Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
pubs.acs.org/chemneuro
Time-Dependent Alterations in the Expression of NMDA Receptor Subunits along the Dorsoventral Hippocampal Axis in an Animal Model of Nascent Psychosis Valentyna Dubovyk†,‡ and Denise Manahan-Vaughan*,† †
Medical Faculty, Department of Neurophysiology, and ‡International Graduate School of Neuroscience, Ruhr University Bochum, 44780 Bochum, Germany ABSTRACT: Psychosis is a mental condition that is characterized by hallucinations, delusions, disordered thought, as well as socio-emotional and cognitive impairments. Once developed, it tends to progress into a chronic psychotic illness. Here, the duration of untreated psychosis plays a crucial role: the earlier the treatment begins, relative to the first episode of the disease, the better the patient’s functional prognosis. To what extent the success of early interventions relate to progressive changes at the neurotransmitter receptor level is as yet unclear. In fact, very little is known as to how molecular changes develop, transform, and become established following the first psychotic event. One neurotransmitter receptor for which a specific role in psychosis has been discussed is the N-methyl-D-aspartate receptor (NMDAR). This receptor is especially important for information encoding in the hippocampus. The hippocampus is one of the loci of functional change in psychosis, to which a role in the pathophysiology of psychosis has been ascribed. Here, we examined whether changes in NMDAR subunit expression occur along the dorsoventral axis of the hippocampus 1 week and 3 months after systemic treatment with an NMDAR antagonist (MK801) that initiates a psychosis-like state in adult rats. We found early (1 week) upregulation of the GluN2B levels in the dorsointermediate hippocampus and late (3 month) downregulation of GluN2A expression across the entire CA1 region. The ventral hippocampus did not exhibit subunit expression changes. These data suggest that a differing vulnerability of the hippocampal longitudinal axis may occur in response to MK801-treatment and provide a time-resolved view of the putative development of pathological changes of NMDAR subunit expression in the hippocampus that initiate with an emulated first episode and progress through to the chronic stabilization of a psychosis-like state in rodents. KEYWORDS: Psychosis, MK801, dorsoventral axis, hippocampus, rodent, NMDA receptors
■
INTRODUCTION Psychosis is often referred to as an abnormal state of mind that is characterized by hallucinatory experiences, delusional thinking, and disordered thoughts.1,2 Moreover, this state is accompanied by impairments in social cognition, inappropriate emotional expressions, and bizarre behavior.3−5 Most often, psychosis develops as part of a psychiatric disorder, of which, it represents an integral part of schizophrenia. It corresponds to the most florid phase of the illness. The very first manifestation of psychosis in a patient is referred to as first-episode psychosis. It reflects a critical transitional stage toward the chronic establishment of the disease, that is presumably mediated by progressive structural and functional abnormalities seen in diagnosed patients.6−8 The earlier the treatment begins after the first-episode (i.e., within the first 1−2 months), the better is the patient’s long-term functional prognosis.9 Conversely, the longer it takes to start the treatment, the more severe are the cognitive and behavioral deteriorations that subsequently develop.7,8,10 Thus, the entire course of the illness depends on the duration of untreated psychosis.11 Despite this knowledge, little is known as to how molecular changes © XXXX American Chemical Society
develop, transform, and become established over the progress of time following the first psychotic event. Although the precise neurobiology of psychosis is unclear, much progress has been made as to a better understanding of which neurotransmitter receptors are specifically involved in the disease. A role for the ionotropic glutamatergic N-methyl-Daspartate receptor (NMDAR) became apparent following observations that receptor antagonists trigger a wide spectrum of psychotic symptoms in healthy humans, and exacerbate them in patients suffering from schizophrenia.12,13 Subsequently, it was shown experimentally that NMDAR hypofunction impairs synaptic transmission between neurons and prevents interneurons from sensing the levels of network excitation, thus leading to disinhibition of principal cells.14 This may relate to changes in the expression of the subunits of NMDARs.15 Special Issue: Synaptic Plasticity Received: January 14, 2018 Accepted: March 27, 2018
