Neuregulin signaling mediates the acute and sustained antidepressant effects of subanesthetic ketamine

This rodent study (n=50) investigated the signaling pathways associated with the rapid antidepressant effects of ketamine (10mg/kg) and found a novel neural plasticity-based mechanism implicated in its acute and sustained effects.

Abstract

“Introduction: Subanesthetic ketamine evokes rapid antidepressant effects in human patients that persist long past ketamine’s chemical half-life of ~2 h. Ketamine’s sustained antidepressant action may be due to modulation of cortical plasticity.

Methods: To determine if the ketamine-mediated amelioration of depression-like behaviors depends on NRG1/ErbB4 signaling in PV inhibitory interneurons, we tested the antidepressant effects of ketamine in the FST using mice, by manipulating NRG1/ErbB4 signaling.

Results: We find that ketamine ameliorates depression-like behavior in the forced swim test in adult mice, and this depends on parvalbumin-expressing (PV) neuron-directed neuregulin-1 (NRG1)/ErbB4 signaling. Ketamine rapidly downregulates NRG1 expression in PV inhibitory neurons in mouse medial prefrontal cortex (mPFC) following a single low-dose ketamine treatment. This NRG1 downregulation in PV neurons co-tracks with the decreases in synaptic inhibition to mPFC excitatory neurons for up to a week. This results from reduced synaptic excitation to PV neurons, and is blocked by exogenous NRG1 as well as by PV targeted ErbB4 receptor knockout.

Discussion: Thus, we conceptualize that ketamine’s effects are mediated through rapid and sustained cortical disinhibition via PV-specific NRG1 signaling. Our findings reveal a novel neural plasticity-based mechanism for ketamine’s acute and long-lasting antidepressant effects.”

Authors: Steven F. Grieco, Xin Qiao, Kevin G. Johnston, Lujia Chen, Renetta R. Nelson, Cary Lai, Todd C. Holmes & Xiangmin Xu

Summary

Subanesthetic ketamine evokes rapid antidepressant effects in human patients that persist long past ketamine’s chemical half-life. We find that ketamine ameliorates depression-like behavior in the forced swim test in adult mice and that this effect depends on parvalbumin-expressing (PV) neuron-directed neuregulin-1 (NRG1)/ErbB4 signaling.

Introduction

Ketamine has gained widespread attention for its potential to treat psychiatric disorders at subanesthetic doses, especially in human patients who are resistant to classical treatments for depression. Ketamine’s mechanism of action at subanesthetic doses is unknown, but it may modulate neural circuit plasticity.

Ketamine modulates the excitatory/inhibitory (E/I) balance in cortical circuits, and this change in E/I balance may initiate long-lasting cortical plasticity in higher-order association cortex such as prefrontal cortex. This study tested the hypothesis that ketamine’s antidepressant effects are due to its modulation of NRG1-directed signaling in PV inhibitory neurons in higher association cortex.

Ketamine’s antidepressant effects depend on PV neuron-directed NRG1/ErbB4 signaling. Ketamine induces sustained PV excitatory input loss and cortical disinhibition.

Antidepressant effects of ketamine depend on NRG1/ ErbB4 signaling

To determine if NRG1/ErbB4 signaling underlies ketamine’s antidepressant effects, we used the FST to measure depression-like behaviors and treated animals with exogenous NRG1. The antidepressant effects of ketamine were found to depend on NRG1/ErbB4 signaling.

NRG1 has been shown to penetrate the blood – brain barrier and functionally activate ErbB4 in the cortex. It blocks the antidepressant effect of ketamine and HNK, a major metabolite of ketamine, in both the acute 30 min and long-term 24 h ketamine treatment groups.

We tested whether NRG1/ErbB4 signaling by PV interneurons is required for the antidepressant effect of ketamine in the FST. The results show that ErbB4 expression is selectively removed in PV-positive interneurons in PV-Cre; ErbB4fl/fl mice, and ketamine is still effective in control mice.

Ketamine increases neural activity of mPFC excitatory neurons in vivo

We performed population calcium imaging of excitatory neurons in vivo in mPFC using head-mounted miniscopes. We found that 95% of the excitatory neurons in mPFC exhibited increased activity 24 h following ketamine administration, and that both calcium event peak amplitudes and integrated calcium event amplitudes significantly increased.

Sustained cortical disinhibition evoked by subanesthetic ketamine

Ketamine may induce cortical disinhibition by reducing interneuron activity. However, neither acute ketamine nor an in vivo treatment of the NMDAR antagonist MK-801 caused appreciable effects on inhibitory synaptic inhibition to L2/3 excitatory neurons in mPFC.

