| Schizophrenia is a devastating psychiatric illness that affects about 1% of the world's population. In recent years, by more and more people neuregulin1 (NRG1) and ErbB4, as members among multiple candidate susceptibility genes for schizophrenia, have become a hotspot of current neuroscience research.Genome-wide linkage analysis and large-scale profiling of gene expression have implicated that NRG1 and ErbB4 are the potential susceptibility genes for schizophrenia in diverse populations from Iceland, Scotland, China, Japan, and Korea. Increase in the expression level of NRG1 isoforms or ErbB4 have been reported in schizophrenia patients. And NRG1-induced activation of ErbB4 in cortex of schizophrenic patients is also increased. Furthermore, NRG1 and ErbB4 mutant mice also exhibit Schizophrenia-like phenotypes.NRG1 belongs to a family of trophic factors, and that are encoded by four individual genes (NRG1-4), of which NRG1 is the best characterized. NRG1 acts by stimulating a family of single-transmembrane receptor tyrosine kinases called ErbB proteins, which also have several isforms (ErbB1-4). Among them ErbB4 is the only autonomous NRG1-specific ErbB that can both interact with the ligand and become activated by it as a tyrosine kinase. Our present knowledge of NRG1 is mostly restricted to early development, like neuronal differentiation, migration, axon guidance and establishment etc.. Although NRG1 and its ErbB4 receptor are expressed highly in the adult rodent and human brain, little is known about their functions. The role of NRG1-ErbB4 signaling and its mechanisms underlying the pathology of schizophrenia are primarily unknown in the adult CNS.The emotional processing abnormalities were observed in patients with schizophrenia, and MRI evidences is suggestive of amygdala involvement. Recent studies showed NRG1 mutant mice exhibits deficits in physiology and behaviors like contextual fear conditioning and social interactions. As we know Fear conditioning is widely used as a model system for understanding how the brain forms and stores information about aversive emotional experiences. Research on the neural basis for the fear conditioning points to the amygdala as a key structure. And changes of long-term potentiation in the amygdala plays and important role in fear conditioning. Among the many mechanism underling LTP in amygdala, GABAergic inhibiton shows a powerful control of the LTP. For amygdala differs from other brain regions by the low firing due to a strong inhibitory tone. And given that NRG1-ErbB4 signaling regulating GABAergic inhibition is a candidate mechanism for the pathology of schizophrenia. NRG1/ErbB4 signaling may also regulate LTP in the amygdala by an GABAergic mechanism and that can also explain the changes in fear conditioning. So we decided to investigate the role of NRG1/ErbB4 signaling for GABAergic inhibition, and to see whether it regulates LTP in the lateral amygdala.Firstly, We recorded input-output relationship for EPSCs and IPSCs and confirmed the result that amygdala exhibits comparatively high GABAergic activity by the factor of IPSC/EPSC ratio. We investigate how the magnitude of the maximal IPSC differs in Lateral amygdala(LA), Hippocampus(CA1) and Cortex(parietal cortex, layer v). LA principle neurons exhibits higher IPSC amplitude (871.40±57.07pA, n=10) than that in Hip. (506.41±65.45 pA, n=10) and cortex (682.44±79.98 pA, n=9). And only between the two groups of LA and Hip. shows significant difference (P<0.05, one way ANOVA). And the IPSC I-O curve in LA lined above the EPSC I-O curve while that fell below in both hippocampus and cortex. Furthermore, the balance of inhibition/excitation in LA was significantly different from that in hippocampus (the 20,25,30,35,40V stimulus shown significant difference, P<0.5, Student T test) and cortex (the 10,15V stimulus shown significant difference, P<0.5, Student T test). Accordingly, LTP does not exist in LA