Conceived and designed the experiments: IPE BPW. Performed the experiments: AB AK. Analyzed the data: AB TH AK SL GC. Contributed reagents/materials/analysis tools: IPE MT BPW. Wrote the paper: BPW. Interpreted data: BPW AB SL IPE GC. Revised manuscript: AB SL IPE GC. Approved final version: AB TH AK SL IPE GC. Provided research support staff: MT.
Current address: Centre for the Cellular Basis of behaviour, The James Black Centre, Institute of Psychiatry, King's College London, London, United Kingdom
Current address: Centre for Immunology, St Vincent's Hospital/University of New South Wales, Sydney, New South Wales, Australia
Current address: Department of Psychiatry, The University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia
The authors have declared that no competing interests exist.
In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human, decreased Dlx1 expression has been associated with psychosis. Using
Abnormalities in cortical GABAergic interneurons have been associated with psychiatric illness
GABAB receptors signal via guanine nucleotide binding proteins (G proteins) whose activity is regulated by guanine nucleotide exchange factors (GEFs) that activate signalling, and GTPase-activating proteins (GAPs) that inhibit signalling. We have shown that expression of a GEF, called mr-gef, is turned on in developing rodent GABAergic neurons
Rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2
Here, we report that mr-gef expression is significantly down-regulated in mice that do not express Dlx1 and Dlx2, suggesting that they form part of a common signalling pathway during GABAergic neuronal development. Further, in the human DLPFC we observed a significant positive correlation between cortical layers II and IV in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but not those with schizophrenia, major depressive disorder or controls. We further observed a significant positive correlation in the two-dimensional (2D) neuronal density between layers II and IV in individuals with bipolar disorder and schizophrenia, possibly reflecting a common pathology in these diseases.
Post-mortem brain sections (14 µm) from Brodmann area 9/46 (BA9/46) of the DLPFC were obtained from the Stanley Foundation Brain Consortium, an established collection of brain samples obtained with full consent of next of kin. The sample consisted of 60 subjects: 15 controls, 15 with schizophrenia, 15 with bipolar disorder, and 15 with major depressive disorder. None of these brains demonstrated evidence of neurodegenerative changes or other pathologic lesions. This study was performed blind to diagnosis on all 60 subjects, however 8 subjects were excluded from analysis due either to severe tissue damage (n = 1) or due to severe difficulty in locating landmarks to compare Nissl and MR-GEF slides because of poor tissue quality in the desired region (n = 7). A summary of demographic, clinical and histological information of the samples used in this study is given in
Sense and anti-sense probes were designed to specifically target a region from within the open reading frame (ORF) of the rodent (mr-gef)
Additional cDNAs used for riboprobe synthesis were Lhx6 (Lhx6 cDNA was a gift from Vassilis Pachnis and Maria Grigoriou, NIMR, London, UK) and Dlx1 (Dlx1 cDNA was a gift from John Rubenstein, University College of San Francisco).
Sections were fixed for 10 minutes in cold 4% paraformaldehyde in 0.1 M phosphate buffer before being washed in Depc-PBS (diethylpyrocarbonate treated phosphate buffered saline), incubated in acetylation solution for 10 minutes and permeabilised by incubation in 1% Triton X-100 (Sigma) for 5 minutes (human tissue) or 30 minutes (mouse tissue). After further washes in Depc-PBS, slides were placed in a hybridisation chamber humidified with 50% formamide and 5xSSC. Sections were pre-hybridised in 500 µl hybridisation buffer (50% formamide, 5xDepc-SSC, 5x Denhardts, 0.25 mg/ml yeast RNA, 0.5 mg/ml herring sperm DNA) for 2–6 hours at room temperature. DIG-labelled RNA probes (100–200 ng/ml diluted in hybridisation buffer) were denatured at 80°C for 5 minutes then cooled on ice. The probe/hybridisation buffer mix was pipetted onto sections and the slides covered with glass coverslips. Hybridisation was carried out at 65°C overnight in a sealed, humidified hybridisation chamber. Sections then went through a series of washes (0.2xSSC at 65°C for 1 hour; 0.2xSSC for 5 mins at room temperature; 0.1 M Tris pH 7.5/150 mM NaCl for 5 mins at room temperature) before being blocked in 10% heat inactivated sheep serum (Sigma, diluted in 0.1 M Tris pH 7.5/150 mM NaCl) for 1–3 hours at room temperature. To detect the DIG-labelled riboprobes, 500 µl of anti-DIG Fab-AP antibody (Roche, diluted 1∶5000) was added to each slide and incubated overnight at 4°C in a humidified chamber. Slides were then washed 3×5 minutes in 0.1 M Tris pH 7.5/150 mM NaCl, followed by 5 minutes in 0.1 M Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2 with 0.24 mg/ml levamisole (Sigma) to inhibit endogenous phosphatase activity. Bound probes were visualised by incubation in the dark in colour solution (50 ng/ml NBT [Roche] and 75 ng/ml BCIP [Merck] with levamisole). Reactions were stopped in water or 1x TE (sense and anti-sense reactions were stopped at the same time). In all cases sense probes showed no specific labelling (see
For human post-mortem analyses, Nissl staining on adjacent sections to those processed for non-radioactive
Tissue sections were viewed using a 20x objective on a Leica BMLB microscope equipped with a Hitatchi colour camera and a Marzhauser x-, y-motorised stage. Similar regions in area BA46 were selected on each slide based on cytoarchitecture. Obvious landmarks were chosen (blood vessels, contour of tissue) in Nissl stained sections to allow the same region to be detected on adjacent MR-GEF stained sections. Composite images from the pial surface to the grey/white matter border were generated as previously described
Post-mortem and demographic variables were included as co-variates where they were found to correlate with outcome at p<0.05. This included assessment of medications via lifetime fluphenazine equivalents, substance abuse and use of ethanol as supplied to us by the Stanley Foundation. In addition, in order to control for potential differential shrinkage of layers 2 and 4 for individual cases we calculated the percentage of the cortical width that each case represented and analysed differences between groups at p<0.05. We found no significant differences in the proportions of layer II and IV to the whole cortical width between any of the psychiatric groups versus controls.
