Document Detail

Reduced connectivity in the self-processing network of schizophrenia patients with poor insight.
Jump to Full Text
MedLine Citation:
PMID:  22912723     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Lack of insight (unawareness of illness) is a common and clinically relevant feature of schizophrenia. Reduced levels of self-referential processing have been proposed as a mechanism underlying poor insight. The default mode network (DMN) has been implicated as a key node in the circuit for self-referential processing. We hypothesized that during resting state the DMN network would show decreased connectivity in schizophrenia patients with poor insight compared to patients with good insight. Patients with schizophrenia were recruited from mental health care centers in the north of the Netherlands and categorized in groups having good insight (n= 25) or poor insight (n = 19). All subjects underwent a resting state fMRI scan. A healthy control group (n = 30) was used as a reference. Functional connectivity of the anterior and posterior part of the DMN, identified using Independent Component Analysis, was compared between groups. Patients with poor insight showed lower connectivity of the ACC within the anterior DMN component and precuneus within the posterior DMN component compared to patients with good insight. Connectivity between the anterior and posterior part of the DMN was lower in patients than controls, and qualitatively different between the good and poor insight patient groups. As predicted, subjects with poor insight in psychosis showed decreased connectivity in DMN regions implicated in self-referential processing, although this concerned only part of the network. This finding is compatible with theories implying a role of reduced self-referential processing as a mechanism contributing to poor insight.
Authors:
Edith J Liemburg; Lisette van der Meer; Marte Swart; Branislava Curcic-Blake; Richard Bruggeman; Henderikus Knegtering; André Aleman
Related Documents :
21272253 - Evidence for severe atherosclerotic changes in chronic hemodialysis patients: comparati...
18404143 - Correlation between serum thyroxine and complements in patients with multiple sclerosis...
14677813 - Increased plasma thioredoxin in patients with acute myocardial infarction.
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-08-09
Journal Detail:
Title:  PloS one     Volume:  7     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2012  
Date Detail:
Created Date:  2012-08-22     Completed Date:  2013-04-23     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e42707     Citation Subset:  IM    
Affiliation:
Department of Neuroscience, University Medical Center Groningen and BCN NeuroImaging Center, University of Groningen, Groningen, The Netherlands. E.J.Liemburg@umcg.nl
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Adult
Awareness*
Brain / cytology,  pathology,  physiology,  physiopathology
Case-Control Studies
Humans
Male
Nerve Net / cytology,  pathology*,  physiology,  physiopathology
Schizophrenia / pathology*,  physiopathology
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
ISSN: 1932-6203
Publisher: Public Library of Science, San Francisco, USA
Article Information
Download PDF
Copyright: 2012 Liemburg et al
License:
Received Day: 2 Month: 3 Year: 2012
Accepted Day: 11 Month: 7 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 9 Month: 8 Year: 2012
Volume: 7 Issue: 8
E-location ID: e42707
ID: 3415395
PubMed Id: 22912723
Publisher Id: PONE-D-12-06361
DOI: 10.1371/journal.pone.0042707

Reduced Connectivity in the Self-Processing Network of Schizophrenia Patients with Poor Insight Alternate Title:Reduced Connectivity in Patients with Poor Insight
Edith J. Liemburg12*
Lisette van der Meer13
Marte Swart13
Branislava Curcic-Blake1
Richard Bruggeman3
Henderikus Knegtering123
André Aleman14
Yu-Feng Zangedit1 Role: Editor
1Department of Neuroscience, University Medical Center Groningen and BCN NeuroImaging Center, University of Groningen, Groningen, The Netherlands
2Rob Giel Research Center, University Medical Center Groningen, Groningen, The Netherlands
3Lentis, Center for Mental Healthcare, Groningen, The Netherlands
4Department of Psychology, University of Groningen, Groningen, The Netherlands
Hangzhou Normal University, China
Correspondence: * E-mail: E.J.Liemburg@umcg.nl
[conflict] Competing Interests: E.J. Liemburg, MSc reports no financial relationships with commercial interests. Dr. L. van der Meer reports no financial relationships with commercial interests. M. Swart, MSc reports no financial relationships with commercial interests. Dr. B. Curcic-Blake reports no financial relationships with commercial interests. Dr. R. Bruggeman reports no financial relationships with commercial interests. Dr. H. Knegtering is on the speakers list of Janssen-Cilag, BMS and Eli Lilly, and received unrestricted research grants from Eli Lilly, Astra Zeneca, Janssen-Cilag and BMS. These companies had no further role in the selection of literature and interpretation of data; in the writing of the review; and in the decision to submit the manuscript for publication. Dr. A. Aleman reports no financial relationships with commercial interests. Lentis Center for Mental Health Care is not a commercial organization, but a local public health care center without profit-making activities. Being an employee of Lentis does not alter those authors’ adherence to all the PLoS ONE policies on sharing data and materials. The authors declare their adherence to all the PLoS ONE policies on sharing data and materials.
Contributed by footnote: Conceived and designed the experiments: LvdM MS RB HK AA. Performed the experiments: LvdM MS. Analyzed the data: EJL BC. Contributed reagents/materials/analysis tools: BC. Wrote the paper: EJL LvdM MS BC RB HK AA.

Introduction

Patients with schizophrenia often have difficulties with social and emotional cognitive processing [1], [2], including self-reflective processes [3]. Such impairments may have important consequences for successful functioning in a social community [1], [4]. Self-referential processing deficits, which may already be present before the onset of the disorder, have been proposed to underlie these social and emotional deficits as well as first rank schizophrenic symptoms, e.g. [5][10]. Such self-related processing deficits may include the formation and maintenance of an accurate representation of one’s traits, abilities and attitudes, or self-reflection [11], [12]. This self-reflective processing is essential in the evaluation of one’s personal behavior as well as in interpersonal communication [2]. More specifically, it has been proposed that self-reflective processing may underlie poor illness insight in patients with schizophrenia [12][14].

