conditioning synaptic response
o The repetitive activation of afferents to layer II resulted in short term frequency dependant enhancement of Glu transmission → could have a role in short term enhancement of transmission in the EC.
o Show that frequency but not amplitude change → suggest a presynaptic event
• Satake et al (2000):
Explored interactions between cerebellar excitatory and inhibitory synapses, Determine whether the CF excitatory transmitter causes heterosynaptic interactions with synaptic inputs converging on rat cerebellar interneurons and PCs, Microelectrodes places on cerebellar cortex were investigated with whole cell recordings from PCs visually identified in rat cerebellar cortex.
o Climbing fibre stimulation triggers glutamate release from CF that targets presynaptic AMPA receptors on GABAergic interneurons, thereby inhibiting presynaptic GABA release (no effect of AP5)
o Both post- and presynaptic actions were mimicked by application of AMPA and inhibited by the AMPA-type glutamate receptor-selective antagonist GYKI 53655.
o We found that a typical excitatory transmitter released from the climbing fiber (CF) in the cerebellar cortex not only excited Purkinje cells directly but also presynaptically inhibited GABAergic transmission from interneurons converging on the same Purkinje cells.
o Both homosynaptic and heterosynaptic actions of the CF transmitter (possibly glutamate) were mediated by activation of AMPA receptors.
o Dual AMPA receptor-mediated functions of excitation and disinhibition may ensure transmission of cerebellar CF signals controlling sensorimotor coordination.
o (may help mediate DSI)
o Indicates a diversity of GluR functions in information processing at central synapses
o - reinforces the output of the cerebellar cortex through PCs
• Satake et al (2004)
This study therefore aimed at further examining the properties of AMPAR-mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum brain slices. Patch electrodes. Stimulation to to evoke interneuron mediated IPSCs. Ca2+ monitored with BAPTA1 and confocal microscope. Drugs used AMPA, MCPG, CPPG, APC, AIDA, DCG-IVE. Purkinje cells (receive GABA inhibitory interneurons from basket and stellate cells); =
o Showed that AMPARs mediate their inhibitory effect by reducing their inhibitory effect by reducing Ca2+ influx through presynaptic voltge gated Ca2+ channels in basket cells
o The climbing fibre-induced inhibition of GABA release from interneurons depends on AMPAR-mediated activation of GTP-binding proteins coupled with down-regulation of presynaptic voltage-dependent Ca2+ channels.
o A Gi/o-protein inhibitor abolished the AMPAR-mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR-mediated excitatory actions on Purkinje cells. Furthermore, Gi/o-coupled receptor agonists and the P/Q-type calcium channel blocker markedly occluded the AMPAR-mediated inhibition of GABAergic transmission.
o Conversely, AMPAR activation inhibited action potential-triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons.
o V gated Ca2+ channel antagonist suppressed GABAa IPSC→ inhibition GABA release from presynaptic terminal of BCs
o Suggests that AMPAR-mediated G-protein signalling is negatively coupled to P/Q VDCC
o By suppressing the inhibitory inputs to Purkinje cells, the AMPAR-mediated presynaptic inhibition could thus provide a feed-forward mechanism for the information flow from the cerebellar cortex.