We injected mice with subanesthetic ketamine 1 h before preparing mPFC slices for IPSC recording, and found that ketamine and its metabolite HNK dramatically reduce evoked IPSC amplitudes in L2/3 excitatory cells. We then found that increased NRG1 signaling reverses these effects.

Ketamine treatment reduced inhibitory input to excitatory pyramidal neurons in the mPFC, and this effect was acutely reversible with bath application of NRG1. Ketamine treatment and NRG1 had no effect on spontaneous IPSCs or paired-pulse ratios.

NRG1/ErbB4 signaling in PV neurons reduced by ketamine

Ketamine downregulates NRG1 mRNA expression in PV interneurons, but not in Emx1+ neurons, in the mPFC. ErbB4 mRNA expression does not change in either PV or Emx1+ neurons after ketamine treatment, except for the 1 week time-point for PV cells where ErbB4 was increased.

We investigated whether ketamine-mediated downregulation of NRG1 mRNA expression in PV neurons in mPFC was associated with increased levels of phosphorylated CREB in excitatory neurons and cortical disinhibition. Ketamine treatment resulted in decreased NRG1/ErbB4-directed signaling and cortical disinhibition.

PV excitatory input loss evoked by ketamine

We measured the excitatory input to PV interneurons in mPFC in control mice and in vivo ketamine-treated adult mice. We found that the excitatory input to PV interneurons was dramatically reduced following ketamine treatment, and this effect was sustained for 1 week.

After ketamine treatment, NRG1 signaling was enhanced in PV interneurons, but not in control PV cells. In contrast, NRG1 enhanced the excitatory synaptic input and the direct uncaging responses of PV interneurons in ketamine-treated mice.

We found that NRG1 did not affect the resting membrane potential or intrinsic membrane excitability in PV cells under control conditions or following ketamine treatment. This allowed us to determine the molecular and circuit locus of ketamine-mediated disinhibition in the mPFC.

Discussion

Our study demonstrates that NRG1/ErbB4 signaling in mPFC PV inhibitory interneurons plays a critical role in mediating the acute and sustained antidepressant effects of subanesthetic ketamine. Ketamine’s antidepressant effects are mediated by its regulation of neural circuit plasticity.

Our work adds to previous studies on ketamine and depression by indicating that ketamine induces neural plasticity-related molecular events. However, it is not clear what circuit mechanisms induce these molecular events.

In this study, we found that PV NRG1/ErbB4 signaling is critical for maintaining excitatory synaptic inputs onto PV cells, and that reductions in PV NRG1/ErbB4 signaling results in cortical disinhibition. This mechanism is important for cortical plasticity not only in juvenile mice, but in adult mice as well.

Ketamine reduces PV NRG1/ErbB4 signaling, which co-tracks a reduction in excitatory input to PV cells, resulting in sustained cortical disinhibition in mPFC. This effect is seen in vivo and in vitro, and is consistent with the proposed NMDAR inhibition-independent mechanism of ketamine’s actions9.

Ketamine-mediated antidepressant effects in the FST depend on PV NRG1/ErbB4 signaling, providing a molecular and circuit mechanism for ketamine’s rapid and sustained effects on cortical plasticity.

Animals

To label PV cells and analyze mRNA expression, PV-IRES-Cre mice were crossed to fsTRAP mice, and Emx1-Cre mice were crossed to fsTRAP mice. PV-Cre mice were hemizygous for both transgenes, and Ai9 tdTomato mice were used to genetically label PV cells.

We randomly assigned mice to groups with treatment of either saline or subanesthetic ketamine. We used the dosage of HNK reported in the published study9 for our in vivo HNK treatment, and we used recombinant NRG1 to treat some mice.

Forced swim test

Mice were habituated in the behavior room for 1 h before testing, and then forced to swim in a transparent glass cylinder at 23 – 25 °C. Their immobility time was recorded for 4 min.

Miniscope imaging experiments

At 2 weeks after AAV1-CaMKII-GCaMP6f injection, a GRIN lens was implanted in the mPFC. A miniscope was fitted into the baseplate and the field of view was in focus to visualize GCaMP6f-expressing neurons and visible landmarks, such as blood vessels.

Mice were habituated to a behavioral arena for 2 days, then 3 days to a behavioral arena with a miniscope fixed onto their head. Then GCaMP6-based calcium imaging of population mPFC neurons was performed in awake freely behaving mice.

The custom-constructed miniscope has a mass of 3 g, uses a single, flexible coaxial cable, and transmits data over SuperSpeed USB to a PC running custom DAQ software. The software simultaneously records the behavior of an animal through a high-definition webcam.