Impaired insight has been considered to be a core feature of schizophrenia [15]. Poor insight in schizophrenia has been associated with poorer global functioning [16][18], greater severity of psychopathology [19], increased relapses and hospitalizations, poorer long term prognosis [20] and reduced treatment compliance [21], [22]. Interestingly, lack of insight in schizophrenia appears to be self-specific, as most patients recognize symptoms in other patients, but fail to do so in themselves [23], [24]. This implies that lack of insight may be caused by disturbed abilities of self-referential processing [12]. Thus, studying the neural link between insight and self-referential processing may reveal important clues with regard to the underlying deficit in patients lacking insight. If patients with schizophrenia have attenuated capacities to reflect on their situation and on other self-relevant information, this could be a barrier for obtaining insight that one suffers from a severe psychiatric disorder.

In terms of brain regions that underlie self-referential processing, research points towards a set of medial brain areas comprising the posterior cingulate cortex (PCC), anterior cingulate cortex (ACC) and the dorsomedial and ventromedial prefrontal cortex (d & vMPFC) [25], [26], together referred to as the cortical midline structures (CMS) [11], [12], [27]. In patients with traumatic brain injury in the CMS [28], patients with mild cognitive impairment [23] and schizophrenia patients [29][32], an association between impaired insight and decreased activation of medial frontal and other CMS regions has been demonstrated.

The CMS show a large overlap with the so-called default mode network (DMN). This is a network of brain areas that is active during rest [33], [34] and involved in processing information related to the self [33], [35]. The brain areas in the network show synchronized slow fluctuations (<0.1 Hz) in the BOLD signal [36], [37]. Areas in this network include the ventral and dorsal medial prefrontal cortex (vMPFC and dMPFC), anterior cingulate (ACC), the posterior cingulate (PCC)/retrosplenial cortex (RspC) and adjacent precuneus, inferior parietal lobule (IPL), medial temporal cortex (MTG) and hippocampal formation [34], [36]. The default mode network appears to encompass subnetworks with distinct functions [34], consisting of an anterior part (ACC/MPFC), a posterior part (PCC, precuneus and IPL), and possibly a ventral part with temporal and ventral prefrontal regions [11], [34]. Studies in schizophrenia patients have found disturbances in DMN structures, with mainly lower medial PFC connectivity compared to healthy controls [38][41] but also altered connectivity within posterior DMN areas, disturbed prefrontal-parietal communication [32], [41][48], or reduced connectivity between other DMN regions [42][45], [48][50]. Of note, some studies showed increased frontal connectivity [51][53].

Structural MRI studies have related poor insight in schizophrenia patients to decreased volume of prefrontal and other DMN regions [54], [55], which may be related to poor self-monitoring [56][59]. Patients or people at risk for psychosis indeed show altered brain activation during self-reflection and theory of mind [29], [30], [32], [60]. Moreover, review studies have shown that schizophrenia patients have a decreased prefrontal and posterior DMN activation in resting state studies [31], [61]. Finally, decreased white matter integrity between DMN areas was also related to poor insight [62]. No studies have as yet investigated resting state connectivity in relationship to poor insight in psychosis.

Connectivity analysis may further the understanding of neural systems beyond the task-activation fMRI designs [63], [64]. Resting state BOLD fluctuations may reflect spontaneous neural activity as most resting state patterns overlap with known brain networks [64], [65], and they may even predict an individual’s task performance or behavior [63]. Moreover, their functional connectivity follows the anatomical outline of white matter bundles [64]. Whereas task-based activation can provide information about the function of separate brain areas, functional connectivity may thus provide information about interaction of brain areas [64]. Resting state research of the DMN is especially interesting with regard to the issue of insight, because we expect a relation between the key function of the DMN, namely self-referential processing, and insight.

Studying resting state fluctuations may have some advantages over task-based fMRI. Experimental control of differences in task performance between groups is not necessary and relatively ill patients groups with limited capacities can be investigated [65], [66]. Only intrinsic differences of the brain, and not differences in cognitive abilities, will explain differences in connectivity. Moreover, resting state functional connectivity may be a more natural, ecologically relevant, measure of brain activation than task-based fMRI [33] as it reflects intrinsic brain interactions [67].

Independent component analysis (ICA) [68] can separate the fMRI signal into spatially independent networks that show shared temporal fluctuations [67], [68]. Independent components (i.e. networks) contain brain areas that show similar fluctuations and are assumed to be functionally linked. The size and strength of the identified networks (components) may differ between individuals and groups sharing a specific trait [67], [68], as may cooperation between different networks [69]. In this study, we will focus on the DMN because this has been related to self-related processing [35]. We expect to identify an anterior and posterior DMN subnetwork as described earlier, as these have been identified previously using ICA [69], [70].

We hypothesize that schizophrenia patients with poor insight may show impaired connectivity of the DMN during rest, which may reflect attenuated self-related processing associated with decreased awareness of symptoms [27]. We therefore compared connectivity of brain areas within anterior and posterior DMN components to the other parts of that component between patients with good and with poor insight. A healthy control group was used as a reference. Moreover, we conducted a group comparison of connectivity strength between the anterior and posterior DMN components, as we hypothesize that impaired connectivity between the anterior and posterior DMN may also contribute to impaired insight.


Methods
Ethics Statement

The study was approved by the local medical ethical committee (Medische Ethische Toetsingscommissie van het Universitair Medisch Centrum Groningen) according to the declaration of Helsinki. All subjects gave oral and written informed consent after the study procedure had been fully explained. All subjects ware capable of signing the informed consent as they were able to live independent, no permanent inpatients, had no care givers taking over responsibilities from them, and all allowed to sign informed consent themselves. All subject data was handled anonymously.

Study Population

The study sample included 44 patients with schizophrenia. Patients were recruited from mental health care centers in the north of the Netherlands, three or four patients came from western parts of the Netherlands. Patients were participants in an fMRI study on neural correlates of auditory hallucinations or a study on cognitive emotional processing; in both studies a resting state scan was part of the research protocol. Diagnosis of schizophrenia according to DSM-IV criteria was confirmed with the SCAN 2.1 diagnostic interview [71]. A healthy control group matched to the patients on age, gender, handedness, and education level was included. This group was included to deduce whether patients showed similar DMN properties as healthy subjects. Healthy controls were excluded in case of psychiatric history, which was confirmed with the screenings questions of the SCAN 2.1 interview. For subject characteristics, see Table 1. Patients were asked to give an overview of the medication they were taking at the moment. The patients reported to use the following medication; antipsychotics: aripiprazole (9x), chlorprotixene (1x), clozapine (15x), haloperidol (4x), olanzapine (9x), paliperidone (1x), penfluridole (1x), perphenazine (1x), pimozide (1x), pipamperone (1x), quetiapine (7x), risperidone (10x), sulpiride (1x), and zuclopentixole (2x); antidepressants: amytriptyline (1x), bupropione (1x), citalopram (3x), clomipramine (1x), fluoxetine (2x), fluvoxamine (1x), mirtazapine (1x), paroxetine (2x), nortriptylin (1x), trazodone (1x), and venlafaxine (2x); benzodiazepines: diazepam (3x), flurazepam (1x), lorazepam (3x), oxazepam (7x), temazepam (5x); other: atenolol (1x), biperiden (6x), carbamazepine (1x), lithiumcarbonate (6x), pantaprazol (2x), promethazine (1x), valproic acid (1x).