A
DOI: 10.1021/acschemneuro.8b00017 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 1. Illustration of the planes used for hippocampal slicing along the dorsoventral axis, and identification of neural and synaptic subcompartments that were subjected to scrutiny. (A) Drawing of the rat brain showing the horizontally sectioned hippocampus along its longitudinal axis. Representative Nissl-stained sections correspond to dorsal (a), intermediate (b), and ventral (c) subdivisions. (B) Schematic illustration of the hippocampal transverse axis depicting its trisynaptic circuit with the laminar view. Abbreviations correspond to o., outer band of the ml of the DG; m., middle band of the ml of the DG; i., inner band of the ml of the DG; gcl, granule cell layer of the DG; pl, polymorphic layer of the DG; so, Stratum oriens of CA3/CA1; pcl, pyramidal cell layer of CA3/CA1; sl, Stratum lucidum of the CA3; sr, Stratum radiatum of CA3/CA1; and slm, Stratum lacunosum-moleculare of CA3/CA1. (C) Example of a dorsal hippocampal section showing the expression of the GluN1 subunit of the NMDAR across its transverse axis. Bottom photomicrograph corresponds to a close-up view of the layers of the CA1 region that were used for the analysis (so, Stratum oriens; pcl, pyramidal cell layer; sr, Stratum radiatum; slm, Stratum lacunosum-moleculare). A similar layer delineation strategy was implemented for the CA3 and DG regions (not shown).
This was the motivation behind the present study: we used the NMDAR antagonist, MK801, to initiate a first episode-like state in adult rats.36 This is one of the best validated pharmacological (animal) models of acute schizophreniform psychosis.37,38 We then explored whether the expression and distribution patterns of NMDAR (GluN1, GluN2A, and GluN2B) subunits become altered immediately following and three months after treatment. Changes were examined along both the hippocampal longitudinal and transverse axes. We observed that changes in the receptor protein expression indeed occur, are both region- and subregion-specific, and exhibit distinct patterns at the two time-points studied. We propose that early changes in NMDAR subunit expression correspond to adaptive restructuring, whereas expression changes 3 months after treatment reflect the chronic establishment of pathological processes (“point of no return”). These findings provide novel insights into the putative time-course of abnormal NMDAR expression changes that follow the first episode of psychosis.
However, to what extent changes in NMDAR may become manifest in the very early stages following the onset of psychosis has not yet been explored. Among brain regions involved in the pathology of psychosis/ schizophrenia, the hippocampus is one structure that displays particularly robust abnormalities. Alterations include various molecular, morphological and functional changes.16−21 The hippocampus is a site of convergence of multimodal sensory information, which becomes assimilated and integrated in space and time within the inner trisynaptic circuit of the hippocampus (dentate gyrus (DG), CA3 and CA1, corresponding to its transverse axis), resulting in the formation of memory representations.22−25 Differences in the roles of the subfields of the transverse axis with regard to spatial processing have been proposed: Whereas, for example, the CA1 region appears to enable specific encoding of elements of spatial content, the DG may support encoding of spatial element that correspond to general orientation (landmarks).26 In addition to this functional differentiation, subregions of the longitudinal axis of the hippocampus may contribute differently to elements of cognitive processing.27−29 Here, it has been proposed that the dorsal hippocampus mediates visuo-spatial information processing; the ventral hippocampus mediates socio-emotional information processing; while the intermediate hippocampus is involved in the processing of mixed information that is received by both dorsal and ventral subdivisions, as well as in rapid learning and flexible use of acquired and updating of stored information.27−29 Impairments in performance on tasks that measure each of these processing domains are routinely reported in patients with psychosis.30−32 Given the importance of the NMDAR for information encoding in the form of synaptic plasticity in both the transverse33,34 and longitudinal axes of the hippocampus,35 this raises the question as to whether different subfields or subregions of the hippocampus are affected by psychosis, and whether putative changes differ as the disease progresses.
