Figure 1.
The hippocampal circuit and experimental setup.
(A) Illustration of the tri-synaptic hippocampal circuit and placement of electrodes for our electrophysiological analyses. Input from the entorhinal cortex enters the dentate gyrus via the perforant pathway forming synapses on the dentate gyrus granule cells. The granule cells project axons, the mossy fibers, to the CA3 pyramidal neurons. The axons from the CA3 cells, the Schaffer collaterals, form synapses on the dendrites of the CA1 pyramidal cells [34]. Arrows indicate the direction of the neurotransmission. In all electrophysiological experiments described in this study the stimulating and recording electrodes were placed as pictured. (B) A representative trace obtained during a recording of the fEPSP. Cursors indicate the region of the rising phase of the fEPSP used to estimate the slope of the response. The slope is linearly related to the synaptic conductance and can be used as a measure of the activation of glutamatergic receptors in the postsynaptic membrane of Schaffer collateral synapses [49]. An arrow indicates the partially blanked stimulation artifact resulting from the brief electrical stimulation transient applied by the bipolar stimulation electrode. The afferent fiber volley (AV) is a result of the action potentials in the population of Schaffer collaterals traveling by the recording electrode and reflects the strength of the afferent input.
Figure 2.
Wobbler mice exhibit increased synaptic excitation.
(A-B) Representative fEPSP traces recorded during I/O-curve studies in presymptomatic (P17-P21) and symptomatic (P45-P60) wobbler mice (right sides) and controls (left sides). The arrows illustrate the first observation of a population spike in the given experiment, and it can be appreciated that the population spikes occur at lower stimulation intensities in wobbler mice. (C) The I/O-curves of both the presymptomatic (P17-P21) (left) and the symptomatic (P45-P60) (right) wobbler mice are shifted compared to the control mice, as evidence of increased synaptic excitation (P17-P21: control: n = 10 slices/5 mice, wobbler: n = 16 slices/6 mice. P45-P60: control: n = 12 slices/7 mice, wobbler: n = 16 slices/7 mice). T-test: *P<0.05; **P<0.01. Error bars represent SEM.
Figure 3.
Population spikes are evoked at lower stimulation intensities, but same fEPSP size, in wobbler mice.
(A) Cut-out from Figure 2A: fEPSP traces showing the first observation of a population spike, resulting from CA1 pyramidal cell firing during I/O-curve recordings in presymptomatic (P17-P21) wobbler mice (right) and controls (left). The arrows point to what was defined as the initial population spikes (corresponding to 0.15 mA and 0.3 mA, respectively, see also arrows in Fig. 2A). The development of the population spikes can be appreciated in Figure 2A. (B) Cut-out from Figure 2B showing fEPSP traces recorded during experiments in wobbler mice (right) at the symptomatic phase (P45-P60) and control mice (left), with arrows illustrating the first observed population spikes (corresponding to 0.1 mA and 0.45 mA, respectively, see also arrows in Fig. 2B). The progression of the population spikes can be seen in Figure 2B. (C) The average stimulation intensity needed to evoke a population spike in P17-P21 (left) and P45-P60 (right) wobbler mice and control are illustrated by the diagrams. The small circles demonstrate the stimulation intensity distribution. (D) The fEPSP slopes corresponding to the first observation of a population spike are similar in the four groups. Small circles illustrate the individual slope measurements. (P17-P21: control: n = 10 slices/5 mice, wobbler: n = 16 slices/6 mice. P45-P60: control: n = 12 slices/7 mice, wobbler: n = 16 slices/7 mice). T-test: *P<0.05; **P<0.01. Error bars represent SEM.
Figure 4.
Normal release probability in wobbler mice.
(A) Representative fEPSP traces recorded during paired-pulse facilitation (PPF) performed in brain slices from a wobbler mouse (left) and a control mouse (right). (B) The fEPSP slope of the second pulse was normalized to the first pulse in the respective sweep, and in each slice, the slopes of three sweeps were averaged for each stimulation interval. No significant difference was seen in the magnitude of PPF between wobbler mice and controls (wobbler: 19 slices/8 mice and control: 18 slices/9 mice). T-test: P<0.05. Error bars represent SEM.
Figure 5.
Wobbler mice do not exhibit presynaptic impairments.
(A) fEPSPs recorded during train stimulation of the Schaffer collaterals, in control mice at P17-P21. Arrows illustrate the pulses employed for the analyses: the slopes of the first three and the last three pulses (pulses 1, 2, 3, 8, 9, and 10 for the types of trains consisting of 10 pulses, and pulses 1, 2, 3, 198, 199, and 200 for the types of trains consisting of 200 pulses) from each type of train were normalized to the slope of the first pulse in the given trains. (B-E) No physiologically relevant differences were observed in short-term synaptic plasticity when comparing wobbler mice and control littermates during the presymptomatic phase (comparing B to C) or the symptomatic phase (comparing D to E). However, a few results reached statistical significance when testing the trains of the same intensity in wobbler mice against controls, as indicated by * (P<0.05) or ** (P<0.01) in the figure (C compared to B) (t-test). Note the shifts in pulse number. (P17-P21: control: n = 5 slices/4 mice, wobbler: n = 6 slices/6 mice. P45-P60: control: n = 10 slices/6 mice, wobbler: n = 9 slices/7 mice). Error bars represent SEM.
Figure 6.
Synaptic depletion is normal in wobbler mice.
Comparisons of the sizes of the very first and very last pulses evoked during the three consecutive trains for each stimulation protocol. Responses (pulses number 10 from the types of trains consisting of 10 pulses, and pulses number 200 from the types of trains consisting of 200 pulses) were normalized to the very first pulse in the first sweep of the three consecutive trains. No physiologically relevant differences were seen between the wobbler mice (B+D) and control littermates (A+C) during the pre-symptomatic phase (P17-P21) (A+B) or the symptomatic phase (P45-P60) (C+D). Two points of statistical significance was however reached in the results, indicated by * (P<0.05) and ** (P<0.01) in the Figure (B) (t-test). Error bars represent SEM.
Figure 7.
Reductions in parvalbumin (PV) positive interneurons in the hippocampus of the wobbler mice.
(A-D) Parvalbumin (PV) positive interneurons (illustrated by arrows) were immunohistochemically stained and counted in slices of the hippocampal formation of presymptomatic (P18-P19) wobbler mice (B) and controls (A). Symptomatic (P56) wobbler mice (D) and controls (C) were also stained. Scale bars represent 200 µm. (E) During the presymptomatic phase (P18-P19) no significant differences were found in the number of parvalbumin positive interneurons per slice in any area of the hippocampal formation between wobbler and control mice. (F) At the symptomatic phase (P56) a reduction was found in all examined areas, except in the hilus, in the wobbler mice. (G) A reduction in parvalbumin positive interneurons is seen in both wobbler mice and control mice between P18-P19 and P56; however the reduction in the wobbler mice was greater in most areas. (H) The distribution of the parvalbumin positive interneurons at the different areas are similar in wobblers and controls at the presymptomatic phase (P18-P19), and remain similar at the symptomatic phase (P56), t-test: P>0.05. (P18-19: control: n = 46 slices/4 mice, wobbler: n = 48 slices/4 mice. P56: control: n = 34 slices/4 mice, wobbler: n = 31 slices/3 mice). T-test: *P<0.05; **P<0.01. Error bars represent SEM.