Venlafaxine stimulates PNN proteolysis and MMP-9-dependent enhancement of gamma power; relevance to antidepressant efficacy

Abstract

Drugs that target monoaminergic transmission represent a first-line treatment for major depression. Though a full understanding of the mechanisms that underlie antidepressant efficacy is lacking, evidence supports a role for enhanced excitatory transmission. This can occur through two non-mutually exclusive mechanisms. The first involves increased function of excitatory neurons through relatively direct mechanisms such as enhanced dendritic arborization. Another mechanism involves reduced inhibitory function, which occurs with the rapid antidepressant ketamine. Consistent with this, GABAergic interneuron-mediated cortical inhibition is linked to reduced gamma oscillatory power, a rhythm also diminished in depression. Remission of depressive symptoms correlates with restoration of gamma power. As a result of strong excitatory input, reliable GABA release, and fast firing, PV-expressing neurons (PV neurons) represent critical pacemakers for synchronous oscillations. PV neurons also represent the predominant GABAergic population enveloped by perineuronal nets (PNNs), lattice-like structures that localize glutamatergic input. Disruption of PNNs reduces PV excitability and enhances gamma activity. Studies suggest that monoamine reuptake inhibitors reduce integrity of the PNN. Mechanisms by which these inhibitors reduce PNN integrity, however, remain largely unexplored. A better understanding of these issues might encourage development of therapeutics that best up-regulate PNN-modulating proteases. We observe that the serotonin/norepinephrine reuptake inhibitor venlafaxine increases hippocampal matrix metalloproteinase (MMP)-9 levels as determined by ELISA and concomitantly reduces PNN integrity in murine hippocampus as determined by analysis of sections following their staining with a fluorescent PNN-binding lectin. Moreover, venlafaxine-treated mice (30 mg/kg/day) show an increase in carbachol-induced gamma power in ex vivo hippocampal slices as determined by local field potential recording and Matlab analyses. Studies with mice deficient in matrix metalloproteinase 9 (MMP-9), a protease linked to PNN disruption in other settings, suggest that MMP-9 contributes to venlafaxine-enhanced gamma power. In conclusion, our results support the possibility that MMP-9 activity contributes to antidepressant efficacy through effects on the PNN that may in turn enhance neuronal population dynamics involved in mood and/or memory. Cover Image for this issue: doi: 10.1111/jnc.14498.

Keywords: MMP-9; depression; gamma oscillations; interneuron; perineuronal net; venlafaxine.

I.. MMP-9 levels and PNN immunoreactivity in hippocampi from venlafaxine treated mice

1A) MMP-9 concentrations in brain lysates from 8 control and 7 venlafaxine (VFX) treated mice. Venlafaxine treatment significantly increased hippocampal concentrations of this enzyme (Control 149.4 +/− 9.9; VFX 253.8 +/− 37.3; p=0.0129; Student’s t-test). IB) A quantitative analysis of PV/PNN ratios from control and venlafaxine treated animals (n=8 animals and N= 13–14 slides for each group) revealed a statistically significant increase in the venlafaxine group (p=0.023; Student’s t-test). PV numbers were not increased (control 8.15 +/− 0.87; venlafaxine 7.93 +/− 0.85; p= 0.84; not shown graphically), suggesting that PNN positivity was selectively reduced. 1C) WFA fluorescence intensity in control and venlafaxine treated mouse hippocampi shows a reduction in the venlafaxine treated group (*p= 0.0187, n= 7–8 mice; N data points each representing the average ROI values for 1–2 images per mouse). 1D-E) PV (red) and PNN (green) immunoreactivity in murine hippocampi in slices from a control and venlafaxine treated animal as indicated. The scale bar represents 75 μm and representative PNN enwrapped PV cells are indicated by arrows. IF-G) High power images of PNN staining in murine hippocampi. In these images the scale bar represents 25 μm.

II.. Venlafaxine increases the power of carbachol-induced gamma activity in ex vivo hippocampal slices

2A) Representative local field potential (LFP) recordings from slices obtained from a saline or venlafaxine treated animal. The LFP was filtered in the theta (4–8Hz), low and high gamma (25–55Hz and 65–85Hz respectively) ranges. 2B) The average low and high gamma power of a 315s window beginning 150s after onset of carbachol perfusion was compared between treatment. For n= 4–6 animals, 1–2 slices per animal for each group, there is a statistically significant increase in low and high gamma power for the venlafaxine treated animals (p = 0.008 and 0.05 respectively; Student’s t test). 2C), we show the ratio of post- to pre- carbachol gamma power for each slice. Again we detect a significant difference between slices from wild type saline and venlafaxine treated animals in low gamma (p=0.0246) and a tendency towards significance in high gamma (p= 0.084).

III.. Venlafaxine stimulated changes in PNN integrity are reduced in MMP-9 null mice

3A-B) PNN and PV staining in slice subsections from treated and untreated MMP-9 null mice. Representative PNN enwrapped PV cells are indicated by arrows, while a PV cell without an appreciable PNN is noted by the arrowhead. Scale bar represents 75μM. 3C) PV/PNN ratio for both groups, with data from wild type mice shown for comparison. For this analysis, n= 4–6 animals per group with 1–3 slides analyzed per animal. As in wild type animals, the number of PV positive cells was not affected by venlafaxine (control 6.87 +/− 0.79, N=14; venlafaxine 5.75 +/− 0.59, N=16; p= 0.26). Though reduced in magnitude as compared to wild type animals, the PV/PNN ratio was increased in the venlafaxine group (*Student’s t test p= 0.017). 3D) WFA intensity in control and venlafaxine treated MMP-9 null mice. Though the PV/PNN ratio was affected by venlafaxine in the MMP-9 nulls, in contrast to what was observed with wild type animals, the intensity of WFA flourescence was not reduced.

IV.. Venlafaxine does not significantly increase the power of carbachol-induced gamma activity in ex vivo hippocampal slices from MMP-9 knockout mice

4A) Representative LFP recordings and filtered data from slices obtained from saline or venlafaxine treated MMP-9 null animals. 4B) Analyses of average low and high gamma power revealed no statistically significant increase due to venlafaxine treatment in MMP-9 null animals (n= 4–6 animals per group and N represents the average value across epochs from 1–2 slices from each n; p > 0.05, Student’s t test). 4C) Post- to pre- carbachol ratio in slices from saline and venlafaxine treated mice. The post to pre carbachol ratio in low or high gamma is not significantly different between control and treatment in MMP-9 null animals.

V.. Hypothetical Overview

Hippocampal levels of serotonin and norepinephrine are increased in response to venlafaxine treatment (far left). These monoamines interact with G-protein coupled receptors expressed on neurons (purple) and non-neuronal cells such as microglia (green) to increase expression of MMP-9. Increased levels of MMP-9 can in turn target PNN components and/or activate additional metalloproteases (MPs) that also target PNN substrates. Reduced PNN integrity can in turn disrupt PV-mediated inhibition of pyramidal cell activity with consequent effects on gamma power.