Synaptic Plasticity I: Evidence for the electrostructural basis of Long-Term Depotentiation (LTD)
MICHMIZOS D., COSTA V., KARLOVASITOU-KONIARI A., ASPRODINI E., BALOYANNIS S.

Since it was first observed in 1973 by Bliss and Lψmo, synaptic plasticity has been regarded as the experimental paradigm most likely to provide us with an understanding of how information is stored in the vertebrate brain. Various types of synaptic plasticity have been demonstrated over these past 30 years, most notably Long-Term Potentiation (LTP) and Long-Term Depression (LTD), and their established characteristics and induction requirements are highly indicative of this plasticity being the substrate for memory formation.

In order to compare the structural modifications of LTP and LTD, young Wistar rats were raised for 16-18 weeks before been sacrificed, their brains removed and immediately immersed into ice-cold Artificial-CerebroSpinal-Fluid (ACSF), continuously superoxygenated and at pH never exceeding 7.34+0.02 after the removal of the olfactory bulbs and the cerebellum, the separation of the hemispheres and the coronal excision of the frontal and occipital brain regions, 400-450 μm slices containing the hippocampal formation were obtained with a Mclwaan-R500 vibratone. the slices were allowed to rest for a least 1.5h in a humidifying interface-type chamber, always saturated with warm (37+0.2 °C) ACSF before the realization of any experimentation (Chen et al, 1999; Brown et al, 1988).

The electrophysiological establishment of LTP or LTD followed the well-attested protocol of placing the stimulating electrode in the Schaffer collaterals and the field-recording electrodes in the stratum radiatum of the CA1 hippocampal region. All recordings were digitized by an Axolab1100 interface that also amplified the signals recorded by the AxoClamp-2A pre-amplifier, and then analyzed by Pclamp 5.03 software. All inputs were programmed through a Master-8 stimulator, including the base stimuli at 10Hz, the High Frequency Stimulation (HFS) at 100Hz for 900 pulses (Stδubli & Zi, 1996; kirkwood et al, 1996; Petersen et al, 1998). The cumulative field potentials clearly demonstrated the induction and consolidation of LTP and LTD respectively, plasticities that were measurably present even after 16-18h.

The potentiated or depotentiated hippocampal slices were consequently fixed for 48h in Sotelo solution, cut into 2x2mm pieces and fixed in OsO4 solution, gradually dehydrated to propylene-oxide and embedded into hardenning Araldit. The ultrathin sections (15-30 nm) of the CA1 hippocampal region obtained with a REICHERT-JUNG Ultracute microtome were mounted and stained with uranyl-oxide and lead-citrate in order to be observed under a ZEISS EM 95-2 election microscope and photographed on monochromatic fine-grain film.

Following the HFS, the synapses became clearly potentiated, that is the postsynaptic response to the stimulus was augmented, whereas LFS induced depotentation, as demonstrated by the instantaneous and cumulative field potential slopes. As expected, the potentiated synapses demonstrated structural modifications, especially in the Post-Synaptic Density (PSD) area that was enlarged in tandem with the presynaptic terminal. These modifications increased their synaptic efficacy and, moreover, reduced the postsynaptic membrance electrical resistance which can account for the increased responsiveness to the base stimulus. Synaptic potentation is known to be accompanied by dendritic growth and numerous growth cones were identified throughout the stratum radiatum. The depotentiated synapses, surprisingly, also demonstrated structural modifications. Specifically, the postsynaptic terminal seems to increase (even in tandem with the presynaptic surfaces) without, however, the accompanying increase in the PSD area that would render the modified synapse increasingly functional and electrically more conductive.

We proposed that, in the case of synaptic depression, the LFS which induses it, causes a limited influx of Ca⁺⁺: its increased concentration, however, is restricted to the perimembranic area of the synapse. this precipitates cytoskeleton modifications which result in the perforation of the PSD and and the increase of the membranic area; however, since the Ca⁺⁺ increase was only brief and transient, and transient, and, thus, no receptions or ion-channels will be uptaken from the dentritic shaft, the increased membranic area will not be rendered conductively functional. In this way, the increased area of the postsynaptic membrance presents an augmented electrical resistance. Even if the limited Ca⁺⁺ influx triggers the local release of some of the receptor pool vesicles, any inserted receptions will not be able to become organized into quantal clusters and, in this way, they will be diffusing any membrance depolarization built-up until they are recycled. the above are responsible for the observed diminished response to the presynaptic stimulus in the case of LTD.

Key words: Memory, hippocampus, synaptic plasticity, long-term depotentiation, LTD.