Neocortical long-term potentiation and long-term depression: site of expression investigated by infrared-guided laser stimulation

Abstract

The synaptic site of expression of long-term potentiation (LTP) and long-term depression (LTD) is still a matter of debate. To address the question of presynaptic versus postsynaptic expression of neocortical LTP and LTD in a direct approach, we measured the glutamate sensitivity of apical dendrites of layer 5 pyramidal neurons during LTP and LTD. We used infrared-guided laser stimulation to release glutamate from its "caged" form with high spatial and temporal resolution. Responses to photolytically released glutamate and synaptically evoked EPSPs were recorded with patch-clamp pipettes from the neuronal somata. LTP and LTD could be induced by electrical stimulation at the same synapses in succession. The NMDA receptor-dependent LTD was accompanied by a decrease in the dendritic glutamate sensitivity, suggesting a postsynaptic expression of neocortical LTD. In contrast, LTP was never accompanied by a change in the dendritic glutamate sensitivity. A possible explanation for this finding is a presynaptic expression of neocortical LTP. Another set of experiments corroborated these results: Photolytic application of glutamate with a frequency of 5 Hz caused a long-lasting Ca2+ and NMDA receptor-dependent decrease in the dendritic glutamate sensitivity. In contrast, LTP of dendritic glutamate sensitivity was never induced by photostimulation, despite several experimental modifications to prevent washout of the induction mechanism and to induce a stronger postsynaptic Ca2+ influx. In conclusion, our findings provide strong evidence for a postsynaptic expression of neocortical LTD and favor a primarily presynaptic locus of neocortical LTP.

Fig. 1.

Infrared-guided laser stimulation.A, Set-up used for infrared-guided laser stimulation. Neurons in brain slices were visualized with transmitted infrared (IR) light and the gradient contrast system. The beam of a UV laser was focused by the objective to a 1 μm spot in the specimen plane. This way, glutamate was photolytically released from caged glutamate with high spatial and temporal resolution. The neuronal responses were recorded with patch-clamp pipettes at the soma.B, Pyramidal neuron of neocortical layer 5 visualized by infrared videomicroscopy. Scale bar, 10 μm.

Fig. 2.

Characterization of infrared-guided laser stimulation. A, B, Caged glutamate does not desensitize glutamate receptors by spontaneous hydrolyzation. Neither the rise time nor the amplitude of evoked EPSPs changed significantly when caged glutamate (caged glu) was added to the extracellular medium. In A, single sweeps are shown. B, Statistical evaluation. The respective values of 15 EPSPs under control conditions (c) and in the presence of caged glutamate were averaged for six neurons. C, D, Spatial specificity of infrared-guided laser stimulation. The laser point was moved laterally away from the dendrite in increments of 2.5 μm. The decrease in glutamate response amplitude is plotted as a function of this distance. In C, a single experiment is shown.D, Only the data of one side were pooled (n = 6 neurons), because the typical Gaussian shape of the other side was sometimes distorted. This may be an effect of invisible dendritic branches.

Fig. 3.

Tetanic release of glutamate causes a decrease in the glutamate response amplitude (photo-LTD). A, Focal photolysis of caged glutamate with a frequency of 5 Hz for 1 min duration (arrow with flash symbol) reliably elicited photo-LTD. For control stimulation, glutamate was released every 20 sec. The data shown are averages for every minute. The 100% value (dashed line) represents the mean of the last 5 min before the onset of the 5 Hz train in all figures. Thetraces show single glutamate responses before (black) and after (gray) 5 Hz stimulation. B, Photo-LTD is not caused by damage to the neuron attributable to the UV radiation. The glutamate responses were not affected by the 5 Hz train when applied in the absence of caged glutamate. In contrast, a second application of the 5 Hz stimulation in the presence of caged glutamate reliably induces photo-LTD. A single experiment is shown. C, Photo-LTD is not induced by the photolytically released caging group. The amplitudes of GABA responses, elicited by photolysis of caged GABA, were not affected by the 5 Hz stimulation. Because the caged glutamate and caged GABA used are protected by the same caging group, photo-LTD cannot be caused by the photolytically released caging group. The traces show single GABA responses before (black), and after (gray) the 5 Hz stimulation.

