Dissociation between CA3-CA1 synaptic plasticity and associative learning in TgNTRK3 transgenic mice

Abstract

Neurotrophins and their cognate receptors might serve as feedback regulators for the efficacy of synaptic transmission. We analyzed mice overexpressing TrkC (TgNTRK3) for synaptic plasticity and the expression of glutamate receptor subunits. Animals were conditioned using a trace [conditioned stimulus (CS), tone; unconditioned stimulus (US), shock] paradigm. A single electrical pulse presented to the Schaffer collateral-commissural pathway during the CS-US interval evoked a monosynaptic field EPSP (fEPSP) at ipsilateral CA1 pyramidal cells. In wild types, fEPSP slopes increased across conditioning sessions and decreased during extinction, being linearly related to learning evolution. In contrast, fEPSPs in TgNTRK3 animals reached extremely high values, not accompanied with a proportionate increase in their learning curves. Long-term potentiation evoked in conscious TgNTRK3 was also significantly longer lasting than in wild-type mice. These functional alterations were accompanied by significant changes in NR1 and NR2B NMDA receptor subunits, with no modification of NR1(Ser 896) or NR1(Ser 897) phosphorylation. No changes of AMPA and kainate subunits were detected. Results indicate that the NT-3/TrkC cascade could regulate synaptic transmission and plasticity through modulation of glutamatergic transmission at the CA3-CA1 synapse.

Figure 1.

Stereological analyses and expression of NMDA and non-NMDA receptor subunits in the hippocampus of TgNTRK3 mice. A, A significant increase in NeuN-positive cell density in CA1, CA2, CA3, and dentate gyrus (DG) was observed in TgNTRK3 mice. B, The density of GFAP-positive cells was reduced in CA1 and CA2 regions of TgNTRK3 mice. C–E, Basal hippocampal expression of NMDA and AMPA receptor subunits and phosphorylated serines NR1⁸⁹⁶ and NR1⁸⁹⁷ from wild-type and TgNTRK3 mice was analyzed by Western blot. C, Graphs show that TgNTRK3 mice express higher levels of NR1 and NR2B subunits. In contrast, basal levels of NR2A (C), phosphorylated serines NR1⁸⁹⁶ and NR1⁸⁹⁷ (D), and GluR1, GluR2–3, GluR4, GluR5, and GluR6–7 subunits (E) were not modified in TgNTRK3 mice with respect to wild types. Results are expressed as a percentage of values collected from TgNTRK3 (white bars) with respect to wild-type (WT) mice (black bars). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

Experimental design for classical conditioning and fEPSP recording. A, Electrodes to record the EMG activity of the left orbicularis oculi (O.O.) muscle were implanted in the upper eyelid. For classical conditioning of eyelid responses, we used a tone (20 ms, 2.4 kHz, 85 dB) as a CS, delivered from a loudspeaker located 30 cm in front of the animal's head. Bipolar stimulating electrodes were implanted on the supraorbitary nerve for US presentation. fEPSPs were recorded (Rec.) at the stratum radiatum of the hippocampal CA1 area after electrical stimulation (St.) of the Schaffer collateral (coll.)/commissural pathway (right hippocampus). Representative examples (3 superimposed traces) of fEPSPs (1) and EMG (2) activities are also illustrated. Sub., Subiculum. B, C, Photomicrographs illustrating the location of recording (CA1; 1, 3) and stimulating (CA3; 2, 4) sites in wild-type (B) and TgNTRK3 (C) mice. Scale bar, 200 μm. D, Dorsal; L, lateral; M, medial; V, ventral; DG, dentate gyrus.

Figure 3.

Learning curves and evolution of the synaptic field potential for wild-type and TgNTRK3 groups. A, At the top is a schematic representation of the conditioning paradigm illustrating CS and US stimuli and the moment at which a single pulse (100 μs, square, biphasic) was presented to Schaffer collaterals (St. Hipp.). An example of an EMG record from the orbicularis oculi (O.O.) muscle, obtained from the eighth conditioning session, is illustrated, as well as an extracellular record of hippocampal activity from the same animal, session, and trial. Note the fEPSP evoked by the single pulse presented to Schaffer collaterals. B, A similar set of records collected from a TgNTRK3 mouse during the eighth conditioning session. Note the presence of a noticeable fEPSP but the absence of an eyelid CR. C, At the top are illustrated superimposed (n = 4) extracellular fEPSP traces collected from the wild-type and TgNTRK3 groups during the second habituation (n = 2) and the ninth conditioning (n = 2) sessions. Evolution of the fEPSP slope in wild-type (black triangles) and TgNTRK3 (white triangles) groups, expressed as the change (in percentage) with respect to mean values collected during the four habituation sessions. Differences between wild-type and TgNTRK3 groups were statistically significant for all of the conditioning and extinction sessions (*p < 0.001; F_(18,162) = 149). D, Evolution of the percentage (in percentage) of CRs during the successive sessions for wild-type (black circles) and TgNTRK3 (white circles) groups. Mean percentage values are followed by ±SEM. Differences between wild-type and TgNTRK3 groups were statistically significant from the third to the 10th conditioning sessions and for the first-third and fifth extinction sessions (*p < 0.01; F_(18,162) = 8.6). E, F, Quantitative analysis of the relationships between the percentage of CRs and fEPSP slopes for the wild-type (E) and TgNTRK3 (F) groups during habituation (black triangles), conditioning (white circles), and extinction (black circles) sessions. Each point represents the mean value collected from a single animal during the corresponding session. Regression lines, and their corresponding equations, are included only for coefficients of correlation, r > 0.6. The corresponding values for p and r, for each regression analysis, are always indicated. Hipp., Hippocampus; St., stimulation; Thr., threshold.

Figure 4.

Paired-pulse facilitation and LTP induction of fEPSPs recorded in the CA1 area after stimulation of Schaffer collaterals. A, At the top are three superimposed extracellular fEPSP paired traces collected from the wild-type and TgNTRK3 groups at interpulse intervals of 50 ms. The data shown are mean ± SEM slopes of the second fEPSP expressed as a percentage from the corresponding value of the first fEPSP, for the six (10, 20, 50, 100, 200, and 500 ms) selected interstimulus intervals. Wild-type mice presented a paired-pulse facilitation similar to that obtained from TgNTRK3 animals at intervals of 20 and 50 ms. B, At the top are examples (4 superimposed traces) of fEPSPs collected from selected animals of each experimental group before and after HFS of Schaffer collaterals. The bottom graphs illustrate the time course of LTP evoked in the CA1 area (fEPSP mean ± SEM) after HFS for wild-type (black circles) and TgNTRK3 (white circles) mice. The HFS was presented after 15 min of control recordings, at the time marked by the dashed line. The fEPSP is given as a percentage of the baseline (100%) slope. Illustrated data were collected up to 2 h after HFS during the first day (Day 1) and for 30 min 24 h later (Day 2). C, The two groups presented a significant increase (ANOVA, two-tailed) in fEPSP slope after HFS when compared with baseline records (* for wild-type and ^● for transgenic, p < 0.01; F_(1,9) = 183). Nevertheless, values collected from the TgNTRK3 group were significantly (^♦p < 0.01; F_(1,9) = 142) larger than those collected from wild-type mice at the indicated times. Data included in each histogram were collected for the intervals (Baseline, 1, 2, and 3) indicated in B.