o - may present a mode of gain control for synaptic transmission
• Schmitz et al 2000:
Hippocampal slices from rats. Field recordings ( in presence of blockers of AMPAR and mGluRs, DA5. Field potential recordings patch pipettes in CA3 or DG. Electrodes in the granule cell layer to stimulate mossy fibres
o Show that KAR activation depolarises hippocampal mossy fibre axons. Demonstrated that glu released from mossy fibre synapses can activate presynaptic KARs (also occurs from neighbouring mossy fibres). Evidence that activation of KARs strongly inhibits transmitter release from mossy fibres
o Show that KAR (kainate receptor) agonists are shown to enhance fibre excitability, and a number of experiments show that this is a direct effect of KARs on presynaptic fibres
o KAR activation inhibits evoked transmitter release from mossy fibre synapses
o These results indicate that KARs, through endogenous release of Glu mediate EPSPs after presynaptic excitability and modulate transmitter releasee
o - many of experiments were frequency of 200Hz, so physiological relevance is questionable (but did see effects at 35 Hz)
• Rodriguez-moreno and Lerma (1998):
Hippocampal slices (contrary to the above) o We have found that the action of kainate on the hippocampal inhibitory postsynaptic current (IPSC) is mediated by a metabotropic process that is sensitive to Pertussis toxin (PTx) and independent of ion channel current. o The downregulation of GABA IPSCs by kainate was also prevented in a dose-dependent manner by calphostin C, a specific inhibitor of PKC, and the inhibition of phospholipase C (PLC) drastically reduced the action of kainate. o These results demonstrate that the activation of kainate receptors triggers a second messenger cascade, which results in the stimulation of PKC, and therefore document a metabotropic action of kainate receptors, which results in the inhibition of GABA release.
• Kakegawa et al (2011):
examined the effect of exogenous d-Ser on Purkinje cells using whole-cell patch-clamp recordings from cerebellar slices acutely prepared from wild-type immature mice on postnatal day, amp of PF EPSP gradually decreased. We loaded peptides that disrupt the interaction between GluA2 and adaptor protein 2 (AP2), an adaptor complex necessary for clathrin-dependent endocytosis, through patch pipettes into wild-type immature Purkinje cells. Furthermore, the application of d-Ser did not cause a reduction in PF-EPSCs after the induction of LTD
o D-Ser serves as an endogenous ligand for Gluδ2 to regulate long-term depression (LTD) at synapses between parallel fibres and Purkinje cells in the immature cerebellum.
o D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibres in immature, but not mature, cerebellum.
o D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells.
o Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.
o - A role for d-Ser in regulation of cerebellar function and LTD in early development
• Hartmann et al (2008):
First unequivocal evidence that TRPC can underlie slow excitatory currents observed at synapses in response to NT release. In cerebellum, looking at parallel fibre Purkinje cell synapses (give fast AMPAR EPSP and slow EPSC with mGluR1 activating TRPC3) We examined synaptic transmission at PF-Purkinje neuron synapses in acute mouse cerebellar slices by combining whole-cell recordings and confocal calcium imaging.
o TRPC3 is needed for mGluR-dependent synaptic signalling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells.
o In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+release signals from intracellular stores are unchanged
o TRPC3 knockout mice exhibit an impaired walking behaviour.
o Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.
o Monitor mGluR1 activity by looking at Ca2+ release from stores
o Can activate mGluR1 specifically with DHPG→ in TRPC3 KO mice, when activate → see Ca2+ increases still released from stores → but lost inward current
o Problem with the study, CPHG was applied to all parts of the slice and so all parts of purkinje cell would have been exposed → so could argue not looking at synaptic activation of mGluR1 but whole cell activation so maybe mGluR1 links to TRPC at the soma not synapse
o So then they synapticaLly stimulate parallel fibres → same results
• Manahan-Vaughan et al (1997):
This study examined the role of (mGluRs) in hippocampal (LTD) in vivo. The group 1 mGluR antagonist ((4CPG – group 1 antagonist), and (MSOPPE-group 2 antagonist). The NMDA receptor antagonist (AP5) was used to examine the NMDA receptor contribution to the observed LTD. Adult male Wistar rats. Stimulating and recording electrodes into the Schaffer collaterals and CA1 stratum radiatum, respectively. After recovery of 5–7 d, the field EPSP was measured from freely moving animals. Drugs were applied either before or after 1 Hz low-frequency train (LFT) or 100 Hz stimulation via a cannula implanted in the lateral cerebral ventricle.
o Nine hundred pulses at 1 Hz produced an LTD that was long-lasting. This LTD was completely inhibited by pre-LFT application of AP5.
o 4CPG partially impaired LTD. MSOPPE completely blocked induction of LTD, although short-term depression remained intact.
o MSOPPE did not block long-term potentiation (LTP) induced by 100 Hz stimulation, whereas 4CPG produced a significant inhibition. When MSOPPE was present, LTD could not be induced either before or after LTP induction.
