Depolarizing GABAA current in the prefrontal cortex is linked with cognitive impairment in a mouse model relevant for schizophrenia

Abstract

Cognitive impairment in schizophrenia (CIAS) is the most critical predictor of functional outcome. Limited understanding of the cellular mechanisms of CIAS hampers development of more effective treatments. We found that in subchronic phencyclidine (scPCP)-treated mice, an animal model that mimics CIAS, the reversal potential of GABA_A currents in pyramidal neurons of the infralimbic prefrontal cortex (ILC) shifts from hyperpolarizing to depolarizing, the result of increased expression of the chloride transporter NKCC1. Further, we found that in scPCP mice, the NKCC1 antagonist bumetanide normalizes GABA_A current polarity ex vivo and improves performance in multiple cognitive tasks in vivo. This behavioral effect was mimicked by selective, bilateral, NKCC1 knockdown in the ILC. Thus, we show that depolarizing GABA_A currents in the ILC contributes to cognitive impairments in scPCP mice and suggest that bumetanide, an FDA-approved drug, has potential to treat or prevent CIAS and other components of the schizophrenia syndrome.

Fig. 1. GABA_A current is depolarizing in ILC pyramidal neurons of scPCP-treated mice.

(A and B) Current/voltage (I/V) curves of the peak GABA_A currents recorded in L5 pyramidal neurons in slices from vehicle (veh)– and scPCP-treated mice. The dotted lines represent polynomial fits to the data. Currents (insets) were recorded in perforated patch clamp in the presence of 3 mM kynurenic acid. (C) Average GABA_A reversal potentials for vehicle- and scPCP-treated mice in ILC L5 cells. Two-tailed Mann-Whitney U test, P = 0.04 and u = 29. The box plots dots represent outliers and + represent means. Orange lines represent the average resting potentials (measured in whole-cell configuration in separate experiments). Numbers above the boxes show the sample size in each group. (D to F) Same as (A) to (C) for prelimbic (PLC) cells; P = 0.86 and u = 39.5.

Fig. 2. ILC pyramidal neurons of scPCP-treated mice display increased excitability.

Cell-attached recordings of layer 2/3 ILC pyramidal cells from a vehicle-treated (left trace) and an scPCP-treated mouse (right trace) without synaptic blockers (A) and in the presence of kynurenic acid (3 mM; B). Slices were electrically stimulated (0.15 to 0.35 mA, 20 Hz, 1 s; red lines) with an electrode in layer 1. Stimulation artifacts are truncated in the figure. (C and D) Scatter plots representing the number of spikes recorded in the 10 seconds following the end of the stimulus without synaptic blockers (C; n = 12 for vehicle-treated mice, and n = 10 for scPCP-treated mice; P = 0.18 and u = 39 by Mann-Whitney U test), and in the presence of kynurenic acid (D; P = 0.07 and u = 58 by Mann-Whitney U test; n = 15 in vehicle versus n = 13 in scPCP). The lines in the plots represent means ± SEM. (E) In the presence of kynurenic acid, the fraction of neurons that kept firing after the end of the stimulus was significantly larger in the scPCP group compared with control (P = 0.029; Fisher’s exact test).

Fig. 3. NKCC1 expression is selectively increased in the ILC of scPCP mice.

(A) In situ hybridization images for NKCC1 (Slc12a2) and KCC2 (Slc12a5) in sections of L5 ILC from vehicle- and scPCP-treated mice. Brown signal [3,3′-diaminobenzidine (DAB)] represents NKCC1 and KCC2 mRNAs; methyl green was used to stain cell bodies. (B) Data quantification. Top: NKCC1; P = 0.0087 and u = 2 for ILC and P = 0.31 and u = 11 for PLC (Mann-Whitney U test). Bottom: KCC2; P = 0.81 and u = 16 for ILC and P = 0.82 and u = 16 for PLC (Mann-Whitney U test). Data from six control and six scPCP mice.

Fig. 4. Bumetanide restores GABA_A reversal potential and rescues cognitive performance of scPCP mice in multiple cognitive tasks.

(A) GABA_A reversal potential measured in an ILC L5 pyramidal cell from a vehicle and (B) scPCP mouse in the presence of bath-applied bumetanide (BMN). (C) GABA_A reversal potentials recorded in ILC slices from vehicle-treated (black box) mice and in slices from scPCP-treated mice recorded either in the absence (blue box) or in the presence (purple box) of bumetanide; P = 0.0057 for vehicle versus scPCP (+saline), P = 0.0058 for vehicle + bumetanide versus PCP (+saline), and P = 0.0032 for scPCP (+saline) versus bumetanide-treated scPCP; F = 6.54, one-way analysis of variance (ANOVA) followed by Bonferroni test. The number of neurons in each group is indicated above the boxes. Orange lines represent the average resting potentials. (D) Discrimination index calculated from NOR tests. Bumetanide (0.1 mg/kg, in saline) was administered by intraperitoneal injection 30 min before testing. P < 0.0001 for scPCP versus either vehicle- or bumetanide-treated scPCP group. Each group, n = 10 (F = 71.49). (E) Y-maze spontaneous alteration behavior test. P < 0.0001 for scPCP versus either vehicle- or bumetanide-treated scPCP group and n = 10 for all three groups (F = 35.78). (F) Operant reversal learning test. The plot shows correct responses in reversal test phase; P < 0.0001 for scPCP versus either vehicle- or bumetanide-treated scPCP group. n = 10 for all groups (F = 95.08). We observed no statistical difference between groups in the initial phase test (not shown).

Fig. 5. Focal administration of bumetanide to the ILC, but not the PLC, improves cognitive performance in scPCP mice.

Bumetanide (0.3 mg/ml; 1 μl) was acutely injected into either the ILC or the PLC of vehicle- and scPCP-treated mice. (A) Effects of bumetanide on cognitive performance (NOR and Y-maze alternation tests) when injected into the ILC; six mice in each group. For NOR test, P < 0.0001 for scPCP versus either vehicle- or bumetanide-treated scPCP group (one-way ANOVA followed by Bonferroni test; F = 73.07). For Y-maze test: P < 0.0001 for scPCP versus either vehicle- or bumetanide-treated scPCP group (F = 39.44). (B) Bumetanide injections into the PLC had no detectable effects on cognitive performance; six mice in each group. NOR: P < 0.0001 for vehicle versus either scPCP- or bumetanide-treated scPCP group (F = 31.66). Y-maze test: P < 0.0001 for vehicle versus either scPCP- or bumetanide-treated scPCP group (F = 28.72).

Fig. 6. NKCC1 knockdown in the ILC improves cognitive performance in scPCP mice.

Adeno-associated viral constructs containing either mouse Slc12a2-shRNA [short hairpin RNA; knockdown (KD)] or a scrambled RNA sequence (SCR) were injected into the ILC. (A) Microphotograph (2×) of the ILC of an injected mouse; green fluorescent protein (GFP) expression identifies the injection sites. (B) Map indicating the injection sites for every mouse treated. Black circles identify vehicle/KD mice, blue squares identify scPCP/SCR, and green circles indicate scPCP + KD. (C) Results of the NOR test. The sample size was five, six, and five mice, respectively. P = 0.0293 for vehicle/KD versus scPCP/SCR and P = 0.0039 for scPCP/KD versus scPCP/SCR, one-way ANOVA followed by Bonferroni test (F = 8.16). (D) Y-maze spontaneous alternation test. Same mice as in (C). P = 0.0001 for vehicle/KD versus scPCP/SCR and P < 0.0001 for scPCP/KD versus scPCP/SCR (F = 28.14).