Abnormal neurobehavioral development in rodents treated with cytokines as neonates
Because of the limitations of the experimental approach in humans, it is essential to establish appropriate animal models to examine the alternative hypotheses of schizophrenia, to understand the pathophysiology of this disease, and to develop novel biological therapies for schizophrenia. To test the cytokine hypothesis of schizophrenia, the impact of cytokine challenge on neurobehavioral development has been evaluated by a series of animal experiments.78,128–132 In these studies, sublethal doses of cytokines have been subcutaneously administered during postnatal day 2 to 10 in rats or mice. Neurobehavioral tests are then performed in the pre-pubertal stage (postnatal three weeks) and/or the post-pubertal stage (postnatal eight weeks). These animal experiments have revealed that neonatal cytokine challenges induce distinct abnormalities in neurobehavioral development, depending upon the type of cytokine administered (Table 1).
Table 1. Neonatal exposure to cytokines and neurobehavioral development in rodents
|3 weeks||8 weeks||3 weeks||8 weeks||3 weeks||8 weeks||8 weeks|
Neonatal EGF treatment accelerates tooth eruption and eyelid opening. This has been described by Cohen133 after isolating EGF, which was originally named the tooth–lid factor. IL-1 and other IL are known as transactivators for ErbB1 via ectodomain shedding of precursors of their ligands.39 In the pre-pubertal stage, horizontal locomotor activity under novel conditions is increased in rats treated with IL-2, whereas it is decreased in rats treated with interferon-γ (IFN-γ) or leukemia inhibitory factor (LIF). However, these abnormalities disappear in the post-pubertal stage. EGF-treated rats exhibit higher vertical locomotor activity only in the post-pubertal stage. Startle responses to 120 dB acoustic stimuli are elevated in rats treated with ErbB1 ligands (EGF, epiregulin and TGF-α) or IL-1α in the post-pubertal stage.
Prepulse inhibition (PPI) is a phenomenon in which a weak prepulse stimulus attenuates the response to a subsequent startling stimulus.134,135 Reduced PPI has been found in patients with neuropsychiatric disorders, including schizophrenia.134,135 Reduction of PPI levels in these patients is thought to reflect impaired function of sensorimotor gating. PPI can be tested in humans and rodents in similar fashions and is widely used in experiments to assess the face validity of animal models of schizophrenia. Neonatal treatment with ErbB1 ligands, IL-1α, NRG-1 or LIF has been found to cause a decrease in PPI in the post-pubertal stage. This PPI abnormality is independent of the increase in acoustic startles, however.130,131 It is of note that second-generation antipsychotics (SGA) such as clozapine and risperidone have been found to reduce PPI deficits in rodents treated with EGF, IL-1α or NRG-1.
Rodents treated with EGF, IL-1α or NRG-1 as neonates display other schizophrenia-like behavioral abnormalities but not learning disability in the post-pubertal stage. Latent inhibition (LI) is the ability to ignore irrelevant stimuli, and is disrupted in patients with schizophrenia.136 Similarly, LI is impaired in mice treated with NRG-1.78 In addition, neonatal NRG-1 treatment induces enhanced sensitivity to methamphetamine. Rats treated with EGF also exhibit hypersensitivity to psychostimulants (cocaine and methamphetamine)137 and the dopamine D2-like agonist quinpirole.138 Social interaction time is increased in rats treated with IL-1α, whereas it is decreased in rodents treated with EGF or NRG-1. Therefore, significant differences in behavioral traits have been reported in studies based on these different cytokine models.
In addition, the neonatal cytokine treatment model of schizophrenia is currently being developed in non-human primates. A cynomolgus monkey treated with EGF as a neonate was found to exhibit behavioral abnormalities such as hyperactivity and self-injury after adolescence.139 These behavioral abnormalities are ameliorated by subchronic treatment with risperidone. The findings of these animal studies indicate that among many cytokines examined, neonatal treatment with EGF, IL-1α or NRG-1 produces the long-lasting schizophrenia-like behavioral abnormalities, some of which are ameliorated by SGA.
Analysis of neurobiological mechanisms underlying schizophrenia using the neonatal cytokine treatment model
Cytokines administered subcutaneously to rat and mouse neonates efficiently penetrate the blood–brain barrier (BBB) and trigger their downstream signaling pathways in the brain.78,128–132,140,141 In comparison, when IL-1α is administered to juveniles (postnatal day 14 to 22), BBB permeation is limited and schizophrenia-like behavioral abnormalities are not manifested in the post-pubertal stage.130 Therefore, it appears to be necessary for peripherally administered cytokines to cross the BBB during the neonatal stage in order for schizophrenia-like behavioral abnormalities to emerge in the post-pubertal stage. An important question is whether cytokines administered directly to the brains of adult rats in which the BBB is fully developed also lead to schizophrenia-like behavioral abnormalities. As might be expected, EGF infusion to the striatum of adult rats has been found to lower PPI and to impair LI.142 However, these deficits are reversible and are extinguished by the cessation of EGF infusion. In contrast, decreased PPI levels are persistent after puberty in rats treated with cytokines as neonates.128,129 As such, peripheral cytokine exposure in fetuses would be expected to lead to similar behavioral impairments.