in common situations, and the amplitude of fEPSP was 102.3±2.5%(n=8 slices,5 animals) after tetanus stimulation for thalamus inputs. In contrast, under the same conditions the LTP in CA1 is 158.5±11.2%(n=14 slices,10 animals). And independent-samples T test showed significant difference between the two areas for the averaged percentage after tetanus (P=0.000).This suggested the high level of GABAergic activity in LA hold the principle neurons to form LTP. To further confirm the theory, we gave thalamic inputs tetanus stimulation in LA under the application of 20μM bicuculline methiodidie(BMI, GABAA receptor blocker) to see whether it affects LTP. We found robust LTP can be induced in the LA in BMI treated slices with an fEPSP amplitude 1h after tetanus amounting to 119.2±1.6%(n=6 slices,4 animals)of its amplitude during baseline, while that in control slices is 102.3±2.5%(n=8 slices,5 animals). And they showed significant difference. This result indicates GABA-mediated inhibition suppressed LTP induction in LA.To further investigate whether NRG1-ErbB4 signaling regulates GABAergic inhibition, we recorded synaptically evoked inhibitory postsynaptic currents (IPSCs) form LA principal cells in slice incubated in the presence of kynurenic acid (1 mM, a broad spectrum ionotropic glutamate receptor antagonist), IPSCs were evoked with a bipolar stimulate electrode placed to thalamus inputs. The amplitude of IPSC was not affected after NRG1 perfusion (2nM, n=9). For each cell, IPSC amplitude was normalized to the value of mean IPSC before bath application of NRG1, after 10 min NRG1 treatment the normalized amplitude was 0.90±0.05(P>0.05). In contrast, ecto-ErbB4 (cloned protein, contain the ecto-domain of ErbB4, binding to NRG1 and inhibit its activity) and AG1478 (a potent and membrane permeable ErbB kinase inhibitor, which is known to prevent receptor autophosphorylation and signaling) remarkably attenuated the amplitude of IPSC, after ecto-ErbB4 and AG1478 treatment the normalized amplitude was 0.65±0.05 (2μg/ml, n=8, P<0.01) and 0.70±0.04(5μM, n=10, P<0.01), respectively. These results showed NRG1 have no effect on eIPSC, but the inhibitors for NRG1/ErbB4 signaling attenuate it, these suggested NRG1/ErbB4 signaling's regulation on GABAergic inhibition is saturated in physiological conditions.In order to investigate whether NRG1/ErbB4 signaling can regulate LTP in the lateral amygdala, we exerted the next experiments of tree groups. Control group(vehicle treated, without Ecto-ErbB4 treatment):It exhibited an FP amplitude 1h after tetanus amounting to 99.87±3.15% (n=8 slices,5 animals) of its amplitude during baseline. NRG1 treated group:105.49±2.47% (n=6 slices,4 animals). ecto-ErbB4 (2μg/ml,7 slices,5 animals) treated group:117.97±3.72%. One way ANOVA for the averaged percentage after tetanus revealed a significant difference of the potentiation under ecto-ErbB4 application(F= 8.092, P= 0.003, P<0.05 compared with control and NRG1 treated group). This result indicates NRG1-ErbB4 signaling controls LTP induction in LA, and the signaling stands in a saturated state only can be negatively regulated.At the same time, ecto-ErbB4(2μg/ml) application show the same effects on LTP when tetanic stimulation was given to the cortical afferents. It exhibited an FP amplitude 1h after tetanus amounting to 123.05±4.82% (n=8 slices,5 animals) of its amplitude during baseline, while that in control slices amounting to 107.59±2.90%(vehicle treated,6 slices,5 animals). Independent-samples T test for the averaged percentage after tetanus revealed a significant difference of the potentiation under ecto-ErbB4 application (P=0.025). This result indicates NRG1-ErbB4 signaling take the same effect for the cortical afferents as that for thalamic afferents, shown NRG1-ErbB4 signaling have no selective effects on the two input pathways.Because blocking GABAA receptors or NRG1/ErbB4 signaling both enhanced the tetanus-induced