Statistical analysis was carried out using a one-way analysis of variance (ANOVA) to test for differences in estimates between psychiatric and control groups using SPSS 18.0 (SPSS, Inc.) at a bonferroni adjusted significance level of p<0.05/2 = 0.025. We determined that this analysis was appropriate after showing that our data was normally distributed for neuronal somal size; both mean and median neuronal somal size was analysed and no significant difference were observed between groups. The percentage of neurons that expressed MR-GEF and the 2D areal density of neurons in layers II and IV was analysed in sections from patient versus control groups. We also carried out a pearson correlation analysis between layers II and IV for the percentage of MR-GEF expressing neurons as well as total neurons (expressing and non-expressing) within groups. The main objective of the statistical analysis was to compare the outcome in each of the patient groups compared with the control group and to identify any relationships that may be specific to the major psychiatric disorder under investigation.
During development, mr-gef expression showed a striking overlap with the LIM-homeobox gene, Lhx6, but a reciprocal pattern of expression with the transcription factor Dlx1 (
Regional expression of mr-gef, Lhx6 and Dlx1 in the E14.5 mouse ventral telencephalon (A–C). mr-gef and Lhx6 are expressed in the SVZ and mantle of the MGE (A,B respectively). mr-gef is expressed in regions adjacent to those in the VZ and SVZ expressing Dlx1 (A and C respectively) and in a band of cells reaching up into the Ctx (arrow A). A similar mr-gef expression pattern is observed in the ventral telencephalon in the
To determine whether Dlx1 and 2 were required for mr-gef expression, we compared mr-gef expression in the forebrain of mice where both
Our observed reduction in striatal and cortical mr-gef expression in
When comparing Nissl stained and MR-GEF labelled serial sections it was apparent that MR-GEF expression in the human DLPFC was not restricted to GABAergic neurons (
Comparison of Nissl stained and MR-GEF labelled serial sections showing that MR-GEF is expressed by cells throughout the cortex and is not confined to GABAergic neurons.
Comparison of the range of somal sizes of MR-GEF expressing neurons in layer II (A) and layer IV (B) of the DLPFC. Abbreviations: BPD, bipolar disorder; MDD, major depressive disorder; SCZ, schizophrenia.
When the percentage of neurons expressing MR-GEF was calculated and compared across all groups and layers no significant difference was observed. Neither was there any significant difference observed in the 2D density of neurons between patient and control groups.
Due to the intimate association between interneurons and pyramidal neurons within the different cortical layers, we assessed the correlation between the percentage of MR-GEF expressing neurons in layers II and IV across the control and patient groups.
We observed a significant positive correlation in the percentage of neurons expressing MR-GEF between layers II and IV in individuals with bipolar disorder (r = 0.704 and p = 0.034) No correlation was observed in individuals with schizophrenia, major depressive disorder or in controls. We also applied the same analysis to the 2D density counts between layers II and IV and observed a significant positive correlation in individuals with schizophrenia (r = 0.586, p = 0.028) and bipolar disorder (r = 0.732. p = 0.025) while no correlation was observed in major depressive disorder or in controls.
During rodent forebrain development many GABAergic neurons express the Rap1 GEF, mr-gef
In humans, neocortical GABAergic interneurons are derived from Dlx-expressing populations in both the neocortex and the ganglionic eminences (GE)
Within the cerebral cortex, different GABAergic neuronal subtypes innervate particular domains on pyramidal neurons and have a specific function in regulating pyramidal neuron activity
When we assessed the correlation in the percentage of MR-GEF expressing neurons between layers II and IV across patient and control groups, we found a significant positive relationship for the percentage of MR-GEF expressing neurons between the two layers in bipolar disorder, possibly suggesting that communication between these two layers is altered in this disease. In addition, the observed significant positive correlation between layers II and IV in 2D neuronal density in individuals with bipolar disorder and schizophrenia may reflect a common pathology in these diseases.
Our observed correlations in both the percentage of MR-GEF expressing neurons and in 2D neuronal density between layers II and IV in bipolar disorder support a growing body of evidence for defects in cortical organisation and communication in this disease. Moreover, since MR-GEF encodes a Rap1 GEF able to activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.
Demographic, clinical and histological data for the 52 analysed patient cases. * One case has no pH information, number given is average of remaining 11 cases. Abbreviations: SCZ, schizophrenia; BPD, Bipolar Disorder; MDD, major depressive disorder; PMI, post-mortem interval.
(0.03 MB DOC)
MR-GEF in situ hybridation on human sections. Cartoon of the human MR-GEF mRNA comprising a short 5′ untranslated region (UTR), an open reading frame (ORF) of 1740 bp and a long 3′ UTR of more than 3 kb (A). Two different probes specific for MR-GEF were designed either to target a portion of the ORF of approximately 800 bp or to target a portion of the 3′ UTR of approximately 1.2 kb (A). In both cases sense probes gave no specific labelling whilst anti-sense gave a specific signal with very little background labelling. A representative image of labelling with sense and anti-sense MR-GEF ORF probes on adjacent sections is shown (B). All experiments described in the text were carried out using MR-GEF ORF sense and anti-sense probes. Scale bar = 200 µm.
(2.98 MB TIF)
We gratefully thank Vassilis Pachnis and Maria Grigoriou (NIMR London, UK) for the kind gift of