■
RESULTS Expression of the NMDAR GluN1 Subunit Is Upregulated 3 Months, but Not 1 Week after MK801 Treatment. Scrutiny of GluN1 1 week after MK801-treatment revealed no changes in protein levels in any of the hippocampal regions and subregions examined (Figure 2A). By contrast, 3 months after treatment, we found a significant upregulation of GluN1 content, specifically in the CA1 region of the intermediate hippocampus (multifactorial ANOVA, IH: F(1,187) = 20.14, p < 0.001) (Figure 2B). Here, the layers affected included Stratum oriens (so), Stratum radiatum (sr), and Stratum lacunosum-moleculare (slm) (Table 1). In other words the entire dendritic tree of pyramidal cells in the CA1 region was affected. A Broad Downregulation in the Expression of GluN2A Subunit Occurs 3 Months, but Not 1 Week after MK801 Treatment. Overall, 1 week after MK801 treatment, we found B
DOI: 10.1021/acschemneuro.8b00017 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 2. GluN1 protein levels display no substantial changes 1 week or 3 months after MK801 treatment. Bar charts illustrate relative change in receptor protein levels across the somato-dendritic layers of the transverse and longitudinal hippocampal axes. (A) No changes were detected 1 week after MK801 treatment. (B) GluN1 levels were increased in the intermediate CA1 3 months after MK801 treatment. Values expressed in arbitrary units (a.u.). Error bars indicate SEM *p < 0.05. Abbreviations correspond to ml, molecular layer of the DG; gcl, granule cell layer of the DG; pl, polymorphic layer of the DG; so, Stratum oriens of CA3/CA1; pcl, pyramidal cell layer of CA3/CA1; sr, Stratum radiatum of CA3/CA1; and slm, Stratum lacunosum-moleculare of CA3/CA1. Photomicrographs provide examples of GluN1-stained sections from the dorsal, intermediate and ventral hippocampal subdivisions that originated from the same vehicle- or MK801-treated animal, and correspond to 1 week (left) or 3 months (right) after treatment.
occurring in the intermediate CA3 region (multifactorial ANOVA, DH: F(1,187) = 25.441, p < 0.001; IH: F(1,187) = 74.716, p < 0.001; VH: F(1,165) = 14.811, p < 0.001). Here, affected layers included the pyramidal cell layer (pcl) and slm of the dorsal CA1; so, pcl and slm of the ventral CA1;
no differences in GluN2A protein levels across the hippocampal dorso-ventral and transverse axes (Figure 3A). By contrast, a substantial reduction appeared three months following treatment (Figure 3B). Changes encompassed the entire CA1 region of the hippocampus, with additional downregulation C
DOI: 10.1021/acschemneuro.8b00017 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience Table 1. Overview of Statistically Significant Differences in the Expression of NMDAR Subunits treatment NMDAR subunit
time point
part
region
layer
GluN1
3 months
IH
CA1
GluN2A
3 months
DH
CA1
IH
CA1
so sr slm pcl slm so pcl sr slm so pcl slm so pcl slm ml pl so pcl sr slm so pcl sr slm ml pl so pcl sr slm so pcl sr slm
CA3
GluN2B
1 week
VH
CA1
DH
DG CA3
CA1
IH
DG CA3
CA1
vehicle 0.94 1.09 0.82 1.67 1.54 1.01 1.47 0.9 1.37 0.83 1.32 1.23 1.08 1.48 1.43 1 0.92 0.92 1.57 0.95 0.7 1.12 1.12 1.17 0.97 1 0.97 0.82 1.34 0.95 0.78 1.07 1.57 1.16 0.95
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.04 0.05 0.08 0.04 0.05 0.06 0.05 0.06 0.04 0.07 0.06 0.05 0.06 0.03 0.07 0.03 0.07 0.05 0.05 0.06 0.1 0.1 0.11 0.09 0.04 0.05 0.05 0.04 0.05 0.06 0.05 0.06 0.05 0.05
MK801
Duncan’s post hoc test p value
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.05 0.02 0.02 0.03 0.002