o These results suggest a differential role for mGluRs in NMDA receptor-dependent hippocampal LTD in vivo. Group 1 mGluRs may play a role in both LTD and LTP (suggest it may be involved in the maintenance?), whereas group 2 mGluRs may be critically involved only in LTD induction
o Group 2 pre and group 1 post. Maybe 2 is critical and m1 is modulatory for LTD
• Mannioni et al (2001):
To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. Rat hippocampal slices, whole-cell patch-clamp recording and Ca(2+)-imaging techniques
o Each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons.
o These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons. Suggests activation of mGluR1 activation leads to somatic calcium transients and neuronal depolarisation whereas mGluR5 activation inhibits Iahp (slow after hyperpolarisation currents and potentiates NMDAR currents)
o The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration (Ca(2+)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral–>CA1 synapse.
o On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor
• Lopes et al (2005):
Expression of K2P channels in Xenopus oocytes, patch clamping cell voltage recordings
o Suggests that hydrolysis of PIP2 mediates inhibition of channel activity by hormones and neurotransmission
o Show that PIP2 hydrolysis modulates voltage dependence of 2 P channels. Demonstrate that agonist-induced PIP2 hydrolysis modulates voltage dependence of both 2 P and c gated KCNQ channels → results that PIP2 is a common gating molecule for K+ channels
o Our results suggest that PIP2 is a common gating molecule for K+ channel families despite their distinct structures and physiological properties.
o Show that (PIP2) gating underlies several aspects of 2-P channel regulation.
o For TREK1, TASK1 and TASK3 channels, PIP2 hydrolysis underlies inhibition by several agonists.
o This finding suggests that the strength of channel PIP2 interactions determines the extent of PLC-induced inhibition.
o - Don’t show that it is linked to MgLURS
• Tong and Jahr (1994)
Showed how powerful Glu transporters can modulate post-synaptic excitabilitu. Looking at spontaneous NT release. Effects of blockers of glutamate transporters on excitatory synaptic transmission to determine whether transporters increase the clearance rate of transmitter from the synaptic cleft on the millisecond time scale. Cultured hippocampal neurons
o When inhibit Glu transporters with THA → responses are much larger
o Found that blockers of glutamate transporters potentiate evoked and spontaneous miniature AMPAR EPSCs in cultured hippocampal neurons
o The transporter blockers Li+ and THA increased the amplitude, but not the decay time, of spontaneous miniature AMPA receptor EPSCs recorded at 34 degrees C but not 24 degrees C.
o The rapidly dissociating AMPA receptor competitive antagonist PDA inhibited evoked AMPA receptor EPSCs less in the presence of THA at both temperatures, implying that transporter blockade slows clearance.
o We suggest that transporters speed glutamate clearance mainly by binding glutamate and that AMPA receptors are not saturated by synaptically released glutamate at 34 degrees C.
o - Doesn’t show how it effect the rate of large release such as with AP-induved NT release
• Kirmse et al (2015):
Here we examine the in vivo effects of GABA in cells of the upper cortical plate using a combination of electrophysiological and Ca(2+)-imaging techniques. Anaesthetised mice
o We report that at postnatal days (P) 3-4, GABA depolarizes the majority of immature neurons in the occipital cortex of anaesthetized mice. Current clamp recordings
o Pharmacological inhibition of NKCC strongly reduced GABA-evoked CaTd, implying a role for secondary active chloride accumulation in GABAergic transmission in vivo
o Blocking GABA(A) receptors disinhibits spontaneous network activity, whereas allosteric activation of GABA(A) receptors has the opposite effect.
o In summary, our data provide evidence that in vivo GABA acts as a depolarizing neurotransmitter imposing an inhibitory control on network activity in the neonatal (P3-4) neocortex.→ depolarizing GABAergic transmission has additional inhibitory functions at the network level, likely via the GABAAR-dependent increase in membrane conductance (that is, shunting inhibition)
o Found that applied GABA did not generate action potentials (APs) in any of the cells.
