Minocycline rescues decrease in neurogenesis, increase in microglia cytokines and deficits in sensorimotor gating in an animal model of schizophrenia

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Abstract

Adult neurogenesis in the hippocampus is impaired in schizophrenic patients and in an animal model of schizophrenia. Amongst a plethora of regulators, the immune system has been shown repeatedly to strongly modulate neurogenesis under physiological and pathological conditions. It is well accepted, that schizophrenic patients have an aberrant peripheral immune status, which is also reflected in the animal model. The microglia as the intrinsic immune competent cells of the brain have recently come into focus as possible therapeutic targets in schizophrenia.

We here used a maternal immune stimulation rodent model of schizophrenia in which polyinosinic–polycytidilic acid (Poly I:C) was injected into pregnant rats to mimic an anti-viral immune response. We identified microglia IL-1β and TNF-α increase constituting the factors correlating best with decreases in net-neurogenesis and impairment in pre-pulse inhibition of a startle response in the Poly I:C model. Treatment with the antibiotic minocycline (3 mg/kg/day) normalized microglial cytokine production in the hippocampus and rescued neurogenesis and behavior. We could also show that enhanced microglial TNF-α and IL-1β production in the hippocampus was accompanied by a decrease in the pro-proliferative TNFR2 receptor expression on neuronal progenitor cells, which could be attenuated by minocycline. These findings strongly support the idea to use anti-inflammatory drugs to target microglia activation as an adjunctive therapy in schizophrenic patients.

Introduction

Schizophrenia is a devastating disorder and constitutes a social and economic burden for patients as well as families and society. During the past decades, an increasing number of studies have associated schizophrenia and inflammation (Müller and Schwarz, 2010, Fineberg and Ellman, 2013). Concomitantly, microglia cells – the intrinsic immune competent cells of the brain – have been pinpointed in the pathophysiology of this neurodevelopmental disorder in both human patients and animal models of this disorder (Blank and Prinz, 2013, Fricker et al., 2013, Harry and Kraft, 2012). In a subpopulation of schizophrenic patients increased microglial cellular density and activity has been found in post-mortem tissue and in vivo (Falkai et al., 1999, Steiner et al., 2006, van Berckel et al., 2008, Busse et al., 2012) as well as in animal models of schizophrenia with varying results depending on brain region and age investigated (Juckel et al., 2011, Garay et al., 2013, van den Eynde et al., 2014).

One way in which activated microglia contribute to pathology is through the production of pro-inflammatory cytokines. An imbalance in cytokine levels may trigger aberrant neurodevelopment in the fetus and lead to neuropathology and psychopathology in the adult offspring. Infection-induced increase of pro-inflammatory maternal cytokines may be one of the key events leading to enhanced risk for neuropsychiatric disorders in the offspring (Gilmore and Jarskog, 1997). Human studies revealed that increased maternal serum levels of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and the chemokine Interleukin-8 (IL-8) during pregnancy are directly associated with a higher risk for schizophrenia in the progeny (Brown et al., 2004, Buka et al., 2001). In line with this finding, Mednick and colleagues reported that fetuses gestating during a viral epidemic are at elevated risk for developing schizophrenia (Mednick et al., 1988). Subsequent prospective studies have shown that maternal infections of various types increase the risk for schizophrenia in the offspring three- to sevenfold (for review see Brown and Derkits, 2010). Rodent studies have confirmed that a maternal immune response is sufficient to induce psychopathology in later life (Biscaro et al., 2012, Ozawa et al., 2006, Meyer et al., 2005, Zuckerman and Weiner, 2005, Shi et al., 2003, Abazyan et al., 2010, Zuckerman et al., 2003, Frick et al., 2013). Injection of pregnant rodents with the viral mimic polyinosinic–polycytidilic acid (Poly I:C) leads to a wide spectrum of schizophrenia-relevant behavioral deficits, such as pre-pulse inhibition of the acoustic startle response (PPI) (Gal et al., 2009, Klein et al., 2013, Kumari et al., 2008, Smith et al., 2007, Nyffeler et al., 2006, Schwarzkopf et al., 1992; for review see Yamada, 2000). Behavioral deficits are associated with schizophrenia-relevant neuropathological deficits including abnormalities in dopaminergic and glutamatergic neurotransmission (Winter et al., 2008; for review see Kirkpatrick, 2013), histopathological (Biscaro et al., 2012, Kühn et al., 2012) and structural changes (Piontkewitz et al., 2011a, Piontkewitz et al., 2012). The relevance of maternal Poly I:C -induced deficits to schizophrenia is further supported by the responsiveness of adult behavioral deficits to neuroleptic treatment (Piontkewitz et al., 2011b). Finally, prenatal Poly I:C -induced behavioral abnormalities exhibit the maturation delay of schizophrenia (Meyer, 2013, Meyer, 2014, Feldon and Weiner, 2009), enabling the elucidation of progressive mechanisms possibly underlying behavioral manifestations as well as preventive interventions. Maternal immune stimulation is thus an excellent model to study pathophysiological and therapeutic aspects relevant to schizophrenia (Meyer and Feldon, 2012, Lipina et al., 2013; for reviews see Meyer, 2013, Meyer, 2014).

