Introduction

Ketamine has been shown to induce a range of long-lasting changes in behavior that cannot be easily explained by its direct pharmacological effects, i.e. the blockade of the glutamatergic N-methyl-D-aspartate receptors. Most notably, a single subanesthetic ketamine treatment produces rapid antidepressant effects in a subset of patients, and the effects sustain long after ketamine has been metabolized ¹. Ketamine is differentially metabolized in females and males, which may partially account for the reported gender-dependent behavioral responses to ketamine ^2,3. In order to get further insights into the sustained neurobiological effects of ketamine, we investigated the resting-state functional connectivity (FC) in urethane-anesthetized female and male rats 24 hours after a single subanesthetic dose of ketamine that initiates molecular-level signaling events implicated in rapid antidepressant effects.

Methods

The animal procedures were approved by the National Animal Experiment Board. Male (n=20) and female (n=20) Wistar rats were used. Ketamine-HCl (50 mg/kg, 2ml/kg, i.p., n=10 in each gender group) or saline (2 ml/kg, n=10 in each gender group) was injected 24 h prior to the functional magnetic resonance imaging (fMRI). For fMRI, rats were anesthetized with urethane (1.25-1.5 g/kg, i.p.), and subsequently fixed to an MRI rat holder with earplugs and bite bar. The rsfMRI data were acquired with single-shot gradient-echo echo-planar imaging sequence with the following parameters: repetition time 1000 ms, echo time 18 ms, 1200 volumes (20 min), field of view 32x32 mm, 15 slices with a thickness of 0.9 mm, matrix size 64x64, and bandwidth 200 kHz. The MRI data were converted to NIfTI (http://aedes.uef.fi), slice-timing corrected, motion-corrected, spatially smoothed, co-registered (SPM8), and band-pass filtered (0.01-0.15Hz). Urethane-induced FC states ⁴ were separated by observing breathing rate ⁵, and only data from the rapid-eye-movement (REM)-like state ⁴ were analyzed (n=5-6 per group). The correlation coefficients between regions of interest were calculated to obtain measures for FC. Connectivity was analyzed from both hemispheres separately to corroborate the findings. Statistical testing was done with two-way ANOVA (uncorrected).

Results

The group-level mean FC matrices are shown in Figure 1, which shows that in both gender groups ketamine-treated rats tended to have suppressed FC compared to controls. Figure 2 shows the p-value matrices obtained from two-way ANOVA analyses, indicating that gender alone was not affecting FC. However, there were several treatment-induced effects on connectivity of medial prefrontal cortex, caudate putamen, and hippocampus in both gender groups. Additionally, significant interactions between treatment and gender were observed, suggesting also different ketamine-induced modulation of FC between gender groups. Figure 3 shows group-level connectivity values obtained from representative brain regions associated with depressive disorders

Discussion and conclusions

Wide range of recent functional neuroimaging studies indicate that the FC of several brain regions, such as prefrontal cortex, striatum, and hippocampus, is significantly modulated in depressive disorders ^6,7. Prefrontal cortex and hippocampus are key nodes in the default mode network (DMN), where hyperconnectivity is present in depressive disorders. In present study, we report ketamine-induced modulation of FC in rats 24 h after the treatment; decreased connectivity was observed in key regions associated with depressive disorders . Importantly, we observed ketamine-induced suppression of FC within the nodes of the DMN, which may be associated with the antidepressant effects of ketamine. In addition to similar ketamine-induced effects in both gender groups, we also observed interaction between gender and treatment in striatal connections, which can shed light on the mechanisms underlying the gender-dependent effects.

Acknowledgements

This work was supported by the Academy of Finland. In addition, we thank Maarit Pulkkinen for technical assistance with the animal preparation.

References

1. Berman RM, Cappiello A, Anand A, et al. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry. 2000;47(4):351-354.
2. Saland SK, Duclot F, Kabbaj M. Integrative analysis of sex differences in the rapid antidepressant effects of ketamine in preclinical models for individualized clinical outcomes. Curr Opin Behav Sci. 2017;14:19-26.
3. Zanos P, Moaddel R, Morris PJ, et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature. 2016;533(7604):481-486.
4. Zhurakovskaya E, Paasonen J, Shatillo A, et al. Global functional connectivity differences between sleep-like states in urethane anesthetized rats measured by fMRI. PLoS One. 2016;11(5):e0155343.
5. Wilson DA, Hoptman MJ, Gerum SV, Guilfoyle DN. State-dependent functional connectivity of rat olfactory system assessed by fMRI. Neurosci Lett. 2011;497(2):69-73.
6. Brakowski J, Spinelli S, Dorig N, et al. Resting state brain network function in major depression - depression symptomatology, antidepressant treatment effects, future research. J Psychiatr Res. 2017;92:147-159.
7. Kaiser RH, Andrews-Hanna JR, Wager TD, Pizzagalli DA. Large-scale network dysfunction in major depressive disorder: A meta-analysis of resting-state functional connectivity. JAMA Psychiatry. 2015;72(6):603-611.