VBM revealed significant atrophy in the following brain areas of WKY rats: the left ventral hippocampus, the right caudate putamen, the right lateral septum, the cerebellar vermis, and the cerebellar nuclei. According to a recent clinical meta-analysis15, the VBM results comparing WKY and Wistar rats are similar to those in human clinical MDD studies, although the largest atrophic cluster was observed in the cerebellum. The results of the two OFT trials and FST indicates the behavioural characteristics of WKY rats as a depression model, namely locomotion disruption and reduced escape activity. Voxel-based correlation analysis revealed areas correlated with escape behaviour in the second OFT. We observed negative correlations in the hippocampus, which agree with the results of a previous animal study35, and the right habenula, as well as a positive correlation in the cerebellar vermis. Our VBM results indicate similar anatomical changes in WKY rats as in humans, validating their use as an endogenous depression model.
Surprisingly, the most significant atrophic cluster was detected in the cerebellum with the moderate threshold. The clusters observed in the cerebellar nuclei and vermis in the 3rd and 9th lobules remained after the stricter threshold was applied. A recent meta-analysis reported an atrophic cluster in the cerebellum of MDD patients12, and other studies have suggested cerebellar involvement in modulating various aspects of mood and cognition36–38. Hypertrophy has been observed in the cerebellum of depressed patients after successful treatment with electroconvulsive therapy39 as well as in the cerebellar vermis after the use of chronic antidepressant medication40. Another recent meta-analysis detected a hypertrophic cluster in the cerebellar vermis in BD patients relative to MDD patients15. The cerebellar atrophy in WKY rats thus reflects MDD but not BD. In addition, positive correlations between atrophic clusters and certain behaviours were detected in the vermis (9th lobule), although we could not confirm positive correlations with other atrophic brain regions, such as the hippocampus. The cerebellar vermis is included in the cerebrocerebellum and receives target projections from motor areas41. According to our OFT results, atrophy in the vermis may lead to disrupted escape activity. Cerebellar atrophy may trigger behavioural disturbances and induce an inability to perform even in the presence of motivation. The role of the cerebellum in emotional processing42 is still being discussed, and the contribution of the cerebellum to depressive symptoms should be investigated further.
We observed a significant cluster extending from the hippocampus to the septal region, somewhat including the thalamus, with the moderate threshold, and a cluster in the left ventral hippocampus remained after the stricter threshold was applied. This demonstrates significant atrophy in the ventral hippocampus and an atrophic trend in the dorsal hippocampus (Supplementary Fig. 3) A previous animal study reported negative correlations between locomotion and grey matter concentration in the ventral hippocampus35. Because the hippocampus is a common region of interest in MDD research, as supported by a recent meta-analysis15, the atrophy observed in the hippocampus and parahippocampal regions in our model indicates similarity to human studies. However, WKY rats have been reported to be vulnerable to stress16, and differential roles of the ventral (stress, emotion) and dorsal (cognition) hippocampi have been discussed43,44, so the atrophy we observed might be stress-related.
Our data demonstrates a negative correlation between escape behaviour and the right habenula as well as volume reduction. The relationship between the volume of the habenula and its effect on escape activity should be investigated further. It has been proposed that the lateral habenula could systematically learn to expect an adverse outcome, and frequent neural firing may lead to a state of continuous disappointment and hopelessness45. A significant volume reduction in the habenula of depressive patients has also been reported46, and the habenula is considered a critical region in MDD research47. Ketamine-induced metabolic reduction in the right habenula of treatment-resistant depression patients has been reported48, and the habenula may be a potential target for future research on treatment-resistant depression.
Brain atrophy observed in the right lateral septum neighbouring the fimbria remained when the stricter threshold was applied, and a negative correlation with escape behaviour was detected. Although the septal area is rarely described in clinical depression studies using MRI, previous animal studies reported that the septal nuclei are involved in stress responses49. In addition, lesion studies of the septum reported aggressive behaviours50,51. Patients with clinical MDD exhibit symptoms of ambivalence, lost motivation, reduced locomotor activity, and increased impulsivity and irritation. Further study is warranted to clarify whether the atrophy that extends from the ventral hippocampus to the septum contributes to the complex symptoms observed in MDD.
Only two clusters including the amygdala were detected using the moderate threshold, although a negative correlation to movement duration was detected in the left amygdala. Some volumetric studies in MDD patients have reported increased amygdala volumes52,53, whereas others have reported reduced volumes54,55. A meta-regression analysis in first-episode MDD patients showed negative correlations between the volume of the right amygdala and Hamilton Depression Rating Scale score10. In another recent study, patients with treatment-resistant MDD exhibited blunted amygdala activity during a facial recognition task56. Further research is thus warranted to determine the relationship between amygdala volume and treatment-resistant MDD.
