EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo.

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

Epidermal growth factor signals attenuate phenotypic and functional development of neocortical GABA neurons.

Phenotypic development of neocortical GABA neurons is highly plastic and promoted by various neurotrophic factors such as neuregulin-1. A subpopulation of GABA neurons expresses not only neuregulin receptor (ErbB4) but also epidermal growth factor (EGF) receptor (ErbB1) during development, but the neurobiological action of EGF on this cell population is less understood than that of neuregulin-1. Here, we examined the effects of exogenous EGF on immature GABA neurons both in culture and in vivo and also explored physiological consequences in adults. We prepared low density cultures from the neocortex of rat embryos and treated neocortical neurons with EGF. EGF decreased protein levels of glutamic acid decarboxylases (GAD65 and GAD67), and EGF influences on neuronal survival and glial proliferation were negligible or limited. The EGF treatment also diminished the frequency of miniature inhibitory postsynaptic currents (mIPSCs). In vivo administration of EGF to mouse pups reproduced the above GABAergic phenomena in neocortical culture. In EGF-injected postnatal mice, GAD- and parvalbumin-immunoreactivities were reduced in the frontal cortex. In addition, postnatal EGF treatment decreased mIPSC frequency in, and the density of, GABAergic terminals on pyramidal cells. Although these phenotypic influences on GABA neurons became less marked during development, it later resulted in the reduced ß- and γ-powers of sound-evoked electroencephalogram in adults, which is regulated by parvalbumin-positive GABA neurons and implicated in the schizophrenia pathophysiology. These findings suggest that, in contrast to the ErbB4 ligand of neuregulin-1, the ErbB1 ligand of EGF exerts unique maturation-attenuating influences on developing cortical GABAergic neurons.

Pathological Implications of Oxidative Stress in Patients and Animal Models with Schizophrenia: The Role of Epidermal Growth Factor Receptor Signaling.

Proinflammatory cytokines perturb brain development and neurotransmission and are implicated in various psychiatric diseases, such as schizophrenia and depression. These cytokines often induce the production of reactive oxygen species (ROS) and regulate not only cell survival and proliferation but also inflammatory process and neurotransmission. Under physiological conditions, ROS are moderately produced in mitochondria but are rapidly scavenged by reducing agents in cells. However, brain injury, ischemia, infection, or seizure-like neural activities induce inflammatory cytokines and trigger the production of excessive amounts of ROS, leading to abnormal brain functions and psychiatric symptoms. Protein phosphatases, which are involved in the basal silencing of cytokine receptor activation, are the major targets of ROS. Consistent with this, several ROS scavengers, such as polyphenols and unsaturated fatty acids, attenuate both cytokine signaling and psychiatric abnormalities. In this review, we list the inducers, producers, targets, and scavengers of ROS in the brain and discuss the interaction between ROS and cytokine signaling implicated in schizophrenia and its animal models. In particular, we present an animal model of schizophrenia established by perinatal exposure to epidermal growth factor and illustrate the pathological role of ROS and antipsychotic actions of ROS scavengers, such as emodin and edaravone.

In vivo administration of epidermal growth factor and its homologue attenuates developmental maturation of functional excitatory synapses in cortical GABAergic neurons.

The ErbB1 ligand family includes epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor, amphiregulin and betacellulin. Previously, we demonstrated that TGFalpha decreases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in cultured neocortical gamma-aminobutyric acid (GABA) neurons. In the present study, we examined in vivo effects of EGF and TGFalpha in the mouse neocortex using electrophysiological and biochemical techniques. In mouse neonates, subcutaneously administered EGF penetrated the blood-brain barrier and activated ErbB1 in the neocortex. Daily administration of EGF or TGFalpha attenuates developmental increases in expression of the AMPA receptor subunits (GluR1 and GluR2/3) in the neocortex of postnatal mice. Immunohistochemistry revealed that the reduction in AMPA receptor expression was significant in the GABAergic neurons, especially those positive for parvalbumin. Using cortical slices prepared from EGF-treated mice, we recorded miniature excitatory postsynaptic currents (mEPSCs) in both GABAergic and pyramidal neurons. Subchronic treatment with EGF decreased the amplitude and frequency of mEPSCs in GABAergic neurons, but its effects were negligible on pyramidal neurons. We conclude that EGF or other ErbB1 ligand(s) attenuates a developmental increase in AMPA receptor expression and function in cortical GABAergic neurons.

Transforming growth factor alpha attenuates the functional expression of AMPA receptors in cortical GABAergic neurons.

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor alpha (TGFalpha), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFalpha treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFalpha treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFalpha effect. Moreover, TGFalpha counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFalpha with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFalpha, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.

Conditioned place preference and locomotor sensitization after repeated administration of cocaine or methamphetamine in rats treated with epidermal growth factor during the neonatal period.

Epidermal growth factor (EGF) and its structurally related proteins are involved in the developmental regulation of various brain neurons, including midbrain dopaminergic neurons. We recently reported EGF and EGF-receptor abnormalities in both the brain tissues and blood of schizophrenic patients. Administration of EGF to neonatal rats transiently increases tyrosine hydroxylase expression and subsequently results in behavioral abnormalities in prepulse inhibition of acoustic startle, locomotor activity, and social interaction after development. The enhanced locomotor and stereotypic responses of the neonatally EGF-treated rats are considered to be an animal model for positive schizophrenia symptoms. In the present study, we investigated psychostimulant sensitivity of neonatally EGF-treated rats. At the adult stage, EGF-treated rats were challenged with cocaine (15 mg/kg) or methamphetamine (1 mg/kg), and conditioned place preference and locomotor activity were examined. The rats that received EGF during the neonatal period had significantly higher conditioned place preference for where cocaine or methamphetamine was administered than controls. The neonatal EGF treatment enhanced behavioral response to methamphetamine and behavioral sensitization to cocaine at the adult stage. Drug-naive controls gradually increased locomotor responses to cocaine during their daily injections, whereas EGF-treated rats exhibited a larger increase in cocaine responses. These results indicate that overactivation of the EGF receptors (ErbB1) during the neonatal period influences future sensitivity to psychostimulants. Our findings indicate a potential link between EGF-receptor activation and drug addiction.