Chapter 4. Functional Division of Hippocampal Area CA1 via Modulatory Gating
4.6 Supplementary Figures
signal in same areas used for c-Fos expression analysis did not differ between groups (Supplementary Figure 4-2A). (B) The number of c-Fos positive cells after novel place exposure was analyzed in the pyramidal layers of area CA1 and CA3 and the granular layer of the DG in the slices (50 m thickness) used in figure 4-1B (n = 6 pairs of animals). Total integrated NeuN signals in same areas used for c-Fos expression analysis did not differ between groups (Supplementary Figure 4-2A). For the analysis of novelty-induced c-Fos expression, a two-way ANOVA was performed with 2 variables:
novelty type (object vs. place) and CA1 subregion (distal vs. proximal), and revealed a significant interaction (p = 0.0008). (C) The number of c-Fos positive cells after novel object exposure was analyzed in each area of the hippocampus on the slices used in figure 4-10A. In the bottom graphs, c-Fos expression was separately analyzed for global and regional expression. In the global c-Fos expression analysis, the total number of c-Fos positive cells in the hippocampus (CA1, CA3, DG) was analyzed. In the regional expression analysis, the number of c-Fos positive cells in each area was normalized to the total number of c-Fos positive cells in the hippocampus, thus, it represents a relative ratio of c-Fos expression in each area of the hippocampus. The total integrated NeuN signals in same areas used for c-Fos expression analysis did not differ between groups (Supplementary Figure 4-2B). Student’s t-test was performed to analyze the significance of global and regional expression differences (*p < 0.05). (D) The number of c-Fos positive cells after novel place exposure was analyzed in each area of the hippocampus as described in C. The total integrated NeuN signals in same areas used for c-Fos expression analysis did not differ between groups (Supplementary Figure 4-2B). For the analysis of
c-Fos expression in distal CA1, a two-way ANOVA was performed with the two variables, novelty type (object vs. place) and drug treatment (saline vs. clozapine), showing a significant interaction (p = 0.0015).
Supplementary Figure 4-2: Control analysis for c-Fos expression experiments
(A) No significant differences were observed in the integrated NeuN signals analyzed same areas as used for c-Fos expression analysis in Figure 4-1. (B) No significant differences were observed in integrated NeuN signals in the same areas as used for c-Fos expression analysis in Figure 4-10. (C) As a control experiment for the data shown in Figure 4-10, a pair of animals was housed in the same cage for at least 2 days, and then,
either saline or clozapine (10 mg/kg) was intraperitoneally injected in each animal. 6 hrs after the injection (no novelty exposure), animals were sacrificed and immunohistchemistry was performed on fixed slices. Intraperitoneal injection itself did not show any significant enhancement of c-Fos expression in CA1 or CA3 pyramidal neurons. There was no significant difference between saline and clozapine treated animals (n = 4 pairs of animals). The total integrated NeuN signals in the same areas used for c-Fos expression analysis did not differ between groups.
Supplementary Figure 4-3: Slice images of c-Fos and NeuN immunostaining after novel object exposure in each area of the hippocampus
Scale bar = 200 m
Supplementary Figure 4-4: Slice images of c-Fos and NeuN immunostaining after novel place exposure in each area of the hippocampus
Scale bar = 200 m
Supplementary Figure 4-5: Acute application of neuromodulators prior to LTP induction
A neuromodulator, DA or NE, was directly applied to the recording chamber 10 sec before LTP induction. The LTP induction protocol was 100 pulses at 100 Hz, repeated twice at a 30 sec interval. Estimated washout time of neuromodulators was about 2 – 3 min. DA and NE differentially modulated the magnitude of LTP at distal TA-CA1 synapses (control: 117 ± 1%, DA: 136 ± 5%, NE: 104 ± 1%, mean percentage of baseline 55 – 60 minute after LTP induction). (n = 7, 6, 6 for each). This is probably due to a reversible baseline shift by acute NE application, and LTP was induced from a depressed baseline level. Then, even if NE’s action on LTP induction itself does not differ from control condition (Figure 4-9), the total proportion of LTP-induced synapses will be smaller (*p < 0.05 relative to ACSF application).
Supplementary Figure 4-6: Presynaptic N-type calcium channel modulation by DA at distal TA-CA1 synapses
(A) Extracellular application of 4-amiopyridine (4AP: 100 M) completely blocked DA-induced depression at distal TA–CA1 synapses (n = 4). 4AP application itself enhanced fEPSP slope and also changed fEPSP waveform. Note that the fiber volley in fEPSP waveform indicated by arrow became wider after 4AP application. However, DA application did not change a fiber volley waveform, suggesting that the site of DA’s action is different from 4AP affecting sites. Instead, 4AP may mask the DA’s effect on N-type calcium channels as previously described (Wheeler et al., 1996). (B) N-type calcium channel dependent action of D1-like receptors at distal TA–CA1 synapses. Field
EPSP at distal TA–CA1 synapses was significantly depressed after N-type calcium channel blocker, -conotoxin GVIA (2.5 M), application, which was directly applied to the recording chamber. After N-type calcium channel blockade, DA-induced partial depression at TA–CA1 synapses was completely blocked by a D2-like receptor antagonist, sulpiride (n = 4). Disappearance of D1-like receptor effects after N-type calcium channel blockade suggests that N-type calcium channel is likely to be a main target of D1-like receptors at distal TA-CA1 synapses.