Our results indicated atrophy from the secondary somatosensory cortex extending to the insular cortex, although this did not remain after the stricter threshold was applied. Therefore, we cannot determine any major contribution of this site in MDD. However, a human study using VBM analysis57 and some meta-analyses7,10,11 have reported insular cortex atrophy in MDD patients, and our VBM data seems to be consistent with these studies. Our voxel-based correlation analysis could not detect a significant cluster in the somatosensory or insular cortices, and no correlation between these areas and behaviour could be confirmed in WKY rats. Insular cortex atrophy is commonly observed in many psychiatric disorders58, so analysis of this region in post-mortem brain studies may help to elucidate genetic spectra across various psychiatric disorders59, including MDD.
To the best of our knowledge, a reduction in pituitary gland volume has not previously been reported in WKY rats. However, it has been demonstrated that isolation paradigms elevate plasma ACTH levels without changes in corticosterone levels30, and we cannot rule out the possibility that isolation exhausted pituitary ACTH in WKY rats, resulting in atrophy. However, our study showed that pituitary atrophy was positively correlated with locomotion behaviour, so future studies are required to determine whether this atrophy is a biomarker of treatment resistance.
In this study, we were unable to confirm any ACC atrophy in WKY rats. Although atrophy in the frontal regions containing the ACC has been reported in MDD patients7,60, a medication wash-out MDD study reported an increase in ACC volume9. Miniscule changes have been observed in medial prefrontal regions in WKY rats16, and we cannot completely rule out small anatomical variations corresponding with strain differences. In addition, deep brain stimulation of the prefrontal region in WKY rats has been shown to rescue disrupted locomotor activity61. The contribution of frontal brain atrophy to depression symptoms remains unclear.
Both the OFT and FST were used to validate depressive behaviour. Although we did not find any literature showing the impact of a 10 min OFT on brain morphology, we cannot rule out the carry-over effect of the OFT upon the FST in this experimental paradigm. OFT has been criticized as an anxiety-detecting measure62, and it has been discussed that immobility during the FST is not indicative of despair, but a coping benefit from learning and memory to promote behavioural adaptation and survival63. We cannot rule this possibility out when interpreting the enhanced preference for passive survival coping strategies in WKY rats; however, our data simultaneously demonstrated low locomotor activity during both the FST and OFT. Disruption of exploratory locomotion during the OFT, where candidates could exhibit coping behaviours to escape from isolation more easily than in the FST, would not relate to survival. Although Wistar rats exhibited prolonged stays in distant blocks, WKY rats did not, although moderate stress is expected to induce this coping behaviour. Our behavioural results thus show reduced escape behaviour both in neutral and threatening situations, which seems to represent the presence of despair. We assume that these results indicate face validity for this model of endogenous depression.
In this study, we housed rats individually, and cannot completely rule out the effect of isolation on brain atrophy. However, there are few reports on microscopic morphological changes induced by social isolation (besides maternal separation) within the first two weeks; only one report describes olfactory bulb atrophy induced by isolation64. Although it has been proposed that a social isolation paradigm might have adverse effects for recovery interventions, such inducing neurogenesis in the hippocampus following exercise65,66, no significant volumetric changes in the hippocampus were reported during two weeks of social isolation using the region of interest-volumetric MRI method64. In another endogenous depression model, the Flinders sensitive line, five weeks of isolation induced erasure of depression-like behaviour67. Whether isolation can be dismissed as stress in animal endogenous depression models is still an interesting question. We suspect that the effect of isolation stress in the first two weeks might be too small to affect brain morphology in animal depression models, although we cannot rule out the possibility that genetic differences might enhance environmental effects.
We used an ex vivo MRI protocol to achieve the quality necessary for VBM. However, ex vivo MRI measurements have been discussed as less preferable for structural brain studies due to potential displacement, disrupted brain tissue integrity, and deformations resulting in artifacts68. This method may cause a global shrinkage in brain volume (~ 10%) and damage is most notable in the cerebellum, olfactory bulb, and cortex69,70. We therefore cannot completely rule out any data noise caused by the fixation method used. Spatially-normalized images in VBM analysis should have a relatively high resolution in humans, and grey matter segmentation is not excessively confounded by partial volume effects4, but these assumptions may not hold in small animals. For in vivo MRI in small animals, it is difficult to prevent motion artifacts and there are resolution disadvantages. In particular, it has been reported that the thalamus and hypothalamus can be more properly segmented using ex vivo MRI data69. There is also an MRI study reporting that the volume of the midbrain, hippocampus, thalamus, and cortex were relatively unaffected by perfuse-fixation70. The significant data observed in parahippocampal regions and cerebellar nuclei in this study might have benefitted from using ex vivo MRI techniques.
This study shows that single-housed WKY rats, as an endogenous depression model, demonstrated morphological similarities to MDD in humans. Correlations were determined to exist between the habenula and cerebellar vermis with inhibited escape behaviour via voxel-based correlation analysis. Although the contributions of the cerebellum36 and habenula47 to MDD have been discussed, there is insufficient research and therapies directly targeting these areas. We believe that WKY rats would be a good model for exploring the mechanisms of MDD and address therapeutic challenges.