Estradiol and luteinizing hormone regulate recognition memory following subchronic phencyclidine: Evidence for hippocampal GABA action

Highlights

• Phencyclidine (PCP) decreased hippocampal GAD67 and caused memory loss in rats.

• Decreasing luteinizing hormone restored hippocampal GAD67 and rescued memory.

• Hippocampal GABA agonism reversed PCP-induced memory loss.

• Hippocampal GABA antagonism or synthesis inhibition mimicked amnesic effects of PCP.

• Estradiol protected memory, and hippocampal GABA antagonism blocked this effect.

Abstract

The cognitive symptoms of schizophrenia are poorly understood and difficult to treat. Estrogens may mitigate these symptoms via unknown mechanisms. To examine these mechanisms, we tested whether increasing estradiol (E) or decreasing luteinizing hormone (LH) could mitigate short-term episodic memory loss in a phencyclidine (PCP) model of schizophrenia. We then assessed whether changes in cortical or hippocampal GABA may underlie these effects. Female rats were ovariectomized and injected subchronically with PCP. To modulate E and LH, animals received estradiol capsules or Antide injections. Short-term episodic memory was assessed using the novel object recognition task (NORT). Brain expression of GAD67 was analyzed via western blot, and parvalbumin-containing cells were counted using immunohistochemistry. Some rats received hippocampal infusions of a GABA_A agonist, GABA_A antagonist, or GAD inhibitor before behavioral testing. We found that PCP reduced hippocampal GAD67 and abolished recognition memory. Antide restored hippocampal GAD67 and rescued recognition memory in PCP-treated animals. Estradiol prevented PCP’s amnesic effect in NORT but failed to restore hippocampal GAD67. PCP did not cause significant differences in number of parvalbumin-expressing cells or cortical expression of GAD67. Hippocampal infusions of a GABA_A agonist restored recognition memory in PCP-treated rats. Blocking hippocampal GAD or GABA_A receptors in ovx animals reproduced recognition memory loss similar to PCP and inhibited estradiol’s protection of recognition memory in PCP-treated animals. In summary, decreasing LH or increasing E can lessen short-term episodic memory loss, as measured by novel object recognition, in a PCP model of schizophrenia. Alterations in hippocampal GABA may contribute to both PCP’s effects on recognition memory and the hormones’ ability to prevent or reverse them.

Introduction

Schizophrenia is a common neuropsychiatric disorder that causes devastating cognitive, negative, and positive symptoms (McGrath et al., 2008). Despite the importance of cognitive symptoms for a patient’s long-term well-being, current treatments for schizophrenia fail to adequately improve them (Green et al., 2004). Better therapies and a greater understanding of the brain mechanisms underlying cognitive loss in schizophrenia are needed.

A dominant model of schizophrenia suggests that hypofunctioning N-methyl-d-aspartate glutamate receptors (NMDARs) play a role in generating schizophrenia symptoms (Moghaddam and Javitt, 2012). Repeated exposure to NMDAR antagonists causes schizophrenia-like symptoms in healthy humans and exacerbates symptoms in those with schizophrenia (Jones et al., 2011; Moghaddam and Javitt, 2012; Nakazawa et al., 2012). Animal models of schizophrenia using repeated (sub-chronic) treatment with NMDAR antagonists such as phencydlidine (PCP) and ketamine have been developed (Jones et al., 2011; Moghaddam and Javitt, 2012), which produce traits that approximate positive, negative, and cognitive symptoms of schizophrenia, including deficits in short-term episodic memory (Jones et al., 2011; Rajagopal et al., 2014).

Current theories propose that NMDA hypofunction disrupts gamma-amino-butyric-acid (GABA) neurons, causing cognitive loss in schizophrenia (Lewis et al., 2012). Experiments suggest that abnormal GABA-containing inhibitory neurons are common in schizophrenia (Nakazawa et al., 2012). Post-mortem analyses reveal that the brains of people with schizophrenia express lower amounts of the rate-limiting enzyme for GABA synthesis, glutamic acid decarboxylase 67 (GAD67) (Benes et al., 2007; Lewis et al., 2012). There is also reduced expression of calcium-binding protein parvalbumin (PV), which marks a subset of GABAergic neurons (Beasley and Reynolds, 1997; Zhang and Reynolds, 2002). These GABAergic abnormalities are found in a number of brain regions thought to be important for declarative and short-term memory, including the hippocampus and frontal neocortex (Benes et al., 2007; Lewis et al., 2012; Zhang and Reynolds, 2002). Studies in NMDAR animal models of schizophrenia have found similar deficits of GAD67 and PV expression in the prelimbic cortex and hippocampus, further supporting their possible mechanistic role in schizophrenia-like cognitive loss (Abdul-Monim et al., 2007; Behrens et al., 2007; Kaalund et al., 2013).

There are indications that estradiol can mitigate memory deficits in schizophrenia and animal models of the disease (for review see McGregor et al., 2017). Clinical trials have suggested that estrogen and analogues may improve some measures of cognition in males and females with schizophrenia (McGregor et al., 2017). Estradiol can also improve recognition memory in the PCP model of schizophrenia when given before or after PCP treatment (Roseman et al., 2012; Sutcliffe et al., 2008). Still, the underlying mechanisms of estradiol’s cognitive rescue remain unknown, and the possibility that estradiol works through GABAergic pathways to restore memory has not been investigated.

Estrogen inhibits release of luteinizing hormone (LH) through negative feedback (Charlton, 2008; Wise and Ratner, 1980), and it is conceivable that estrogen’s beneficial effects in schizophrenia are due to LH inhibition. High LH levels are associated with memory decline in humans, and rodent studies support a causal relationship (Burnham et al., 2016; Burnham and Thornton, 2015; Casadesus et al., 2007). Nevertheless the molecular mechanisms underlying LH’s amnesic effects remain uncharacterized, and the therapeutic possibilities of LH inhibition in schizophrenia are unknown.

Here we tested whether modulating estradiol (E) or LH could mitigate loss of short-term episodic memory in the subchronic PCP model of schizophrenia, and if these hormones act by counteracting PCP’s putative effects on GABA neurons (Roseman et al., 2012; Wise and Ratner, 1980; Ziegler and Thornton, 2010; Janhunen et al., 2015; Jones et al., 2011; Rajagopal et al., 2014). Rats with altered E and LH levels were injected with subchronic PCP then tested in the novel object recognition task (NORT). NORT is widely used to assess episodic memory loss in animal models of schizophrenia, and performance in the task depends on specific functioning of cortical and hippocampal regions altered in the disorder (Benes et al., 2007; Broadbent et al., 2004; Cohen et al., 2013; Lewis et al., 2012; Oliveira et al., 2010; Rajagopal et al., 2014; Zhang and Reynolds, 2002). To assess whether GABAergic changes could account for the drug and hormone effects on novel object recognition, we measured GAD67 levels and counted PV-expressing cells in the prelimbic cortex and dorsal hippocampus. We then infused a GABA receptor agonist, antagonist, or synthesis blocker into the dorsal hippocampus of rats before NORT.