neurophysiology tutorial 2

• the paper (Takahashi et al, 1996) looks at the mechanisms of pre-synaptic inhibition of mGluRs
• rat brainstem slices were used which contained the Calyx of Held (fastest CNS synapse). the activities at the presynaptic calyx of held and the postsynaptic target in the medial nucleus of the trapezoid body (MNTB) were simultaneously recorded
• an issue with this procedure is that the rats were aged 8-18 days old as it is is easier to image smaller brain slices. however, rats are not able to hear until day 12-14. this means that a relay synapse in the auditory pathway, that is not working, is being used in this experiment. the response of mature rats might be different
• presynaptic inhibition mediated by the mGluR family has been implicated in autoreceptor inhibition at the mammalian glutamatergic synapses
• presynaptic mGluRs are crucially involved in the induction of long term depression at hippocampal mossy fibre CA3 synapses
• mechanisms of mGluRs that have been suggested to be involved with the presynaptic inhibition are suppression of presynaptic calcium conductance, augmentation of potassium conductance or inhibition of exocytotic machinery downstream of calcium influx
• investigating potential mechanisms for inhibition require direct access to both the pre and post synaptic sites of mammalian synapse, has previously been conducted in invertebrate synapses and chick autonomic ganglia
• the data shown in Figure 1A was collected using simultaneous, whole cell recording from the calyx of held and it's postsynaptic target. both were held at -70 mV under a voltage clamp. depolarisation was for 20 seconds and voltage step was approximately 0 mV.
• the middle trace of figure 1A shows the presynaptic calcium current when a sustained depolarisation takes place. the upward spiking is the capacitive current (charging of the membrane) and then there is a huge inward Ca2+ current that is sustained as long as the depolarisation is evoked. after the depolarisation has ended, there is a small capacitive current and then outward flow of calcium ions
• the bottom trace of Figure 1A shows the postsynaptic EPSC. the slow closure seen means that the signal has been propagated and the channels are gradually closing. depolarising command pulse causes a sustained EPSC
• Figure 1B shows the calcium I-V relation. the top part of the trace show Ca2+ currents evoked by depolarisation from -80 mV to -45 mV to -15 mV. the extracellular Ca2+ concentration was 1 mM. the reversal potential of calcium was found to be +50 mV (where it crosses the x axis). current reached a peak at -10 to -20 mV which indicates that the calcium current was a HVA (high voltage activated type).
• Figure 1C shows the effects of block by w-agatoxin IVA (200 nM with cytochrome C) which is a P/Q-type calcium channel blocker presynaptically. the residual current of 8% was abolished by 100uM Cd2+. voltage step from -80 mV to -20 mV and depolarisation for approximately 30 ms. initial current is approximately 1.1 nA. Agatoxin was applied for 10 minutes, Cd2+ was applied for 5 minutes.
• Figure 1D shows the relationship between the concentration of external calcium and the normalised calcium current. Ca2+ currents were evoked by a 50 ms pulse from -80 to -20 mV OR 1 ms pulse from -70 to +30 mV. peak amplitude of Ca2+ currents was normalised to that of 1mM. apart from the 2 data points at 2 mM, the data represents means and SEMs derived from 4 calyces. solid line indicates a 1 to 1 linear relationship.
• presynaptic NT release was induced by voltage dependent Ca2+ channels elicited in the presynaptic terminal under voltage clamp
• sustained EPSCs were seen during long, depolarising pulses. result from asynchronous transmitter release. Short Ca2+ currents produce fast EPSCs that were comparable in time course with those evoked by nerve stimulation
• L type calcium channel blocker nicardipine had no effect on reduction in current. N type Ca2+ blocker w-contoxin GVIA also had little or no effect on the Ca2+ current at the calyx of held synapse.
• the amplitude of the presynaptic Ca2+ current decreased with reduced external calcium concentration or with increased external magnesium concentration.