Measures

The most important measure of the study was connectivity of brain areas within the anterior and posterior DMN component to the rest of that component. Differences in connectivity within a component were compared between groups by doing a voxel-wise group comparison of the spatial maps of individual subjects. Connectivity between components was also determined by correlating the time courses of the anterior and posterior DMN component. These were converted to Z-scores and compared between groups.

Design

The primary goal was to compare connectivity measures between patients with good and poor insight. A matched healthy control group was used as a reference. If possible, differences were statistically compared, but as described below, in some cases only qualitative comparison was possible.

All schizophrenia patients were interviewed with the Positive and Negative Syndrome Scale (PANSS) [72]. The PANSS interview measures three domains of symptoms, namely positive and negative symptoms and general pathology. Each item can be rated from 1 (not present) –7 (extreme). The interviews were performed by experienced and trained raters. Based on the rating of the interview item that measures illness insight (G12), patients were categorized into two groups with good insight (score 1–2, which are in the normal range) or poor insight (>2). Even though this is only one single item, strong correlations with more thorough measures of insight such as the Scale to Assess Insight (SAI; r = 0.88), Scale to Assess Insight – Expanded (SAI-E; r = 0.90), or the Insight and Treatment Attitudes Questionnaire (ITAQ; r = 0.90) have been demonstrated [73], [74], confirming that the PANSS G12 item reliably rating insight.

Education level was rated according to a six point scale defined by Verhage [75], which ranges from primary school (1) to university level (6). Handedness was confirmed by the Edinburgh handedness inventory [76]. Age and education level were compared between controls and the two patients groups with a Kruskal-Wallis H test (α<0.05). Between patient group differences in PANSS subscales were tested with a Mann-Whitney U test. For the PANSS General pathology subscale the Insight item G12 was subtracted from the total score, because this item was a selection criterion for both groups. A Chi-square test for independence (α<0.05) was used to test for differences in gender and handedness. All statistical tests were performed with Statistical Package for Social Sciences (SPSS) 16. Exclusion criteria for the study consisted of MRI incompatible implants, possible pregnancy, claustrophobia and non-native Dutch speakers.

MRI Procedure

All subjects underwent a resting state fMRI scan. They were instructed to close their eyes, relax, and to stay awake. Subjects were reminded of this just before the scan started. A 3 T Philips Intera MRI scanner (Best, The Netherlands) equipped with a 8-channel SENSE head coil was used to acquire 200 whole brain echo-planar functional images (EPÌs), TR 2.3 s and TE 28 ms. The volumes contained 39 (old sequence) or 43 (after scanner upgrade) interleaved slices (3.8×3.8×3 mm) with a 0 mm slice gap and a 85° flip-angle (FOV = 220×117×220 mm). The duration of the scan was 460 seconds. A high-resolution, transverse T1 anatomical was also acquired for overlay of statistic images (160 slices; voxel size 1×1×1 mm; FOV 256×220×256 mm).

Analysis

The raw images were converted to ANALYZE format and analyzed using Statistical Parametric Mapping (SPM8; FIL Wellcome Department of Imaging Neuroscience, London, UK) running on Matlab 7.1. Images were first corrected for slice-time differences and realigned to the first functional image. The mean image created during realignment was co-registered to the anatomy, together with the functional images, and the anatomy and functional images were normalized (voxel size 3×3×3 mm) to the T1 template of SPM. Finally, images were smoothed with a 10 mm FWHM isotropic Gaussian kernel. Additional filtering was not necessary, because artifacts will generally represented by separate components in ICA [67], [68].

After the preprocessing, images were processed in Group ICA FMRI Toolbox (GIFT; http://icatb.sourceforge.net/gift/gift_startup.php) [68]. For referential purposes, a separate ICA was conducted on the group of healthy control subjects. Healthy subjects were not included in the ICA of patients but treated separately, because subtle differences in spatial maps of patients, only distinguished based on insight score, may disappear due to inclusion of a group with different network properties, such as healthy controls [68].

The mean number of independent components (I s) was estimated using Maximum Description Length (MDL) and Akaike’s criteria [77], to prevent splitting or merging of components [65]. Images were intensity normalized before ICA estimation, which implied scaling the time courses to a mean of 100. The intensity normalized images (patients and controls separately) were decomposed into a set of spatially independent components (for every subject) by the Infomax algorithm. A component consists of a time course showing the temporal fluctuations of that component, and a spatial map that shows the contribution of every voxel to that component. Stability of the components, i.e. whether a component has the tendency to split or merge with another component, was validated by running the ICASSO toolbox implemented in GIFT using twenty iterations with both random iterations and bootstrapping [78].

Selection of the components of interest for both healthy controls and patients, namely the anterior DMN (including the ACC/MPFC) and posterior DMN (PCC/precuneus/IPL), was done by selecting components showing a large spatial overlap with a priori defined anatomical masks. Thus, the spatial component could also involve other brain areas, but involvement of the areas defined by the masks was crucial. These anatomical masks of the ACC/MPFC (to select the anterior DMN component) and of the PCC/precuneus (for posterior DMN component selection) were created with WFU–pickatlas (http://www.nitrc.org/projects/wfu_pickatlas). Masks provided by WFU pickatlas are based on brain regions defined by Talairach and Tournoux (1998) that were implemented in this toolbox after conversion to MNI space [79], [80].

Spatial maps of selected anterior and posterior DMN components were visually compared between patients and controls to establish whether similar networks were present in both groups. Statistical comparison of image maps of two different ICÀs is unjustified, because the outline of image maps may differ between groups due to the separate ICA unmixing procedure of the image time courses in both groups.