Fig. 4.

Photo-LTD is Ca²⁺ and NMDA receptor dependent. A, Addition of the Ca²⁺ chelator BAPTA to the pipette solution blocks photo-LTD. Only an insignificant reduction in glutamate response amplitude remains. B, The block of NMDA receptors by MK801 and a high external Mg²⁺ concentration prevents the induction of photo-LTD. The reduction in glutamate response amplitude is statistically insignificant. Dashed lines represent the 100% value. C, Approximately 40% of the amplitude of the photolytically evoked glutamate response is mediated by NMDA receptors at resting membrane potential. Thetraces show single glutamate responses before (black) and after (gray) blockade of the NMDA receptors. The faster kinetics of the AMPA receptor-mediated component is clearly recognizable if the sweeps are scaled.

Fig. 5.

Neocortical synaptic LTD. A, Electrical 5 Hz stimulation (arrow) induces LTD of EPSPs. This form of synaptic plasticity is associated with a decrease in the dendritic glutamate sensitivity. To 6 of the 14 neurons, the 5 Hz train of light flashes (arrow with flash symbol) was applied. No additional photo-LTD could be elicited in these neurons (gray diamonds), indicating a complete occlusion of photo-LTD by synaptic LTD. Without LTD induction, the glutamate response was stable for the entire time of recording (60 min, white squares). Thedashed line represents the 100% value. B, Traces of glutamate responses and EPSPs before and 35 min after electrical 5 Hz stimulation. Depolarizations elicited by photostimulation had a time-to-peak (62 ± 3 msec) that was approximately three times as long as the time-to- peak of electrically evoked EPSPs (20 ± 2 msec; n = 7 neurons).C, Comparison of the reduction in EPSP amplitude (white bar) and glutamate response peak (black bar) during synaptic LTD. Forty-eight minutes after electrical LTD induction, both potentials show no difference (p > 0.5). The gray barindicates the mean reduction in the glutamate response amplitude of the neurons, which were also stimulated by a 5 Hz light tetanus.D, Photo-LTD and synaptic LTD are not associated with a change in the glutamate response kinetics. Traces show single glutamate responses before and after the 5 Hz stimulation. The unchanged kinetics of the glutamate responses is clearly recognizable if the sweeps are scaled. c, Control.E, Synaptic LTD is NMDA receptor dependent. Blockade of NMDA receptors by MK801 and a high extracellular Mg²⁺ concentration prevented the induction of synaptic LTD and the decrease in dendritic glutamate sensitivity (n = 7 neurons). The arrow indicates electrical 5 Hz stimulation (see A). The dashed line represents the 100% value.

Fig. 6.

Neocortical synaptic LTP. A, Electrical stimulation (arrow) induces LTP of EPSPs. During LTP, the dendritic glutamate sensitivity does not change. Averages of 12 neurons are shown. B, Dendritic glutamate sensitivity during synaptic LTP and LTD successively induced at the same synapses. LTP and LTD were induced by electrical 5 Hz stimulation (n = 7 neurons). The stimulation intensities used for LTP and LTD induction were 2.5× and 2× the threshold of spike generation, respectively. The dendritic glutamate sensitivity during LTP remains constant. In contrast, LTD is associated with a decrease in the glutamate response amplitude. The traces show single EPSPs and glutamate responses. Dashed lines represent the 100% value. C, Statistical evaluation of the experiments shown in A and B. A high significant difference (p < 0.01) from the control value is indicated by the stars. As a control value for LTD, the mean of the last 5 min (16–20 min) before LTD induction was used. During synaptic LTP, the glutamate response amplitude shows no significant difference (p= 0.1).

Fig. 7.

Perforated patch-clamp recordings. To prevent a potential washout of the induction mechanism of LTP, the cells were recorded using perforated patch-clamp techniques. Photolytic release of glutamate induced photo-LTD in all neurons independently of the induction protocol used. The dashed line represents the 100% value.