Miniscope videos were first concatenated, downsampled by a factor of two, and then motion-corrected using the NoRMCorre MATLAB package. Then, the calcium activity of individual neurons was extracted using the newly developed method of extended constrained non-negative matrix factorization for endoscopic data (CNMF-E).

The deconvolved calcium event activity of each neuron was obtained using OASIS57 and SCOUT58. The amplitudes of the calcium events were calculated using the calcium peak values and the integrated area under the calcium event signal.

Translating ribosome affinity purification (TRAP)

Polysomal mRNA was purified from prefrontal cortical lysate by using a brain block and scalpel. Pooled cortex from 2 to 5 mice was grinded to powder on dry ice, sonicated for 5 s in ice-cold lysis buffer, and then centrifuged for 10 min at 20,000 g to pellet insoluble material. Two mouse monoclonal anti-GFP antibodies were then added to the cell-lysate supernatant, and the mixture was incubated overnight. RNA was eluted from beads and purified using RNeasy Micro Kit (Qiagen) per the manufacturer’s instructions.

Quantitative Real-Time Polymerse Chain Reaction (qPCR)

Purified RNA was converted to cDNA using Superscript® III reverse transcriptase, and quantitative changes in cDNA levels were determined by real-time PCR using the Power SYBR Green Master Mix. The expression of mouse NRG1, ErbB4, and GAPDH were determined using primers at 500 nM.

Immunohistochemistry

Animals were deeply anesthetized with Uthasol and perfused with PBS containing 4% paraformaldehyde and phosphatase inhibitor. Coronal sections of the brain were taken and stained with immunohistochemical staining and analysis.

Free floating sections of brain were rinsed five times with 1 PBS, incubated in a blocking solution for 1 h at room temperature on a shaker, and then incubated with a primary antibody diluted in blocking solution for 36 h at 4 °C.

Immunostained sections were examined using a confocal microscope, and image tiles, overlaying, maximum projections, and subset z-stack selections were performed using the Zeiss image processing software.

Individual cell fluorescence measurements are performed in final output images using Adobe Photoshop software, and the corrected total fluorescence per cell is calculated in an Excel sheet by applying the measurements obtained from the analyzed cell with the formula: Corrected total cell fluorescence = Integrated density / mean fluorescence of background reading.

Electrophysiology and laser-scanning photostimulation

Coronal sections of mouse mPFC were cut with a vibratome and incubated in sucrose-containing ACSF for 30 min before being transferred into slice-recording chambers with standard ACSF.

We used a differential interference contrast/fluorescent Olympus microscope to record whole-cell potentials from L2/3 PV interneurons and pyramidal neurons within the mPFC. The recordings were made in oxygenated ACSF at room temperature with patch pipettes filled with an internal solution containing 126 mM K-gluconate.

Data were recorded using data acquisition boards, a custom-modified version of Ephus software 5, and a cesium-based internal solution. IPSCs were measured in L2/3 excitatory pyramidal neurons by preferentially activating L5 to L2/3 feedforward projections to L2/3 inhibitory neurons.

During photostimulation experiments, a laser unit was used to deliver 355 nm UV laser pulses for glutamate uncaging. A high-resolution digital CCD camera was used to visualize the cortical slice image and guide and register photostimulation sites.

A standard stimulus grid was used to tessellate mPFC from pia to white matter, and glutamate uncaging laser pulses were delivered sequentially in a nonraster, nonrandom sequence. EPSCs and IPSCs were isolated by voltage clamping PV and pyramidal cells at 70 mV.

Direct responses are excluded from local synaptic input analysis, but are used to assess glutamate-mediated excitability/responsiveness of recorded neurons. Synaptically mediated responses override the relatively small direct responses at some locations, and are identified and included in synaptic input analysis. LSPS evoked EPSCs are quantified across the 16 x 16 mapping grid for each cell, and 2 – 4 individual maps are averaged per recorded cell. The total synaptic input strength is measured for individual cells, and is then plotted as the average integrated amplitude per pixel location. For experiments with NRG1, MK-801, ketamine or HNK, the reagent(s) were added into the ACSF solution with the specified concentrations.

Statistical analysis

Data analysis was conducted using Matlab and R. Several appropriate statistical tests were applied, including ANOVA, t-test, Kruskal-Wallis test, and Friedman’s test.

Linear mixed effect (LME) model

We used the LME model to analyze calcium imaging data from 24 mice. The model had a random effect variable called mouse IDs, and we fit an LME with nested random effects.

Normality and nonparametric tests

When normality does not hold or sample sizes are small, parametric results might not be accurate. We verified the results using nonparametric tests, and the trends and conclusions remain valid.

Multiple comparisons

When there are more than one hypothesis tests, the Bonferroni correction is used to control familywise type I error rates.