After that, for the patients the reconstructed individual spatial maps of the anterior and posterior DMN component were entered in a two sample t-test random-effects analysis comparing the good versus poor insight group. This analysis shows brain areas that are differently connected to the rest of the anterior or posterior DMN component. A statistical threshold was applied of p<0.001, as has been done previously [83]. The analysis was restricted to areas that significantly contributed to the ICA component, as previously described by [70]. This was done because ICA components maps have values close to zero in areas where the time course of that component is not represented. Voxel intensities in these areas are mainly determined by noise properties and may in group comparison lead to false-positive clusters. Since we formulated a specific hypothesis comprising specific brain areas and used a mask to restrict the search volume, and because a comparison between two groups of patients was performed, cluster correction was not applied to avoid type II errors [81]. In an additional analysis, a voxel-wise linear regression was performed with the time courses of each voxel in the component maps of the DMN against the PANSS G12 Insight scores.

In another analysis, a correlation was calculated between the anterior and posterior DMN component time courses of all subjects. The correlations were converted to Z-scores by a Fische s Z transformation with Z = ½*ln((1+r)/(1−r)),where r represents the correlation. These data were loaded in SPSS. The Z-scores between the time courses of the anterior and posterior DMN of all patients were compared to those of controls and the Z-scores of patients with poor insight to those of patients with good insight using Mann-Whitney U tests (α = 0.05).

Two additional analyses were performed. First, because there was a significant difference in the PANSS positive subscale between groups, this subscale was added as a covariate to the group comparison. Second, as DMN regions have been shown to deactivate during task-performance, we also investigated whether the regions that we identified in the ICA group comparison overlapped with regions that showed task-related deactivation. For this, we analyzed a language task involving valence evaluation (positive, negative) of visually presented words that was performed by subjects during scanning. Deactivation of the DMN during task performance was shown by contrasting the fixation cross of the task with task blocks. The clusters showing a difference in DMN connectivity between the good and poor group were then overlayed on the task-related deactivation (Figure S1).


Results

Twenty five patients were classified as having good insight, and nineteen patients were classified as having poor insight. The demographical characteristics of these two groups were compared, also with respect to the controls when applicable (see Table 1). The PANSS Positive subscale was significantly different between groups, but there was a significant correlation between PANSS G12 and the Positive symptom subscale (r = .36; p = .015), implying that patients with more positive symptoms had poorer insight. Therefore, the Positive symptom subscale was added as a covariate in the group comparisons, but this did not change the results. There was no significant difference in age, gender, handedness, education level and most PANSS scores, though the PANSS Positive subscale was significant.

The component estimation resulted in an estimate of 32 components for the patients, and 30 for the healthy controls. The identified anterior default mode component encompassing the ACC/MPFC showed a spatial overlap correlation with the anatomical mask created by WFU Pickatlas of 21% for healthy controls (left side Figure 1) and of 56% for patients (left side Figure 2). Overlap of other components was <10%, indicating that the components of interest (anterior and posterior DMN) could be identified with high specificity. Visual inspection showed that the component map of the healthy controls had a more extended and stronger network contribution than the patients. The posterior component showed an overlap of 31% for healthy controls (right side Figure 1), and of 57% for patients (right side Figure 2).

The anterior and posterior components were compared with a two-sample t-test. Patients with good insight showed stronger connectivity of the ACC to the rest of the anterior DMN component compared to patients with poor insight: t = 4.37, Z = 3.94, cluster size = 18, p<0.001, xyz = −12 39 3 (Figure 3, left side). Subsequently, a voxel-wise regression between the image maps and the insight score was calculated. This revealed a cluster in the same location. In the two sample t-test of the posterior DMN component, a significant cluster was identified in the precuneus (t = 3.94, Z = 3.62, cluster size = 20, p<0.001, xyz = 24 −72 24, see Figure 3, right side). The linear regression with insight score resulted in the same cluster. There was no significant cluster in the poor vs. good insight t-test comparison for both components.

A correlation between the time courses of the anterior and posterior DMN component was calculated and converted to Z-scores. These Z-scores were compared between healthy controls and all patients, and between patients with good and poor insight. Z-scores are plotted per group in Figure 4. Whereas the Z-scores for the healthy controls were all above zero (with the exception for one subject), part of the patients showed a negative Z-scores with an overall mean around zero and a larger variation (SDcontrols = 0.20, SDpatients = 0.66). This difference was significant (U = 388, z = −3.0, p = 0.003). Patients with poor insight showed the largest variation in Z-scores (SDgood insight = 0.56, SDpoor insight = 0.79), but did not differ significantly from the patients with good insight (U = 214, z = −0.56, p = 0.58).

Finally, adding the PANSS positive symptoms subscale as a covariate to the group comparison between good and poor insight did not change the results. In addition, during the valence evaluation task deactivation of DMN regions was observed. The clusters that differed significantly between good and poor insight groups overlapped with the DMN regions showing significant deactivation during the task (Figure S1).


Discussion

In this study, the relationship between insight (awareness of illness) in schizophrenia and functional connectivity of regions in the default mode network (DMN) was investigated in patients with schizophrenia. The DMN connectivity pattern of patients clearly overlapped with the network in healthy control subjects, though the network was less extended (in accordance with e.g. [31], [46], [48][50], [61]. Importantly, patients with poor insight showed a lower connectivity within the anterior cingulate and precuneus compared to patients with good insight. Group differences were found in DMN regions that indeed deactivated during task performance, supporting our interpretation. Moreover, although the poor insight group showed significantly more positive symptoms, these did not explain the group differences Connectivity between anterior and posterior DMN was lower in all patients compared to controls, but there was no significant difference between patients with good and poor insight.