• L-AP4 is an agonist of mGlur subtypes 4, 6, 7 and 8. was applied at 50 to 100 uM. presynaptic Ca2+ current was suppressed, particularly at an early phase about the pea by 25.2%. this effect was reversible after L-AP4 washout. a specific agonist of mGluR subtypes 2 and 3 (DCG-IV) at 3uM had no effect. therefore, can be deduced that one or more mGluR subtypes (4,6,7 and 8) are involved in current suppression. mGluR mediated depression was not accompanied by a shift in cottage dependence of the Ca2+ current as shown in figure 2A.
• figure 2A shows the effect of L-AP4 on presynaptic calcium current. depolarisation step was from -80 mV to -15 mV for a period of 10 ms. shows the HVA activity seen again. The reversal potential of -50 mV remained the same with LP4 but the maximum amplitude seen changed from approximately -1.75 nA to -1.3 nA both at approx -15 mV (not a change in voltage dependence)
• Figure 2B shows that L-AP4 had no effect on the presynaptic potassium current. the outward potassium currents were evoked by 20 mV incremental depolarising steps from -80 mV holding potential. in the presence of 1uM TTX. potassium currents before and after 50 uM L-AP4 superimpose directly on top of each other. potassium current was activated at depolarisation of -60 mV and above.
• paired recordings from a presynaptic terminal and its postsynaptic target showed concurrent suppression of presynaptic calcium cirremt and EPSC during application of L-AP4. inhibition seen was 24.3 % for calcium currents and 40% for EPSCs
• to determine whether calcium current suppression could account for EPSC reduction, the relationship between the Ca2+ current amplitude and respective EPSCs were examined. this was done in the reduction of extracellular Ca2+ and during L-AP4 application. extracellular calciumw as reduced with Mg replacement. presynaptic Ca2+ currents and EPSCs were dimished in parallel as shown in Figure 3B.
• Figure 3a shows the presynaptic calcium curents evoked by a 1 ms pulse from -70 to +30 mV and the resultant EPSCs at -70 mV after 2 mM calcium externally was replaced with 5 mM Mg or after application of 50 uM L-AP4.
• Figure 3B shows time plots of presynaptic calcium currents and EPSCs. recording was done for approximately 50 seconds with no calcium and 100 seconds for application of L-AP4. parallel reduction is seen under these conditions.
• Figure 3C shows the peak amplitude of presynaptic Ca2+ current plotted against the amplitude of EPSCs in double log co-ordinates. the linear regression slopes for the extracellular calcium reduction and L-AP4 application were 2.06 and 1.82 respectively. a student's t test showed that they were statistically significant
• previous studies showed, by using Ca sensitive dyess, that presynaptic Ca2+ entry into a population of nerve terminals can be suppressed by mGluR agonists.
• suppression of presynaptic Ca2+ entry can be the result of suppression of presynaptic Ca2+ channels or augmentation of potassium channels.
• at the calyx-MNTB synapse, L-AP4 had no effect on voltage gated potassium currents in the preterminal. therefore, the potassium channels do not appear to be involved in the presynaptic mGluR modulation at this synapse.
• the results of this study emphasized the need for cautious interpretation of results from indirect methods when studying presynaptic mechanisms since previous work on the EPSCs at the calyx-MNTB synapse suggested that mGluRs had no effect on the presyanptic Ca2+ current, looking at paired pulse experiments
• examined whether mGluRs may additionally modulate the exocytotic machinery downstream of Ca2+ entry by comparing the Ca2+ current EPSC relation under 2 conditions: reduction of extracellular calcium and application of L-AP4. close agreement of these input-output relations suggests that presynaptic inhibition by mGluRs is largely caused by the suppression of presynaptic Ca2+ currrent at the calyx-MNTB synapse.
• direct modulation of the exocytotic machinery by mGluRs had been suggested from the reduced frequency of spontaneous miniature synaptic current by mGluR agonists. miniature freuwncy does not always display a direct relation to evoked NT release during presynaptic modulation.
• washout influencing downstream mechanisms cannot be excluded from the whole cell study