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. 2013 Dec 11;33(50):19724-33.
doi: 10.1523/JNEUROSCI.1584-13.2013.

Development of GABA circuitry of fast-spiking basket interneurons in the medial prefrontal cortex of erbb4-mutant mice

Jian-Ming Yang  1 , Jing Zhang, Xiao-Juan Chen, Hong-Yan Geng, Mao Ye, Nicholas C Spitzer, Jian-Hong Luo, Shu-Min Duan, Xiao-Ming Li
Affiliations

Development of GABA circuitry of fast-spiking basket interneurons in the medial prefrontal cortex of erbb4-mutant mice

Jian-Ming Yang et al. J Neurosci. .

Abstract

erbb4 is a susceptibility gene for schizophrenia and ErbB4 signals have been hypothesized to function in a number of cortical developmental processes (Silberberg et al., 2006; Mei and Xiong, 2008). Several recent studies show that the expression of ErbB4 is mainly restricted to GABAergic interneurons (Yau et al., 2003; Woo et al., 2007), specifically, to parvalbumin-positive (PV) fast-spiking (FS) interneurons (Vullhorst et al., 2009; Fazzari et al., 2010), a large majority of which are PV FS basket cells (Kawaguchi, 1995; Taniguchi et al., 2013). However, in the medial prefrontal cortex (mPFC), a brain region that is closely associated with neuropsychiatric disorders including schizophrenia, little is known about the roles of ErbB4 signals during the development of GABAergic circuitry particularly that associated with PV FS basket cells. Here, using molecular genetics, biochemistry, and electrophysiology, we deleted ErbB4 receptors in GABAergic forebrain neurons during the embryonic period and demonstrated that in the mouse mPFC, ErbB4 signals were dispensable for the development of GABAergic synapses by PV FS basket cells. Interestingly, they were required for the final maturation rather than the initial formation of glutamatergic synapses on PV FS basket cells. Furthermore, activity-dependent GABAergic PV FS pyramidal neuron transmission was decreased, whereas activity of pyramidal neurons was increased in KO mice. Together, these data indicate that ErbB4 signals contribute to the development of GABAergic circuitry associated with FS basket cells in component- and stage-dependent manners in the mPFC in vivo, and may suggest a mechanism for neuropsychiatric disorders including schizophrenia.

Keywords: ErbB4; GABAergic circuitry; development; medial prefrontal cortex (mPFC); parvalbumin (PV) interneurons; schizophrenia.