The result of reduced connectivity in the precuneus and ACC of the DMN in poor insight patients was in accordance with our expectations that poor insight would be related to decreased DMN connectivity [12], [28], [31], [35], although it may only concern part of the network. Studies assessing the overlap between self-referential processing and DMN activation, demonstrated that the ACC was consistently activated [82], [83] and thus seems to be particularly important for self-referential thought. Lesion studies demonstrated that lesions in/around this area can result in a diminished self-referential processing [84] and in a dysfunction of emotional self-control [85]. This suggests that reduced connectivity in this region may indeed result in abnormal self-referential processing. Whereas the ACC may be specifically involved in self-related processing, research has shown that precuneus activation is less self-specific and also activates during thinking about other persons [12], [82], [83]. Instead, the precuneus has been hypothesized to be involved autobiographical and episodic memory retrieval and mentalizing, which has been confirmed by several studies [12], [31], [35], [86], [87]. Consistent with this, structural neuro-imaging results point towards a relationship between impaired insight and reduced grey [58], [59] and white matter [62] in this region among others. Taken together, this suggests that hampered self-processing through a lack of integration of self-related information may underlie impaired insight in schizophrenia [12].

Schizophrenia patients had a lower, i.e. more negative, correlation between time courses of the anterior and posterior DMN. Though the mean connectivity was not significantly lower in patients with poor insight compared to good insight, the variation appeared to be higher in patients with poor insight. Disturbed connectivity between the frontal and posterior DMN could possibly have a modulating effect on insight. Patients with schizophrenia have shown decreased connectivity between the medial frontal cortex and other brain regions during self-reflective processing [32], [88]. Reduced communication between self-reflection areas may result in less transfer of self-related information (i.e. autobiographical or interoceptive information) of posterior areas to the anterior self-reflective areas.

One limitation of the study may be that insight was rated based on one item of a standardized interview. However, as we discussed above, this G12 item correlates highly with other more thorough measures of insight, suggesting that it can adequately index insight. Furthermore, it can be argued that subjects were not involved in self-reflective processing during resting state conditions. However, other studies have shown that self-referential processing is one of the major processes taking place during resting state [34], [35], [89], [90]. And as this is spontaneous self-referential processing, it was exactly the type of processing we were interested in. More research is needed to elucidate the contribution of different cortical midline structures in more detail.

In conclusion, schizophrenia patients with relatively preserved insight showed stronger connectivity than patients with poor insight in the anterior cingulate cortex and precuneus, both key regions in self-reflective processing. These findings tentatively support the hypothesis that poor insight may be related to impaired self-related processing.


Supporting Information Figure S1

Group comparison of good vs. poor insight patients overlayed on task related deactivation. The anterior component shown on the left with the increased ACC connectivity in the good insight group, and the posterior component on the right with the increased precuneus connectivity (p<0.001; k >10; masked with component image map), the task-related deactivation was defined by contrasting the fixation cross of a language task with task blocks.

(TIF)


Click here for additional data file (pone.0042707.s001.png)


The authors acknowledge Anita Sibeijn-Kuiper and Judith Streurman for their assistance with fMRI scanning.


