Role of reuniens nucleus projections to the medial prefrontal cortex and to the hippocampal pyramidal CA1 area in associative learning

Abstract

We studied the interactions between short- and long-term plastic changes taking place during the acquisition of a classical eyeblink conditioning and following high-frequency stimulation (HFS) of the reuniens nucleus in behaving mice. Synaptic changes in strength were studied at the reuniens-medial prefrontal cortex (mPFC) and the reuniens-CA1 synapses. Input/output curves and a paired-pulse study enabled determining the functional capabilities of the two synapses and the optimal intensities to be applied at the reuniens nucleus during classical eyeblink conditioning and for HFS applied to the reuniens nucleus. Animals were conditioned using a trace paradigm, with a tone as conditioned stimulus (CS) and an electric shock to the trigeminal nerve as unconditioned stimulus (US). A single pulse was presented to the reuniens nucleus to evoke field EPSPs (fEPSPs) in mPFC and CA1 areas during the CS-US interval. No significant changes in synaptic strength were observed at the reuniens-mPFC and reuniens-CA1 synapses during the acquisition of eyelid conditioned responses (CRs). Two successive HFS sessions carried out during the first two conditioning days decreased the percentage of CRs, without evoking any long-term potentiation (LTP) at the recording sites. HFS of the reuniens nucleus also prevented the proper acquisition of an object discrimination task. A subsequent study revealed that HFS of the reuniens nucleus evoked a significant decrease of paired-pulse facilitation. In conclusion, reuniens nucleus projections to prefrontal and hippocampal circuits seem to participate in the acquisition of associative learning through a mechanism that does not required the development of LTP.

Figure 1. Experimental design.

(A) EMG recording electrodes were implanted in the orbicularis oculi (O.O.) muscle of the upper left eyelid. In addition, bipolar stimulating electrodes were implanted on the ipsilateral supraorbital nerve for presentation of unconditioned stimulus (US). The conditioned stimulus (CS) consisted of a tone delivered from a loudspeaker located 30 cm from the animal's head. Animals were also implanted with stimulating electrodes in the thalamic reuniens nucleus and with recording electrodes in the medial prefrontal cortex (top diagram) or the hippocampal CA1 area (bottom diagram). (B–D) Photomicrographs illustrating the location of recording electrodes in the mPFC (B) and in the hippocampal CA1 area (D), as well as the stimulation (C) site (arrows). Calibration bars 500 µm. Abbreviations: D, L, M, V, dorsal, lateral, medial, and ventral. E, 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 the reuniens nucleus (St. Reu.). An example of an EMG record from the orbicularis oculi (O.O.) muscle obtained from the 9th conditioning session is illustrated, as well as an extracellular record of hippocampal activity from the same animal, session, and trial. Note the fEPSPs evoked by the pulse presented to the reuniens nucleus.

Figure 2. Input/output curves and paired-pulse stimulation of the reuniens-mPFC and reuniens-CA1 synapses using paired-pulse stimulation.

(A) Relationships between the intensity (in mA) of pairs of stimuli (40 ms of interstimulus interval) presented to the reuniens nucleus and the amplitude of the fEPSPs evoked in the mPFC by the 1st (black triangles) and the 2nd (white triangles) pulses. Data are represented as mean ± s.e.m. *, P<0.01 for differences in the amplitude of fEPSPs evoked by the two pulses. The best three-parameter sigmoidal fits to the two set of data are included (1st pulses, continuous line: y = 0.75/(1+exp[−(x-0.71)/0.16]); r = 0.97; P<0.001; 2nd pulses, dashed line: y = 1.09/(1+exp[−(x-0.73)/0.13]); r = 0.98; P<0.001). *, P<0.01. (B) The same as for A, but representing data collected from the reuniens-CA1 synapse. *P<0.01 for differences between fEPSPs evoked by the two pulses. Sigmoidal fits to the two set of data are also included (1st pulses, continuous line: y = 0.53/(1+exp[−(x-0.67)/0.23]); r = 0.96; P<0.001; 2nd pulses, dashed line: y = 1.16/(1+exp[−(x-0.58)/0.23]); r = 0.97; P<0.001). *, P<0.01. (C) Representative records (average of three traces) of fEPSPs evoked at the reuniens-mPFC synapse by paired-pulse stimulation at five different (20, 40, 100, 200, and 500 ms) interpulse intervals. Stimulus intensity was 0.7 mA, i.e., ≈50% of the asymptotic value for this synapse (see arrow in A). (D) Paired-pulse depression and facilitation of fEPSPs recorded from the reuniens-mPFC (white circles) and the reuniens-CA1 (black circles) synapses. The pairs of pulses were set at 0.7 mA (see arrows in A and B). The data shown are mean ± s.e.m. amplitudes of the 2nd fEPSP expressed as a percentage of the 1st [(2nd/1st) ×100] for the five interstimulus intervals used in this study. Note that peak facilitation was at 40 ms of interval for the reuniens-CA1 synapse and at 100 ms for the reuniens-mPFC synapse. *, statistical differences for reuniens-CA1 synaptic facilitation at 40 ms of interval, with respect to the 500 ms one; #, statistical differences for reuniens-mPFC synaptic facilitation at 100 ms interval, with respect to both the 20 and 500 ms ones, P<0.05. Tukey's post-hoc comparison test.