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Figures

Figure 1.
Figure 1.
Light micrograph of a neurobiotin-stained EGFP-expressing FS basket cell in P60 G42 mice. Arrows indicate the axonal arbors that form “basket” terminals. Scale bar, 50 μm.
Figure 2.
Figure 2.
Conditional knock-out of the erbb4 gene in interneurons resulted in an absence of ErbB4 protein in the PFC. A, ErbB4 protein was almost completely abolished in P0 KO mice. B, ErbB4 protein level was below the detection limit in EGFP-positive neurons in P16 KO G42 mice. Scale bar, 10 μm. C, Full-length blots of ErbB4 immunoreactivity demonstrate specificity.
Figure 3.
Figure 3.
The PFC developed normally in KO mice. A, Coronal brain sections immunostained with NeuN (top), and PV (bottom) in the P30 PFC of control and KO mice. Roman numerals (I, II, III, V, VI) indicate cortical layers. Scale bar, 100 μm. B, Quantification of the laminar distribution of PV neurons did not differ significantly between control and KO mice. C, Western blots of the PFC using antibodies against gephyrin, GAD65, GAD67, α1, α2, β2, β-actin, and GAPDH revealed similar protein expression levels in P30 KO mice compared with their control littermates. D, The same as for C in P60 mice. E, Relative protein quantification of gephyrin, GAD65, GAD67, α1, α2, and β2 showed no significant difference between control and KO mice. All values were normalized to control, and the data in control were given a value of 1. F, The same as for E in P60 mice. Mean ± SEM.
Figure 4.
Figure 4.
GABAergic synapses on PNs formed by FS basket cells developed lacking ErbB4. A, uIPSCs were induced in PNs by eliciting action potentials in FS neurons from a control (upper) and a KO (lower) P17 mouse. Traces are the average of 50 trials. B, Short-term plasticity was not changed. Mean ± SEM. NS, not significant. C, vGAT- and PV-labeled presynaptic terminals around PN somata immunostained with NeuN antibody in P30 and P60 mice. Each image is a single confocal plane. Scale bars, 5 μm (P30); 10 μm (P60). D, Representative traces of mIPSCs from P60 PN neurons. (E, F) Cumulative plots of mIPSC frequencies (E) and amplitudes (F).
Figure 5.
Figure 5.
ErbB4 signaling was not required for the development of chemical connections among FS basket cells. A, uIPSCs were induced in postsynaptic neurons by eliciting APs in the chemically coupled presynaptic neurons via injection of depolarizing currents from a control (top) and a KO (bottom) FS-FS pair in P16 mice. Traces are the average of 50 trials. Left, Insets, The firing patterns of both postsynaptic FS interneurons. Recordings were done in the presence of mefloquine (50 μm; to block gap junctions). B, Short-term plasticity was unaltered in KO mice. Mean ± SEM. NS, Not significant. C, vGAT-labeled presynaptic terminals around PV neuron somata in P30 mice. Each image is a single confocal plane. Scale bar, 5 μm. D, Representative traces of mIPSCs recorded from FS basket cells in P60 mice. E, F, Cumulative plots of mIPSC frequencies (E) and amplitudes (F).
Figure 6.
Figure 6.
The lack of ErbB4 was not compensated for by ErbB2. A, Expression levels of ErbB2 and Akt protein in P30 control and KO mice were similar. B, There was no significant difference in the protein expression levels of ErbB2 and Akt in both genotypes. Mean ± SEM; n = 6 mice per genotype.
Figure 7.
Figure 7.
The initial formation of glutamatergic synapses on FS basket cells was not affected by ErbB4 signaling. A, uEPSCs were induced in postsynaptic FS neurons by eliciting APs in presynaptic PNs from a control (top) and a KO (bottom) PN-FS pair in P17 mice. Traces are the average of 50 trials. B, Short-term plasticity was not changed in KO mice; mean ± SEM. C, vGluT1-labeled presynaptic terminals on PV neuron somata in P16 mice. Each image is a single confocal plane. Scale bar, 10 μm. D, Representative traces of mEPSCs recorded from FS basket cells in P18 mice. E, F, Cumulative plots of mEPSC frequencies (E) and amplitudes (F).
Figure 8.
Figure 8.
The maturation of glutamatergic synapses on FS basket cells was disrupted in the absence of ErbB4 signaling. A, vGluT1-labeled presynaptic terminals around PV neuron somata in P40 mice. Each image is a single confocal plane. Scale bar, 10 μm. B, Representative traces of mEPSCs recorded from FS neurons in P60 mice. C, D, Cumulative plots of mEPSC frequencies (C) and amplitudes (D). mEPSC frequencies were significantly reduced in KO mice. E, There is a trend of decrease in the amplitude of eEPSCNMDAR in KO mice.
Figure 9.
Figure 9.
Activity-dependent GABAergic FS-PN transmission was reduced in KO mice. A, Representative traces of sIPSCs recorded from PNs in P60 mice. B, sIPSC frequency but not amplitude was significantly decreased in KO mice. C, Representative traces of AP firing elicited in response to 500 ms suprathreshold current of 450–650 pA recorded from FS basket cells in P60 mice; Vh, −70 mV; increment, 50 pA. D, Plots of firing frequency (spikes/pulse) revealed significant differences between Cont and KO mice at 600 pA and 650 pA depolarizing levels. E, Representative traces of spontaneous firing rate of FS neurons in P60 mice; mean ± SEM in B, D; *p < 0.05.
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