References
1. Pinkham AE,, Hopfinger JB,, Ruparel K,, Penn DL, (Year: 2008) An investigation of the relationship between activation of a social cognitive neural network and social functioning. Schizophr Bull34: 688–69718477583
2. Atkinson RL,, Robinson NM, (Year: 1961) Paired-associate learning by schizophrenic and normal subjects under conditions of personal and impersonal reward and punishment. Journal of Abnormal and Social Psychology62: 322–32613684985
3. Amador XF, David AS (2004) Insight and psychosis: Awareness of illness in schizophrenia and related disorders. Oxford: Oxford University Press.
4. Pinkham AE,, Penn DL,, Perkins DO,, Lieberman J, (Year: 2003) Implications for the neural basis of social cognition for the study of schizophrenia. Am J Psychiatry160: 815–82412727681
5. Frith CD (1995) The cognitive neuropsychology of schizophrenia. East Sussex: Psychology Press.
6. Frith CD,, Corcoran R, (Year: 1996) Exploring ‘theory of mind’ in people with schizophrenia. Psychol Med26: 521–5308733211
7. Parnas J,, Handest P, (Year: 2003) Phenomenology of anomalous self-experience in early schizophrenia. Compr Psychiatry44: 121–13412658621
8. Nelson B,, Sass LA,, Thompson A,, Yung AR,, Francey SM,, et al. (Year: 2009) Does disturbance of self underlie social cognition deficits in schizophrenia and other psychotic disorders?Early Interv Psychiatry3: 83–9321352181
9. Sass LA,, Parnas J, (Year: 2003) Schizophrenia, consciousness, and the self. Schizophr Bull29: 427–44414609238
10. Raballo A,, Saebye D,, Parnas J, (Year: 2011) Looking at the schizophrenia spectrum through the prism of self-disorders: An empirical study. Schizophr Bull37: 344–35119528205
11. Northoff G,, Heinzel A,, de Greck M,, Bermpohl F,, Dobrowolny H,, et al. (Year: 2006) Self-referential processing in our brain – a meta-analysis of imaging studies on the self. Neuroimage31: 440–45716466680
12. Van der Meer L,, Costafreda S,, Aleman A,, David AS, (Year: 2010) Self-reflection and the brain: A theoretical review and meta-analysis of neuroimaging studies with implications for schizophrenia. Neuroscience and Biobehavioural Reviews34: 935–946
13. Flashman LA, Roth AM (2004) Neural correlates of unawareness of illness in psychosis. In: Amador XF, David AS, editors. Insight and Psychosis: Awareness of Illness in Schizophrenia and Related Disorders. Oxford: Oxford University Press.
14. Lysaker PH,, Carcione A,, Dimaggio G,, Johannesen JK,, Nicolo G,, et al. (Year: 2005) Metacognition amidst narratives of self and illness in schizophrenia: Associations with neurocognition, symptoms, insight and quality of life. Acta Psychiatr Scand112: 64–7115952947
15. David AS (2004) The clinical importance of insight: An overview. In: Amador X, David AS, editors. Insight and Psychosis. Oxford: Oxford University Press. 359–392.
16. Dickerson FB,, Boronow JJ,, Ringel N,, Parente F, (Year: 1997) Lack of insight among outpatients with schizophrenia. Psychiatric Services48: 195–1999021849
17. Pyne JM,, Bean D,, Sullivan G, (Year: 2001) Characteristics of patients with schizophrenia who do not believe they are mentally ill. Journal of the Nervous and Mental Disease189: 146–153
18. Stefanopoulou E,, Laurente AR,, Saez Fonseca JA,, Huxley A, (Year: 2009) Insight, global functioning and psychopathology amongst in-patient clients with schizophrenia. Psychiatr Q80: 155–16519526340
19. Mintz AR,, Dobson KS,, Romney DM, (Year: 2003) Insight in schizophrenia: A meta-analysis. Schizophrenia Research61: 75–8812648738
20. Schwartz RC, (Year: 1998) The relationship between insight, illness and treatment outcome in schizophrenia. Psychiatr Q69: 1–229536472
21. Kemp R,, David A, (Year: 1996) Psychological predictors of insight and compliance in psychotic patients. British Journal of Psychiatry169: 444–4508894195
22. Yen CF,, Chen CS,, Ko CH,, Yeh ML,, Yang SJ,, et al. (Year: 2005) Relationships between insight and medication adherence in outpatients with schizophrenia and bipolar disorder: Prospective study. Psychiatry Clin Neurosci59: 403–40916048445
23. Ries ML,, Jabbar BM,, Schmitz TW,, Trivedi MA,, Gleason CE,, et al. (Year: 2007) Anosognosia in mild cognitive impairment: Relationship to activation of cortical midline structures involved in self-appraisal. Journal of the International Neuropsychological Society13: 450–46117445294
24. Startup M, (Year: 1997) Awareness of own and others’ schizophrenic illness. Schizophrenia Research26: 203–2119323352
25. Johnson SC,, Baxter LC,, Wilder LS,, Pipe JG,, Heiserman JE,, et al. (Year: 2002) Neural correlates of self-reflection. Brain125: 1808–181412135971
26. Kelley WM,, Macrae CN,, Wyland CL,, Caglar S,, Inati S,, et al. (Year: 2002) Finding the self? an event-related fMRI study. J Cogn Neurosci14: 785–79412167262
27. Northoff G,, Bermpohl F, (Year: 2004) Cortical midline structures and the self. Trends in Cognitive Sciences8: 102–10715301749
28. Schmitz TW,, Rowley HA,, Kawahara TN,, Johnson SC, (Year: 2006) Neural correlates of self-evaluative accuracy after traumatic brain injury. Neuropsychologia44: 762–77316154166
29. Brüne M,, Lissek S,, Witthous H,, Peters S,, Nicolas V,, et al. (Year: 2008) An fMRI study of theory of mind in schizophrenic patients with “passivity” symptoms. Neuropsychologia46: 1992–200118329671
30. Carter CS,, MacDonald III AW,, Ross AW,, Stenger VA, (Year: 2001) Anterior cingulate cortex activity and impaired self-monitoring of performance in patients with schizophrenia: An event-related fMRI study. America Journal of Psychiatry158: 1423–1428
31. Kuhn S, Gallinat J (2011) Resting-state brain activity in schizophrenia and major depression: A quantitative meta-analysis. Schizophr Bull.
32. Holt DJ,, Cassidy BS,, Andrews-Hanna JR,, Lee SM,, Coombs G,, et al. (Year: 2011) An anterior-to-posterior shift in midline cortical activity in schizophrenia during self-reflection. Biol Psychiatry69: 415–42321144498
33. Raichle M,, MacLeod A,, Snyder A,, Powers W,, Gusnard D,, et al. (Year: 2001) A default mode of brain function. PNAS98: 676–68211209064
34. Buckner RL,, Andrews-Hanna JR,, Schacter DL, (Year: 2008) The brain’s default network anatomy, function, and relevance to disease. Ann N Y Acad Sci1124: 1–3818400922
35. Gusnard DA,, Raichle ME, (Year: 2001) Searching for a baseline: Functional imaging and the resting human brain. Nature Reviews Neuroscience2: 685–694
36. Raichle ME,, Gusnard DA, (Year: 2005) Intrinsic brain activity sets the stage for expression of motivated behaviour. J Comp Neurol493: 167–17616254998
37. Beckmann CF,, DeLuca M,, Devlin JT,, Smith SM, (Year: 2005) Investigations into resting-state connectivity using independent component analysis. Philos Trans R Soc Lond B Biol Sci360: 1001–101316087444
38. Bassett DS,, Bullmore E,, Verchinski BA,, Mattay VS,, Weinberger DR,, et al. (Year: 2008) Hierarchical organization of human cortical networks in health and schizophrenia. J Neurosci28: 9239–924818784304
39. Cole MW,, Anticevic A,, Repovs G,, Barch D, (Year: 2011) Variable global dysconnectivity and individual differences in schizophrenia. Biol Psychiatry70: 43–5021496789
40. Liu Y,, Liang M,, Zhou Y,, He Y,, Hao Y,, et al. (Year: 2008) Disrupted small-world networks in schizophrenia. Brain131: 945–96118299296
41. Zhang D,, Guo L,, Hu X,, Li K,, Zhao Q,, et al. (Year: 2011) Increased cortico-subcortical functional connectivity in schizophrenia. Brain Imaging Behav6: 27–3522002475
42. Mannell MV,, Franco AR,, Calhoun VD,, Canive JM,, Thoma RJ,, et al. (Year: 2010) Resting state and task-induced deactivation: A methodological comparison in patients with schizophrenia and healthy controls. Hum Brain Mapp31: 424–43719777578
43. Lui S,, Li T,, Deng W,, Jiang L,, Wu Q,, et al. (Year: 2010) Short-term effects of antipsychotic treatment on cerebral function in drug-naive first-episode schizophrenia revealed by “resting state” functional magnetic resonance imaging. Arch Gen Psychiatry67: 783–79220679586
44. Woodward ND,, Rogers B,, Heckers S, (Year: 2011) Functional resting-state networks are differentially affected in schizophrenia. Schizophr Res130: 86–9321458238
45. Lynall ME,, Basset DS,, Kerwin R,, McKenna PJ,, Kitzbichler M,, et al. (Year: 2010) Functional connectivity and brain networks in schizophrenia. Journal of Neuroscience30: 9477–948720631176