Figure 3. Learning curves and evolution of synaptic field potentials evoked in the PFC by electrical stimulation of the reuniens nucleus for controls (A, B) and following two HFS sessions (C, D).

(A) Evolution of fEPSPs evoked at the reuniens-PFC synapse across the successive habituation, conditioning, and extinction sessions. At the top are illustrated selected fEPSPs recorded in the PFC during the indicated sessions, following a single pulse presented to the reuniens nucleus. Note that no significant change in fEPSP amplitude was observed across conditioning. Calibrations as indicated. (B) Evolution of the percentage (%) of conditioned responses during the successive sessions. Mean % values are followed by ± s.e.m. (C) Evolution of fEPSPs evoked at the reuniens-PFC synapse across training, following two HFS sessions presented 30 min before the first two conditioning sessions. Note that these HFS sessions did not evoke any noticeable LTP in fEPSPs recorded in the PFC. (D) Evolution of the percentage (%) of conditioned responses during the successive sessions following the two HFS sessions. Note the small increase in the percentage of conditioned responses. *, P<0.001, significant differences with respect to habituation values for B and D. #, P<0.05; ##, P<0.01, significant differences between the percentage of conditioned responses achieved without (B) or following two HFS sessions (D).

Figure 4. Learning curves and evolution of synaptic field potentials evoked in the CA1 area by the electrical stimulation of the reuniens nucleus for controls (A, B) and following two HFS sessions (C, D).

(A) Evolution of fEPSPs evoked at the reuniens-CA1 synapse across the successive habituation, conditioning, and extinction sessions. At the top are illustrated selected fEPSPs recorded in the CA1 area during the indicated sessions, following a single pulse presented to the reuniens nucleus. Note that no significant change in fEPSP amplitude was observed across conditioning. Calibrations as indicated. (B) Evolution of the percentage (%) of conditioned responses during the successive sessions. Mean % values are followed by ± s.e.m. (C) Evolution of fEPSPs evoked at the reuniens-CA1 synapse across training, following two HFS sessions presented 30 min before the first two conditioning sessions. Note that these HFS sessions did not evoke any noticeable LTP in fEPSPs recorded in the CA1 area. (D) Evolution of the percentage (%) of conditioned responses during the successive sessions following the two HFS sessions. Note the small increase in the percentage of conditioned responses. *, P<0.001, significant differences with respect to habituation values for B and D. #, P<0.05; ##, P<0.01, significant differences between the percentage of conditioned responses achieved without (B) or following two HFS sessions (D).

Figure 5. Results collected from the object recognition task.

A representation of the attention devoted to a familiar object or to a novel one by control mice (black bars) and by animals following a single HFS session (gray bars), during an object recognition task, for the training (T = 0) session, and 1 h (T = 1) and 24 h (T = 24) afterwards. The object presentation sequence is schematized at the bottom. Values are mean ± s.e.m. of the percentage of the total attention exhibited in each session. *, Statistical differences between percentages of attention, P<0.05; **, P<0.01.

Figure 6. Effects of two HFS sessions on the reuniens-CA1 and reuniens-mPFC synapses.

(A, B) Evolution of fEPSPs evoked in the PFC (A) and in the CA1 area (B) by paired-pulse stimulation of reuniens nucleus before and after two HFS sessions. Each animal was presented with two HFS sessions (see shaded areas) each consisting of five 200 Hz, 100 ms trains of pulses at a rate of 1/s. This protocol was presented six times, at intervals of 1 min. The 100 µs, square, biphasic pulses used to evoke LTP were applied at the same intensity used for the single pulse presented following HFS presentation. The evolution of LTP was checked using a pair of pulses (1st, black circles; 2nd, white circles) with an interstimulus interval of 40 ms. Recording was carried out for 72 h. Note that fEPSP amplitudes evoked by the 1st and the 2nd pulses reached values below baseline following the two HFS sessions for both synapses. *, P<0.05 for fEPSPs evoked by the 1st pulse; #, P<0.05 for fEPSPs evoked by the 2nd pulse.