LTP in LA. Thus, we hypothesized that ErbB4 inhibitor enhance the LTP induction by decreasing GABAA mediated inhibition in the LA. To test this hypothesis, we examined the effects of application of only ecto-ErbB4(2μg/ml) or both ecto-ErbB4(2μg/ml) and 20μM BMI on tetanus-induced LTP in amygdal slices, and to see whether they can produce synergistic or additive effects. We found that there are greatly enhanced tetanus-induced LTP in both two groups, and they showed almost the same potentiation extent on LTP. For the ecto-ErbB4 and BMI treated group, it exhibited an FP amplitude 1h after tetanus amounting to 120.76±5.68% (n=8 slices,4 animals) of its amplitude during baseline, while that in only BMI treated group amounting to 119.23±1.61%(20μM,6 slices,4 animals)). Independent-samples T test for the averaged percentage after tetanus revealed no significant difference between the two groups(P=0.825). These results suggested that NRG1/ErbB4 signaling regulates LTP induction through the GABAergic inhibition.Beside the effects of ecto-ErbB4 on LTP, it had no effect on basal synaptic transmission. We performed complete input-output (I-O) curves at a series of increasing stimulation intensities, after 20 min of ecto-ErbB4 superfusion additional I-O curves were performed at the same stimulation, and between them no obvious changes were detected. Also, ecto-ErbB4 superfusion has no influence on PPF that reflect an impact on presynaptic mechanisms.Next, we used a kind of ErbB4 knockout mice (ErbB4-/- HER4heart). We want to see how genetic alterations of NRG1-ErbB4 signaling influents GABAergic inhibition and LTP induction in the LA. Again, we recorded input-output relationship for EPSCs and IPSCs to investigate whether ErbB4 knockout mice have impaired GABAergic inhibition by the factor of IPSC/EPSC ratio. We first compared the maximal IPSCs between knockout mice and WT mice. The LA principle neurons of ErbB4-/-HER4heart mice exhibits smaller IPSC amplitude (619.09±72.65pA, n=18) than that in WT control (809.10±83.69pA, n=12). But the difference between them was not significant (P>0.05, student T test). Also the IPSC I-O curve in LA from ErbB4-/-HER4heart mice fell below the EPSC I-O curve while that lined above in the WT control mice. Even more important, the balance of inhibition/excitation in LA from ErbB4-/-HER4heart mice was significantly different from that in WT control mice (the15,20,25,30,35,40,50V stimulus shown significant difference, P<0.5, Student T test). This result further supported that NRG1-ErbB4 signaling plays and important role in regulating GABAergic inhibition.In the next step, we evaluated the difference of LTP induction between the ErbB4 knockout mice and WT mice. Tetanic stimulation of thalamic afferents can not induce LTP in the LA in control slices of WT mice. It exhibited an FP amplitude 1h after tetanus amounting to 105.93±5.09% (n=7 slices,4 animals) of its amplitude during baseline. In contrast, in the ErbB4-/-HER4heart mice (6 slices,4 animals), we found robust LTP can be induced in the LA with an FP amplitude 1h after tetanus amounting to 121.94±3.42% of its amplitude during baseline. It was significantly bigger than that in WT mice (P＜0.05). We further applied BMI for ErbB4-/-HER4heart mice slices to see whether they have synergic effects, the BMI treated group (5 slices, 3 animals) exhibited and LTP of 119.22±4.76%. And significant difference cannot be seen between ErbB4-/-HER4heart mice and BMI treated slices. This evidence confirmed the result that NRG1/ErbB4 signaling regulate LTP induction through GABAergic inhibition.In conclusion, our results here showed the saturated NRG1/ErbB4 signaling activity holds the robust GABAergic inhibition and suppressed the LTP induction in the lateral amygdala. This mechanism might explain the impairments of contextual fear conditioning in NRG1 mutant mice, and contribute to emotional dysfunctions in subjects with schizophrenia. |