46. Skudlarski P,, Jagannathan K,, Anderson K,, Stevens MC,, Calhoun VD,, et al. (Year: 2010) Brain connectivity is not only lower but different in schizophrenia: A combined anatomical and functional approach. Biol Psychiatry68: 61–6920497901
47. Wolf ND,, Sambataro F,, Vasic N,, Frasch K,, Schmid M,, et al. (Year: 2011) Dysconnectivity of multiple resting-state networks in patients with schizophrenia who have persistent auditory verbal hallucinations. J Psychiatry Neurosci36: 366–37421791169
48. Jang JH,, Jung WH,, Choi JS,, Choi CH,, Kang DH,, et al. (Year: 2011) Reduced prefrontal functional connectivity in the default mode network is related to greater psychopathology in subjects with high genetic loading for schizophrenia. Schizophr Res127: 58–6521277171
49. Ongur D,, Lundy M,, Greenhouse I,, Shinn AK,, Menon V,, et al. (Year: 2010) Default mode network abnormalities in bipolar disorder and schizophrenia. Psychiatry Res183: 59–6820553873
50. Rotarska-Jagiela A,, Van de Ven VG,, Oertel-Knochel V,, Uhlhaas PJ,, Vogeley K,, et al. (Year: 2010) Resting-state functional network correlates of psychotic symptoms in schizophrenia. Schizophr Res117: 21–3020097544
51. Shen H,, Wang L,, Liu Y,, Hu D, (Year: 2010) Discriminative analysis of resting-state functional connectivity patterns of schizophrenia using low dimensional embedding of fMRI. Neuroimage49: 3110–312119931396
52. Salomon R,, Bleich-Cohen M,, Hahamy-Dubossarsky A,, Dinstien I,, Weizman R,, et al. (Year: 2011) Global functional connectivity deficits in schizophrenia depend on behavioral state. J Neurosci31: 12972–1298121900576
53. Salvador R,, Anguera M,, Gomar JJ,, Bullmore ET,, Pomarol-Clotet E, (Year: 2010) Conditional mutual information maps as descriptors of net connectivity levels in the brain. Front Neuroinform4: 11521151357
54. Shad MU,, Muddasani S,, Prasad K,, Sweeney JA,, Keshavan MS, (Year: 2004) Insight and prefrontal cortex in first-episode schizophrenia. Neuroimage22: 1315–132015219603
55. Parellada M,, Boada L,, Fraguas D,, Reig S,, Castro-Fornieles J,, et al. (Year: 2011) Trait and state attributes of insight in first episodes of early-onset schizophrenia and other psychoses: A 2-year longitudinal study. Schizophr Bull37: 38–5120884756
56. Shad MU,, Muddasani S,, Keshavan MS, (Year: 2006) Prefrontal subregions and dimensions of insight in first-episode schizophrenia–a pilot study. Psychiatry Res146: 35–4216361089
57. Orfei MD, Piras F, Macci E, Caltagirone C, Spalletta G (2012) The neuroanatomical correlates of cognitive insight in schizophrenia. Soc Cogn Affect Neurosci.
58. Morgan KD,, Dazzan P,, Morgan C,, Lappin J,, Hutchinson G,, et al. (Year: 2010) Insight, grey matter and cognitive function in first-onset psychosis. Br J Psychiatry197: 141–14820679268
59. Cooke MA,, Fannon D,, Kuipers E,, Peters E,, Williams SC,, et al. (Year: 2008) Neurological basis of poor insight in psychosis: A voxel-based MRI study. Schizophrenia Research103: 40–5118539438
60. Modinos G,, Renken R,, Ormel J,, Aleman A, (Year: 2011) Self-reflection and the psychosis-prone brain: An fMRI study. Neuropsychology25: 295–30521443341
61. Hill K,, Mann L,, Laws KR,, Stephenson CM,, Mimmo-Smith I,, et al. (Year: 2004) Hypofrontality in schizophrenia: A meta-analysis of functional imaging studies. Acta Psychiatrica Scandinavia110: 243–256
62. Antonius D,, Prudent V,, Rebani Y,, D’Angelo D,, Ardekani BA,, et al. (Year: 2011) White matter integrity and lack of insight in schizophrenia and schizoaffective disorder. Schizophr Res128: 76–8221429714
63. Fox M,, Raichle M, (Year: 2007) Spontaneous fluctuations in brain activity observed with functional magnetic resonance imaging. Nature Reviews, Neuroscience8: 700–711
64. Van den Heuvel MP,, Hulshoff Pol HE, (Year: 2010) Exploring the brain network: A review on resting-state fMRI functional connectivity. Eur Neuropsychopharmacol20: 519–53420471808
65. Smith SM,, Fox PT,, Miller KL,, Glahn DC,, Fox PM,, et al. (Year: 2009) Correspondence of the brain’s functional architecture during activation and rest. Proc Natl Acad Sci U S A106: 13040–1304519620724
66. Fransson P, (Year: 2006) How default is the default mode of brain function?: Further evidence from intrinsic BOLD signal fluctuations. Neuropsychologia44: 2836–284516879844
67. Van de Ven VG,, Formisano E,, Prvulovic D,, Roeder CH,, Linden DEJ, (Year: 2004) Functional connectivity as revealed by spatial independent component analysis of fMRI measurements during rest. Hum Brain Mapp22: 165–17815195284
68. Calhoun V,, Adali T,, Pearlson G,, Pekar J, (Year: 2001) A method for making group inferences from funcional MRI data using independent component analysis. Hum Brain Mapp14: 140–15111559959
69. Jafri M,, Pearlson G,, Stevens M,, Calhoun V, (Year: 2008) A method for functional network connectivity among spatially independent resting-state components in schizophrenia. Neuroimage39: 1666–168118082428
70. Garrity AG,, Pearlson GD,, McKiernan K,, Lloyd D,, Kiehl KA,, et al. (Year: 2007) Aberrant “default mode” functional connectivity in schizophrenia. Am J Psychiatry164: 450–45717329470
71. Giel R, Nienhuis F (1996) Scan 2.1: Scedules for clinical assessment in neuropsychiatry (in dutch). Geneve/Groningen: WHO.
72. Kay S,, Fitzbein A,, Opler L, (Year: 1987) The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophr Bull13: 231–267
73. Sanz M,, Constable G,, Lopez-Ibor I,, Kemp R,, David AS, (Year: 2011) A comparative study of insight scales and their relationship to psychopathological and clinical variables. Psychological Medicine28: 437–4469572100
74. Drake RJ,, Lewis SW, (Year: 2003) Insight and neurocognition in schizophrenia. Schizophrenia Research62: 165–17312765757
75. Verhage F (1984) Intelligentie en leeftijd: Onderzoek bij nederlanders van twaalf tot zevenenzeventig jaar. Assen: Van Gorcum.
76. Oldfield R, (Year: 1971) The assessment and analysis of handedness: The edinburgh inventory. Neuropsychologia9: 97–1135146491
77. Li Y, Adali T, Calhoun VD (2006) Sample dependence correction for order selection in fMRI analysis. Washington, D.C.: in Proc. ISBI.
78. Himberg J,, Hyvarinen A,, Esposito F, (Year: 2004) Validating the independent components of neuroimaging time series via clustering and visualization. Neuroimage22: 1214–122215219593
79. Lancaster J,, Woldorff M,, Parson L,, Lotti M,, Freitas C,, et al. (Year: 2000) Automated talairach atlas labels for functional brain mapping. Hum Brain Mapp10: 120–13110912591
80. Lancaster J,, Summerlin J,, Rainey L,, Freitas C,, Fox P, (Year: 1997) The talairach daemon, a database server for talairach atlas labels. Neuroimage5: S633
81. Tie Y,, Whalen S,, Suarez RO,, Golby AJ, (Year: 2008) Group independent component analysis of language fMRI from word generation tasks. Neuroimage42: 1214–122518621548
82. Whitfield-Gabrieli S,, Moran JM,, Nieto-Castanon A,, Triantafyllou C,, Saxe R,, et al. (Year: 2011) Associations and dissociations between default and self-reference networks in the human brain. Neuroimage55: 225–23221111832
83. Qin P,, Northoff G, (Year: 2011) How is our self related to midline regions and the default-mode network?Neuroimage57: 1221–123321609772
84. Philippi CL,, Duff MC,, Denburg NL,, Tranel D,, Rudrauf D, (Year: 2012) Medial PFC damage abolishes the self-reference effect. J Cogn Neurosci24: 475–48121942762
85. Allman JM,, Hakeem A,, Erwin JM,, Nimchinsky E,, Hof P, (Year: 2001) The anterior cingulate cortex. the evolution of an interface between emotion and cognition. Ann N Y Acad Sci935: 107–11711411161
86. Cavanna AE, (Year: 2007) The precuneus and consciousness. CNS Spectrums12: 545–55217603406
87. Cavanna AE, (Year: 2006) The precuneus: A review of its functional anatomy and behavioural correlates. Brain129: 564–58316399806
88. Wang L,, Metzak PD,, Woodward TS, (Year: 2011) Aberrant connectivity during self-other source monitoring in schizophrenia. Schizophrenia Research125: 136–14221147519
89. Spreng RN,, Grady CL, (Year: 2009) Patterns of brain activity supporting autobiographical memory, prospection, and theory of mind, and their relationship to the default mode network. J Cogn Neurosci22: 1112–112319580387
90. Wicker B, (Year: 2003) A relation between rest and the self in the brain?Brain Research Reviews43: 224–23014572916