Schizophrenia has the most robust clinical evidence for a disease-related reduction in grey and white matter including smaller hippocampal volume assessed in chronic schizophrenic patients (Wexler et al., 2009) and animal models (Meyer, 2013, Meyer, 2014, Piontkewitz et al., 2012, Lipska, 2004). Hippocampal involvement is likely to be associated with neuropsychological impairments of schizophrenia (Harrison, 2004) as well as with its psychotic symptoms (Ewing and Winter, 2013, Floresco and Jentsch, 2011).

Hippocampal structural pathology in schizophrenia might be due to aberrant neurodevelopment and abnormal neural plasticity. One particular example of cell-based brain plasticity is the generation of new neurons in the hippocampus throughout life (Altman and Das, 1965, Eriksson et al., 1998). Neurogenesis has been linked with hippocampal-dependent function (for reviews see Deng et al., 2010, Bruel-Jungerman et al., 2007). Recently, microglial activity has been shown to be important for the homeostasis of neurogenesis, predominantly through the phagocytosis of apoptotic neuronal progenitor cells (Sierra et al., 2010) and balancing apoptotic and proliferative events via TNF-α signaling (Chen and Palmer, 2013). Baseline microglial activity and cytokine levels in the hippocampus are needed to maintain baseline neurogenesis while an immune response accompanied by an increase in pro-inflammatory cytokines is thought to be detrimental for neurogenesis. Consequently, anti-inflammatory drugs have been shown to ameliorate the decrease of neurogenesis caused by pro-inflammatory cytokines (for review see Kohman and Rhodes, 2013). In schizophrenic patients Miyaoka and colleagues demonstrated significant and robust clinical improvements using the tetracycline minocycline – a potent inhibitor of microglial activation (Miyaoka et al., 2008, Seki et al., 2013). Minocycline has been used successfully in some clinical trials since as an adjunctive therapy to antipsychotics for schizophrenia (for review see Dean et al., 2012). How minocycline affects microglia function in vivo and neurogenesis is still not fully understood.

We here evaluated the effects of minocycline treatment on neurogenesis in parallel to microglia density, activation and cytokine production in the hippocampus compared to other brain regions in the Poly I:C rat model of schizophrenia. We correlated these data with the effects of minocycline treatment on sensorimotor gating deficits – a behavioral phenotype relevant to schizophrenia.

Section snippets

Animals

All experimental protocols conformed to the guidelines of the European Communities Council Directive (86/609/EEC) for care of laboratory animals and were approved by the local ethic committee (Landesdirektion Dresden). Wistar Rats (Harlan laboratories) were housed in a temperature and humidity controlled vivarium with a 12-h light–dark cycle (lights on: 6 a.m. to 6 p.m.). They had access to food and water ad libitum.

Poly I:C injections

Rats were mated at about an age of three months and the first day after

The down regulation of neurogenesis in Poly I:C offspring is normalized by minocycline treatment

To label proliferating cells we injected BrdU i.p. four weeks prior to sacrifice. We counted the BrdU positive cells in the subgranular zone of the dentate gyrus (DG) of the hippocampus and phenotyped them using doublecortin (DXC), a marker of the late mitotic stage and NeuN, a marker of mature neurons (Fig. 1). In line with reports from us and others (Meyer et al., 2010, Wolf et al., 2011), we could detect a decrease in the total number of BrdU positive cells in the Poly I:C H2O offspring (2213

Discussion

Hippocampal adult neurogenesis in the dentate gyrus contributes to brain plasticity with a pool of new neurons, which mature and integrate into functional circuits. We and others have previously shown that adult hippocampal neurogenesis is down-regulated in the offspring of in utero immune challenged animals (Wolf et al., 2011, Meyer et al., 2010). Studies conducted on schizophrenic patients reveal smaller hippocampal volume, which correlates with the positive symptoms of schizophrenia, and

Acknowledgments

The authors are grateful for excellent technical assistance by Sophie Neuber, Doris Zschaber and Nadine Scharek. The work was supported by the by BMBF under the framwork of EraNet Neuron (DBS F20_rat) and the German research council (DFG) through SFB TR 43.

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    DM & ADI/CW & SAW contributed equally as first or last authors respectively.

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