Age-Dependent Specific Changes in Area CA2 of the Hippocampus and Social Memory Deficit in a Mouse Model of the 22q11.2 Deletion Syndrome

Abstract

Several neuropsychiatric disorders are associated with cognitive and social dysfunction. Postmortem studies of patients with schizophrenia have revealed specific changes in area CA2, a long-overlooked region of the hippocampus recently found to be critical for social memory formation. To examine how area CA2 is altered in psychiatric illness, we used the Df(16)A(+/-) mouse model of the 22q11.2 microdeletion, a genetic risk factor for developing several neuropsychiatric disorders, including schizophrenia. We report several age-dependent CA2 alterations: a decrease in the density of parvalbumin-expressing interneurons, a reduction in the amount of feedforward inhibition, and a change in CA2 pyramidal-neuron intrinsic properties. Furthermore, we found that area CA2 is less plastic in Df(16)A(+/-) mice, making it nearly impossible to evoke action potential firing in CA2 pyramidal neurons. Finally, we show that Df(16)A(+/-) mice display impaired social cognition, providing a potential mechanism and a neural substrate for this impairment in psychiatric disorders.

Figure 1. The density of parvalbumin-expressing INs in the hippocampus is decreased in area CA2 of adult Df(16)A^+/− mice

(A) Immunohistochemical staining for parvalbumin in the different hippocampal areas of the hippocampus from a 8-week old wild-type and Df(16)A^+/− mouse. The different hippocampal layers are indicated (SO: stratum oriens, SP: stratum pyramidale, SR: stratum radiatum, SL: stratum lucidum). (B) Quantification of the density of parvalbumin positive soma (PV+) in the different strata of areas CA1, CA2 and CA3 of 8-week old WT and Df(16)A^+/− mice (n = 6 mice for both genotypes). (C) Immunohistochemical staining for parvalbumin in the different hippocampal areas of the hippocampus from a 4-week old wild-type and Df(16)A^+/− mouse. (D) Quantification of the density of PV+ soma in the different strata of CA1, CA2 and CA3 areas in 4-week old WT (n = 6) and Df(16)A^+/− mice (n = 6). Error bars show SEM. See also figure S1.

Figure 2. Inhibitory transmission onto CA2 PNs is decreased in adult Df(16)A^+/− mice

(A) Top; Schematic representation of the experimental conditions in which a compound EPSP/IPSP sequence was recorded in CA2 pyramidal neurons (PNs) following stimulation of Schaffer collaterals (SC). Bottom; Sample traces of the compound EPSP/IPSP (Control, black traces) and the EPSP obtained after blocking inhibition (SR/CGP, grey traces) in CA2 PNs in response to stimulation of SC inputs in WT and Df(16)A^+/− mice. Below: the inhibitory component obtained after subtracting the control traces from the traces with GABA receptor blockers is shown in grey. (B) Summary graph of the input-output curves of the PSP in control conditions and after blocking inhibition (SR/CGP) in response to SC stimulation in adult WT (n = 9) and Df(16)A^+/− mice (n = 19). (C) Summary graph of the fold-increase in PSP amplitude after blocking inhibition in WT and Df(16)A^+/− mice. (D) Summary graph of the input-output curves of the IPSP amplitude obtained by subtraction of control traces from the traces with GABA receptors blockers in WT and in Df(16)A^+/− mice. (E) Summary graph of the input-output curves of the PSPs in response to SC stimulation in control conditions and following blockade of inhibitory transmission in young (4–5-week old) WT (n = 6) and Df(16)A^+/− mice (n = 5). (F) Summary graph of the input-output curves of the IPSP in response to CA3 input stimulation obtained by subtracting control traces from traces with GABA receptor blockers in 4–5-week old WT and Df(16)A^+/− mice. Error bars show SEM. See also figure S2.

Figure 3. Synaptic transmission of distal inputs onto CA2 PNs is not altered in adult Df(16)A^+/− mice

(A) Top; Schematic representation of the experimental conditions in which a compound EPSP/IPSP sequence was recorded in CA2 PNs following stimulation of distal inputs. Bottom; sample traces of the compound EPSP/IPSP (Control, black trace) and the EPSP obtained after blocking inhibition (SR/CGP, grey trace) in CA2 PNs in response to stimulation of distal inputs in WT and Df(16)A^+/− mice. The inhibitory component obtained after subtracting the control traces from the traces with GABA receptor blockers is shown in grey. (B) Summary graph of the input-output curves of the PSP amplitude in response to distal input stimulation in control conditions and after blocking inhibition in adult WT (n = 8) and Df(16)A^+/− mice (n = 18). (C) Summary graph of the fold-increase in PSP amplitude after blocking inhibition in WT and Df(16)A^+/− mice. (D) Summary graph of the input-output curves of the IPSP obtained after subtraction of the control traces from traces with GABA receptor blockers in WT and in Df(16)A^+/− mice. (E) Summary graph of the input-output curves of PSPs in response to distal input stimulation in control conditions and after blocking inhibitory transmission in 4–5-week old WT (n = 6) and Df(16)A^+/− mice (n = 6). (F) Summary graph of the input-output curves of the IPSP in response to distal input stimulation obtained by subtracting control traces from traces with GABA receptor blockers in 4–5-week old WT and Df(16)A^+/− mice. Error bars show SEM.