Figures

[Figure ID: pone-0042707-g001]
doi: 10.1371/journal.pone.0042707.g001.
Figure 1  DMN in healthy controls.

Components map of the DMN of healthy controls showing the anterior DMN on the left and the posterior part on the right (p<.001; k >10).



[Figure ID: pone-0042707-g002]
doi: 10.1371/journal.pone.0042707.g002.
Figure 2  DMN in schizophrenia patients.

Components map of the DMN of patients controls showing the anterior DMN on the left and the posterior part on the right (p<.001; k >10).



[Figure ID: pone-0042707-g003]
doi: 10.1371/journal.pone.0042707.g003.
Figure 3  Differences in DMN connectivity between patients with good an poor insight.

Group comparison of good vs. poor insight patients with the anterior component on the left showing the ACC, and the posterior component on the right showing the precuneus (p<0.001; k>10; masked with component image map).



[Figure ID: pone-0042707-g004]
doi: 10.1371/journal.pone.0042707.g004.
Figure 4  Connectivity between the anterior and posterior DMN in patients and controls.

Z-scores of connectivity between the anterior and posterior DMN for healthy controls and schizophrenia patients with good and poor insight.



Tables
[TableWrap ID: pone-0042707-t001] doi: 10.1371/journal.pone.0042707.t001.
Table 1  Demographical data.
Good insight (n = 25) Poor insight (n = 19) Controls (n = 30) Statistical test score (Z or X2) p-value
Mean age (SD) 33.4 (11.2) 35.9 (11.9) 33.4 (10.5) .69 .71
Mean education (SD) 3.52 (1.3) 3.53 (1.2) 4.1 (1.1) 1.1 0.59
Gender (M/F) 9/16 7/12 15/15 .0 0.51
Handedness (L/R) 3/22 2/17 6/24 .0 1.0
PANSS G12 (SD) 1.3 .5 3.7 .8 5.9 <.005
PANSS Positive (SD) 14.3 (4.8) 17.1 (4.8) 1.96 .050
PANSS Negative (SD) 14.3 (4.3) 14.4 (4.8) .21 .83
PANSS General -12 (SD) 25.8 (8.3) 28.1 (7.4) 1.34 .18
Illness duration years (SD) 10.5 9.6 8.9 8.2 .46 .67
No antipsychotic (%) 0 21.1 6.1 .11
Typical (%) 8.0 10.5
Atypical (%) 68.0 47.4
Typical + atypical (%) 2.0 10.5

Overview of demographical data of the good insight and poor insight groups and the control group; The PANSS general item is shown without item G12. The fifth column shows the Z (Mann-Whitney) or Chi-square (Kruskal-Wallis and Chi-square test for independence) values of the statistical comparisons and the fifth the p-values.



Article Categories:
  • Research Article
Article Categories:
  • Biology
    • Neuroscience
      • Neuroimaging
        • Fmri
      • Behavioral Neuroscience
      • Neuropsychology
Article Categories:
  • Medicine
    • Mental Health
      • Psychiatry
        • Personality Disorders
      • Psychology
        • Cognitive Psychology
          • Memory
          • Reasoning
          • Recall
        • Neuropsychology
        • Personality
Article Categories:
  • Social and Behavioral Sciences
    • Psychology
      • Cognitive Psychology
      • Neuropsychology
      • Personality


Previous Document:  Perceptual characterization and analysis of aroma mixtures using gas chromatography recomposition-ol...
Next Document:  Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed ...