This vehicle-controlled animal study (n=24) investigated the effects of esketamine (4.5mg/0.3kg) on astrocyte plasticity in the hippocampus of a depression-model rat strain. Results indicate that ketamine can rapidly modify the shape of astrocytes (sub-type of glial cells) so that they can optimally modulate the synaptic micro-environment, neurogenesis, and vascularization, which is otherwise impaired under depression.

Abstract

“Background and purpose: Astroglia contribute to the pathophysiology of major depression and antidepressant drugs act by modulating synaptic plasticity; therefore, the present study investigated whether the fast antidepressant action of ketamine is reflected in a rapid alteration of the astrocytes’ morphology in a genetic animal model of depression.

Experimental Approach: S‐Ketamine (15 mg·kg⁻¹) or saline was administered as a single injection to Flinders Line (FSL/ FRL) rats. Twenty‐four hours after the treatment, perfusion fixation was carried out and the morphology of glial fibrillary acid protein (GFAP)‐positive astrocytes in the CA1 stratum radiatum (CA1.SR) and the molecular layer of the dentate gyrus (GCL) of the hippocampus was investigated by applying stereological techniques and analysis with Imaris software. The depressive‐like behaviour of animals was also evaluated using forced swim test.

Key Results: FSL rats treated with ketamine exhibited a significant reduction in immobility time in comparison with the FSL‐vehicle group. The volumes of the hippocampal CA1.SR and GCL regions were significantly increased 1 day after ketamine treatment in the FSL rats. The size of astrocytes in the ketamine‐treated FSL rats was larger than those in the FSL‐vehicle group. Additionally, the number and length of the astrocytic processes in the CA1.SR region were significantly increased 1 day following ketamine treatment.

Conclusions and Implications: Our results support the hypothesis that astroglial atrophy contributes to the pathophysiology of depression and a morphological modification of astrocytes could be one mechanism by which ketamine rapidly improves depressive behaviour.”

Authors: Maryam Ardalan, Ali H. Rafati, Jens R. Nyengaard & Gregers Wegener

Summary

Introduction

Major depressive disorder is a serious and costly psychiatric condition, and glial cells play a dynamic role in modifying brain structure. Glial cells are believed to contribute to the pathophysiology of major depression. Astrocytes are glial cells that play a key role in the maintenance of brain homeostasis by supplying energy to the neurons, recycling neurotransmitters, and by connecting to the blood vessels promoting their development.

Currently, the treatment of major depression is not ideal due to the slow onset of action and partial therapeutic effect of traditional antidepressant drugs. However, the rapid-acting intervention ketamine improves depressive symptoms especially suicidal ideation in patients with treatment-resistant depression.

Animals

This study was performed on adult male Flinders sensitive line (FSL) rats and Flinders resistant line (FRL) rats. The animals were housed in groups of two in a temperature-controlled environment (20 – 22°C), with a normal 12 h light : dark cycle.

immunohistochemistry

Free-floating sections were washed in TBS containing 0.1% Triton X-100 for 30 min, blocked endogenous peroxidase for 30 min, and then immunostained with rabbit anti-GFAP antibody/HRP for 2 h. The sections were mounted on gelatin-coated slides and counterstained with 0.25% thionin solution.

Stereological quantification of the volumes of the hippocampal regions

Two subregions of the rat hippocampus were estimated using the Cavalieri estimator with point counting on Nissl-stained sections using the newCAST software, an Olympus light microscope and a motorized microscope stage.

A systematic set of Z-stacks was captured by an Olympus scanner VS120 using a 60 oil objective. The Z-stacks were 20 – 25 m high and acquired in steps of 0.5 m.

Morphological analysis of astrocytes

The morphology of astrocytes was quantified using Imaris software (Version 7.7, Bitplane A.G., Zurich, Switzerland). The length of the processes was measured based on the radial distance from the reference point in 10 m.

A morphological analysis of 30 GFAP positive astrocytes was performed in the CA1.SR area of the hippocampus from each animal. The complexity of the astrocytic branches was quantified by identifying the branch endpoints and bifurcations.

Stereological quantification of the volume of astrocytes

The volume of astrocytes was investigated in the CA1.SR and molecular layer of the dentate gyrus on GFAP stained sections.

The volume of GFAP-immunopositive astrocytes was calculated by applying 3D nucleator and sampling 50 – 80 astrocytes per animal with a 100 oil-immersion objective lens.

Statistical analysis

Statistical analysis was performed on data collected from four groups of animals. A two-way ANOVA followed by a post hoc Bonferroni test was used to compare the behaviour of animals, hippocampal volumes and morphological alterations of the astrocytes.

Results

Ketamine had an antidepressant-like effect on immobility time in rats. The duration of immobility time was higher in the FSL-vehicle rats compared to the FRL-vehicle rats.

Strain and ketamine treatment significantly influenced the volume of GFAP positive astrocytes in the CA1.SR area of the hippocampus. Additionally, astrocytes in the CA1.SR area of the hippocampus were significantly larger in FSL rats with ketamine treatment than FSL rats without treatment.

A significant strain x treatment interaction was observed for the volume of CA1.SR and GCL, and a significant influence of ketamine treatment on the volume of CA1.SR and GCL was observed.

Rapid effect of ketamine treatment on the morphology of astrocytes in the CA1.SR subregion of the hippocampus

Sholl analysis revealed that astrocytes in the CA1.SR subregion of the hippocampus had significantly different branching patterns between FSL and FRL rats, and that acute ketamine treatment was associated with a significant increase in the length of the astrocytic branches.

Discussion

Ketamine modulates several structural parameters related to astroglial plasticity in the hippocampus in the following 24 h and has an ameliorative effect on the immobility behaviour in patients with severe depression.

A reduction in the volume of the hippocampus was shown in patients with major depressive disorder, and this reduction was primarily attributed to changes in the volume of the CA1 and DG areas.

Conventional antidepressant drugs normalize the structural plasticity of the hippocampus by altering the volume of the area, the number of cells, and the volume of the cells. We found evidence that ketamine increases the number of granule cells in the hippocampus of depressed animals, and reduces the rate of apoptosis, which could explain the increase in the volume of the hippocampus after ketamine treatment.

Depressed animals have reduced morphology of astrocytes in the CA1.SR area, which is important for modulating excitatory glutamate hippocampal synapses by regulating the amount of glutamine, an essential factor for releasing neuronal glutamate and D-serine factor.

Ketamine had a rapid effect on the soma size and astrocytic arborization in the CA1.SR area of the hippocampus in a rat model of depression. This effect might be due to the rapid activation of astrocytes and substantial stimulation of BDNF synthesis. Earlier studies reported that fluoxetine affected the expression of 5-HT receptor subtypes on astrocytes and that fluoxetine reduced the volume of astrocytes in the hippocampus. However, our study demonstrated that fluoxetine did not affect astrocyte volume.

Astrocytic Ca2+ is increased, which has a significant effect on the synaptic plasticity. Additionally, some astrocytic branches and cell bodies in the cerebral cortex of adult rats contain NMDA receptors, which may explain the fast synaptic plasticity observed after ketamine treatment.

The study showed that the size of GFAP-positive astrocytes in the MDG subregion of the hippocampus was significantly smaller in the FSL rats than in the control group, and that ketamine reversed this effect. This result indicates that the morphological changes of astrocytes in depression inhibit VEGF signalling.

Conclusion

Ketamine can modify the morphology of astrocytes to optimally modulate the synaptic micro-environment, neurogenesis and vascularization.

Author contributions

A group of researchers designed and carried out a study to investigate the influence of gender on perception of facial expressions.