Figure 4. Adult CA2 PNs in Df(16)A^+/− mice have a more hyperpolarized resting potential

Summary graphs and diagrams illustrating how the measurements were made of the resting membrane potential (A), the membrane resistance (Rm) (B), the membrane capacitance (C), the depolarizing sag during a hyperpolarizing current step (D), and the action potential threshold (E) measured in CA2 PNs in WT and in Df(16)A^+/− mice at different postnatal weeks. The number of cells is shown for each data point. (F) The fluoxetine-sensitive current evoked by a voltage ramp for WT (n = 13) and Df(16)A^+/− mice (n = 13). Grey shading indicates the SEM. Inset, the estimated conductance of this current, likely due to TREK channels. (G) A plot of the TREK-1 conductance versus Resting membrane potential for all of the recorded cells, showing the correlation between the TREK conductance and resting membrane potential (Pearson’s R = −0.53). Error bars show SEM. See also figure S3.

Figure 5. Action potential firing of CA2 PNs is decreased in adult Df(16)A^+/− mice

(A) Sample traces of the depolarization of CA2 PNs in response to stimulation of SC inputs (5 pulses at 100Hz) in WT and Df(16)A^+/− mice. (B) Summary graph of the number of action potentials (left) and of the percentage of cells firing at least one action potential (right) during the train of stimulation of CA3 inputs at different intensities (10, 20 and 30V) in WT (n = 12) and Df(16)A^+/− mice (n = 18). (C) Sample traces of the depolarization of CA2 PNs in response to a stimulation of distal inputs (5 pulses at 100Hz) in WT and in Df(16)A^+/− mice. (D) Summary graph of the number of action potentials (left) and of the percentage of cells firing at least one action potential (right) during the train of stimulation of distal inputs at different intensities (10, 20 and 30V) in WT (n = 12) and in Df(16)A^+/− mice (n = 17). Error bars show SEM. See also figure S4.

Figure 6. Long-term depression at inhibitory transmission onto CA2 PNs is decreased in adult Df(16)A^+/− mice

(A) Summary graph of normalized IPSCs recorded before and after delivery of tetanic stimulation (100 pulses at 100Hz twice) in WT (n = 9) and Df(16)A^+/− mice (n = 9). Sample traces corresponding to the time points before (a) and after (b) tetanus are shown on top. (B) The paired-pulse ratio for individual experiments before (a) and after (b) tetanic stimulation for WT and Df(16)A^+/− mice. Error bars show SEM.

Figure 7. The dis-inhibitory increase in PSP amplitude and in action potential firing is impaired in adult Df(16)A^+/− mice

(A) Summary graph of the change in the amplitude of the PSP recorded in whole-cell current clamp configuration in response to SC input stimulation following a high frequency stimulation (100 pulses at 100Hz repeated twice). The lasting increase in the depolarizing component of the PSP in WT mice (black filled circles, n = 6) is significantly smaller in Df(16)A^+/− mice (red filled circles n = 8). This increase is dependent on inhibitory transmission and is blocked by GABA receptor blockers (SR/CGP) both in WT (black open circles, n = 4) and in Df(16)A^+/− mice (red open circles, n = 5). Cells were held at −70 mV. (B) Summary graph of the change in the amplitude of the field PSP recorded extracellularly in response to CA3 input stimulation following a high frequency stimulation (HFS: 100 pulses at 100Hz repeated twice) in WT (black, n = 6) and in Df(16)A^+/− mice (red, n = 5). (C) Summary graphs of the amplitude of the population spike monitored extracellularly in the somatic layer of area CA2 in response to SC input stimulation before (a) and after (b) HFS in WT (left, n = 14) and in Df(16)A^+/− mice (right, n = 14). Error bars show SEM.

Figure 8. Social memory is impaired in adult Df(16)A^+/− mice

(A) Top: Experimental setup for the direct interaction test in which a different stimulus animal was presented in trials 1 and 2. Bottom left: the two groups (Df(16)A^+/−, n = 8; WT, n = 8) engaged in social interaction with the two stimulus animals for a similar amount of time. Bottom right: Df(16)A^+/− mice and their WT littermates have similar difference scores when interacting with two novel juvenile mice (One-way ANOVA F(1,15) = 0.469, P = 0.504). (B) Top: experimental setup for the direct interaction test in which the same stimulus animal was presented for both trials 1 and 2. Bottom left: Unlike WT, Df(16)A^+/− mice fail to show significant recognition of the familiar animal (two-way RM ANOVA: Genotype x Trial F(1,14) = 13.503, P = 0.0025). Social memory in the WT mice is evidenced by a decrement in social investigation on trial 2, which is not the case for the Df(16)A^+/− mice. Bottom right: the difference score of the Df(16)A^+/− group was not only less than that of the WT group, but even had a negative value, indicating that the Df(16)A^+/− show no social memory when tested with this paradigm. Error bars show SEM.