Neonatal rodents have immature brains compared with human neonates. With respect to the neurodevelopmental schedule, the neonatal period (postnatal day 2 to 10) in rats appears to be equivalent to the middle gestational period of human fetuses.143 If it is possible to extrapolate the findings on the above cytokine models to humans, human fetuses exposed to high concentrations of cytokines would subsequently exhibit neurobehavioral developmental dysfunctions. The levels of cytokines including EGF, IL-1β and IL-6 in the amniotic fluid are elevated in patients who experience premature rupture of membranes during pregnancy, one of the most serious obstetric complications, even when intrauterine infection cannot be confirmed.144 It is likely that cytokines in human fetuses transmit peripheral immune/inflammatory signals through the developing BBB to their immature brains, and perturb the structural and functional development of the brain (e.g. GABA neurons and dopaminergic neurons).
One of the most common approaches in modeling the gene–environmental interactions relevant to schizophrenia is to compare the neurobehavioral consequences of neonatal manipulations (e.g. hippocampal lesions and viral infection) among different strains of rodents.145 Neonatal cytokine treatment induces distinct phenotypes depending on genetic background (Table 2).140,141 Among four strains examined (C3H/He, C57BL/6, DBA/2 and ddY), DBA/2 mice are the most sensitive to the cytokines, exhibiting pervasive behavioral alterations such as accelerated horizontal locomotor activity, elevated startle responses, and reduced PPI. It is of note that the strain-dependent behavioral sensitivity to neonatal treatment with EGF or IL-1 is correlated with basal ErbB1 phosphorylation or IL-1-triggered acute signaling in the brain, respectively. These complex gene–cytokine interactions might explain a portion of the pathological heterogeneity of schizophrenia.
Table 2. Effects of neonatal EGF/IL-1α treatment on neurobehavioral development in different mouse strains
To understand the neurobiological mechanisms underlying schizophrenia, the effects of neonatal EGF treatment on developing neurons have been investigated using electrophysiological and biochemical techniques. In the striatum, EGF increases in dopamine metabolism and TH expression have been revealed.35,128 A similar neurotrophic effect of NRG-1 on midbrain dopaminergic neurons has also been reported.78 In the ventral tegmental area, EGF enhanced excitatory synaptic input to dopaminergic neurons.146 The elevation of glutamate receptor expression may result in higher excitability of dopaminergic neurons,146 which is implicated in hyper-dopaminergic function associated with schizophrenia. In the dentate gyrus, EGF attenuates GABAergic synaptic outputs to granule cells and decreases the protein levels of vesicular GABA transporters.147 Both in vivo and in vitro, ErbB1 ligands such as EGF, HB-EGF and TGF-α, all reduce the protein expression of GluR1, most prominently in parvalbumin-positive GABAergic neurons.148–150 These findings indicate a potential pathological link between hyper-ErbB1 signaling and GABAergic dysfunction and hyper-dopaminergic dysregulation. In the neocortex, EGF and its homologue amphiregulin attenuate the expression of synaptic scaffolding proteins such as glutamate receptor interacting protein 1 and synapse-associated protein 97 kDa (SAP97).151 These findings are consistent with the results of a postmortem brain study showing that the postsynaptic proteins SAP97 and GluR1 are decreased in the PFC of patients with schizophrenia.152 These reports suggest that aberrant synaptic development triggered by cytokines may be associated with this disease as well.
The findings of animal experiments and postmortem brain studies suggest that perturbed ErbB1 signaling in either prenatal or perinatal stages may induce aberrant development or function of dopaminergic and GABAergic neurons, which is strongly implicated in the neuropathology of schizophrenia.153 Interestingly, ventral forebrain-specific Hb-egf knockout mice exhibit schizophrenia-like behavioral abnormalities, life-long decreases in the ErbB1 signal cascade, and reductions in protein levels of NR1 and postsynaptic protein-95 in the PFC.154 Brain function might be impaired in states of both hyper- and hypo-ErbB1 signaling, resulting in counterintuitive behavioral similarities between the neonatal EGF treatment model and Hb-egf knockout mice. Further elucidation of the mechanisms of cytokine signaling involved in altering brain structure and function will facilitate understanding of the pathophysiology of schizophrenia.
Search for novel antipsychotic candidates based on the cytokine hypothesis of schizophrenia
According to the cytokine hypothesis of schizophrenia, anti-inflammatory agents may have beneficial efficacy in the treatment of patients. Minocycline, a second-generation tetracycline, has been found to attenuate PPI deficits in an animal model using the NMDA receptor antagonist MK801 (dizocilpine).155,156 In a six-month, double-blind, randomized, placebo controlled trial, concomitant treatment with minocycline and SGA produced greater improvement in the negative and cognitive symptoms of patients with early-phase schizophrenia than SGA alone.157 Celecoxib, a cyclooxgenase-2 inhibitor, ameliorates impairments in PPI and LI induced by the striatal administration of EGF in adult rats.142 In an eight-week, double-blind, randomized and placebo-controlled trial, celecoxib added to risperidone surpassed risperidone in the treatment of positive and general psychopathological symptoms of patients with chronic schizophrenia.158 The results of these clinical trials suggest that anti-inflammatory agents may serve as promising adjunctive drugs in the treatment of schizophrenia.
Potential candidates for novel antipsychotics have been identified by studies using the neonatal cytokine treatment model of schizophrenia. Subchronic oral administration of emodin, a broad tyrosine kinase inhibitor, has been found to suppress acoustic startle responses and abolish PPI deficits in the neonatal EGF treatment model.159 These findings suggest that the effects of emodin on abnormal sensorimotor gating in the neonatal EGF treatment model might be ascribed to its inhibitory action on EGF/ErbB signaling. In addition, a quinazoline derivative, which is an ErbB1 inhibitor and was developed as an anticancer agent, has similar therapeutic effects in this model.160 Intensive research into the molecules involved in modulating cytokine signals may aid the development of novel classes of antipsychotics that can provide optimum outcomes for patients suffering from schizophrenia.