Presynaptic modulation of synaptic efficacy in the rat hippocampus

Modifications of synaptic efficacy play a crucial role in information processing in the brain. In particular, they are thought to be very important for the refinement of neural circuitry, information storage, learning and memory. Therefore, investigating the mechanisms that modulate synaptic transmission is of fundamental importance for understanding brain functions. In the present study, patch-clamp recordings were performed in order to further investigate synaptic transmission in the hippocampus, focusing on different presynaptic mechanisms that may affect synaptic efficacy. In the last decade, silent synapses and their activation during postnatal development and plasticity processes, such as LTP, have attracted particular attention. These synapses are called silent because they do not respond at rest but are active at positive membrane potentials and can be converted into functional synapses by pairing presynaptic stimulation with postsynaptic depolarisation. A widely accepted interpretation is that Nmethyl- D-aspartate (NMDA) but not a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors are functionally expressed on the subsynaptic membrane. Thus, wakening of silent synapses during LTP induction would be due to the activation of new functional AMPA receptors. However, most of these studies have been done in the CAl region of the hippocampus. Therefore, the first part of this study was focused on testing whether a similar kind of connections is present also in the CA3 region during postnatal development. Excitatory postsynaptic currents (EPSCs) were recorded from CA3 pyramidal neurones, following minimal stimulation (0.05 Hz) of Mossy fibres (MF) inputs in acute hippocampal slices from new-born rats. Although 16% of synapses appeared silent at 24 °C, AMP A receptors were found to be functionally expressed on CA3 neurones. In fact, different tools known to increase the probability of release, such as paired-pulse stimulation (50 ms interval) or increasing the temperature (from 24°C to 32°C), were able to activate silent synapses. Similar results were obtained at Schaffer collateral (SC)-CA1 connections where 36% of synapses were found silent, but AMPAmediated synaptic responses could be detected after increasing the probability of release by paired-pulse stimulation, by raising the temperature or by the application of cyclothiazide (CTZ), a drug known to block AMPA receptor desensitisation and to increase transmitter release. Overall, these results show that, in both CA3 and CA 1 region of the hippocampus, AMP A receptors are expressed and functional on the subsynaptic membrane already at early stages of postnatal development. However, some synapses may appear silent because of a very low probability of glutamate release, as they can be converted into functional ones by factors that enhance release probability. Although it cannot be excluded that "latent AMP A receptors" can become functional following activity dependent processes, these results clearly indicate that in the neonatal hippocampus a proportion of glutamatergic synaptic connections are "presynaptically" rather than "postsynaptically" silent. Moreover, conducting synapses could be switched off by increasing the frequency of stimulation from 0.05 to 0.1-1 Hz, suggesting a critical role for use-dependent modulation of synaptic efficacy. To better investigate how activity can regulate synaptic efficacy, eventually leading to synaptic depression and silencing, hippocampal organotypic slice cultures were used. Taking advantage of the high connectivity between neurones, double-patch clamp recordings between interconnected pairs of CA3 pyramidal cells were performed to study the properties of sho1t-term depression occurring in these synapses under different frequencies of presynaptic firing. In stationary conditions (0.05-0.067 Hz) pairs of presynaptic action potentials (50 ms apart) evoked EPSCs whose amplitude fluctuated from trial to trial with occasional response failures. Increasing stimulation frequency from 0.05-0.067 Hz to 0.1-1 Hz induced low-frequency depression (LFD) of EPSC amplitude with a gradual increase in the failure rate, suggesting the involvement of presynaptic mechanisms. Overall, 75% of cells became almost "silent" at 1 Hz, whereas recovery from depression could be obtained by lowering the frequency of stimulation to 0.025 Hz. Surprisingly, when the firing rate was sequentially shifted from 0.05 to 0.1 and 1 Hz, changes in synaptic efficacy were so strong that the paired-pulse ratio (PPR) shifted from paired-pulse facilitation (PPP) to paired-pulse depression (PPD). These results can be explained by a model that takes into account two distinct release processes, one dependent on the residual calcium and the other on the size of the readily releasable pool (RRP) of vesicles. According to this model, the depletion of the RRP of vesicles accounts for LFD and for the unexpected shift from PPF to PPD. The possibility of switching between functional and non-functional synapses might play a crucial role in controlling the communication between neurones as well as network synchronisation. The CA3 hippocampal region is known to act as the pacemaker for the generation of synchronised activity, mainly because of the dense network of collaterals of axons interconnecting pyramidal neurones. This network is under control of both extrinsic factors, such as neurotransmitters and neuromodulators, and active conductances. Thus, in the last part of the study, the role of a voltage-dependent, fast activating and slowly inactivating potassium current similar to 10 in controlling temporal coding and synaptic strength at CA3-CA3 connections was investigated. As 10 , this current is responsible for the delayed appearance of the first spike upon prolonged membrane depolarisation and for action potential repolarisation. Moreover, it could be blocked by low concentrations of 4-aminopyridine (4-AP), a drug known to generate interictal discharges that can propagate from the CA3 region to the entire hippocampus. Interestingly, the 10 -like current, was down-regulated by intracellular calcium, as demonstrated by the observation that Cd++, a blocker of voltage-dependent calcium channels, significantly increased the delay of the first spike generation. Suppressing lo by low concentration of 4-AP reduced this delay and, by broadening the presynaptic action potential, increased synaptic strength. Thus, it is likely that modulation of this current by fluctuations ·in resting membrane potential or intracellular calcium concentration, for example during activitydependent processes, may play a critical role in determining information coding and network synchronisation in the CA3 region.