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“Negative allosteric modulation of mGlu7 disrupts fear memory reconsolidation and glutamatergic signaling in rat and human brain tissue”

Authors: Alexandru Cristian Ciobanu1, David Mota Caseiro1, Ruifang Niu1, Rodrigo Triana del Rio1, Cédric Leroux1, Alessio Stefanelli1, Carmen Flores Nakandakare1, Etienne Pralong2, Roy T. Daniel2, Robert Lütjens3, Erwin H. van den Burg1*, Ron Stoop1*

Affiliations:
1Center for Psychiatric Neurosciences, Lausanne University Hospital Center (CHUV), Prilly-Lausanne, Switzerland.
2Section of Neurosurgery, Department of Clinical Neurosciences, Lausanne University Hospital Center (CHUV), Lausanne, Switzerland.
3Addex Therapeutics, Chemin des Mines 9, Campus Biotech, CH-1202, Geneva, Switzerland.

This dataset presents raw data in Excel files and video files that have been used to compose the above-mentioned manuscript. It includes data from behavioral studies and in-vitro patch clamp studies. Combined, the data have led to the conclusion that a novel negative allosteric modulator of metabotropic glutamate receptor 7 can impair the reconsolidation of fear memories in rats, and that it modulates glutamatergic signaling in thalamus-to-lateral amygdala synapses through presynaptic action. The modulator is named ADX71743, and this name is used in all the files included in the dataset.

Description of file content:
Ciobanu_Fig1.xlsx
This file provides the raw data of fear-related behavior (which is freezing behavior) of individual rats indicated by number that have been fear conditioned to a tone of 5 kHz. These rats have been canulated, targeting the lateral amygdala. The first tab gives the percentage of freezing during the acquisition of fear memory. The learning consists of pairing the tone (indicated by CS, short for Conditioned Stimulus) with a mild electric shock of 0.5 mA (indicated by US, short for Unconditioned Stimulus) to the feet of the animals. The group of animals that will receive vehicle (control) solution or ADX71743 later in the paradigm are already separated. Hab is Habituation, the time the animals spend in the fear conditioning chamber before the learning procedure starts.
The second tab shows the freezing levels in percentage of time during habituation (Hab) and fear memory recall (CS). After the recall, the animals were either infused with vehicle solution (control) or ADX71743, as detailed in the manuscript.
The third tab shows the freezing levels in percentage of time during habituation (Hab) and the five presentations of the tone three days later in a fear memory retention test. Animals have again been divided into the two experimental groups (vehicle controls and ADX71743-treated animals).
These data are the basis of Figure 1 of the manuscript.

Ciobanu_FigS1.xlsx
These data are similar to those for Fig.1 described above, but details freezing levels for two animals in which the cannulas had been placed outside the lateral amygdala. In these animals, ADX71743 treatment was without effect.

Ciobanu_FigS2.xlsx
These data support a similar fear conditioning experiment as in Fig.1 described above, but in this case the fear conditioning was to two different tones, which were 5 kHz and 15 kHz. The first tab of the excel file shows the fear learning to both tones.
The second tab concerns freezing levels during the recall of fear memory of cannulated (in the LA) rats, subdivided over two groups. The first group was exposed to the 5 kHz tone, followed by ADX71743 injection into the LA on the day after learning (Indicated by Day 2). One day later (Indicated by Day 3), these rats were exposed to the 15 kHz tone, and received vehicle solution. For the second group of rats, this is the reverse.
The third tab (recall per day and treatment) are the same data but sorted differently to make clear how the figure panels have been created.
The fourth tab (Fear retention per CS) shows the rats’ freezing levels when exposed to the 5 kHz and 15 kHz tone during the fear memory retention test.
The fifth and final tab (fear retention average) shows for each rat the freezing levels during the retention test, averaged across the five tone presentations for both tones separately.
Also indicated is that rat number 3 had misplaced cannulas (i.e. outside the LA), and here ADX71743 after the 5 kHz tone was without effect. This rat is still shown in the figure, but has been excluded from the statistical analyses.

Ciobanu_FigS3.xlsx
The data presented over the three tabs follow a similar structure as the for those that are described in Ciobanu_Fig1.xlsx. The difference is that in S3, the animals received ADX71743 4h after fear memory recall, instead of after 10 min after fear memory recall in Fig.1.
On the fear retention tab, two additional rats have been included that had received ADX71743 10 min after fear recall on day 2. The values here represent the averaged freezing levels across the five tone (CS) presentations to compare the effects of time in one experiment (in addition to comparing with the freezing levels of rats during the retention test presented in figure 1).

Ciobanu_Fig2.xlsx
The data presented here reflect freezing levels of rats that had received vehicle control solution or ADX71743 subcutaneously in an otherwise similar fear conditioning experiment.
The first tab (Fear learning) gives the freezing levels in percentage of time during the fear memory acquisition step of the protocol. Rats have been divided over four groups: Vehicle + Recall, ADX71743 + Recall, Vehicle – Recall, ADX71743 – Recall (Column A).
The second tab (Fear recall) presents the freezing levels in percentage of time during the fear memory recall (CS).
The third tab (Fear retention) gives the freezing levels in percentage of time for each rat during the fear memory retention test.
The fourth tab (Fear reinstatement) gives the freezing levels in percentage of time for each rat that had received an additional foot shock on Day 11, and have been exposed to the CS on Day 12. This was to assess whether the fear memory can be reactivated after fear memory had been disrupted by ADX71743 + recall (as explained in detail in the manuscript).

Ciobanu_FigS4.xlsx
The data reflect freezing levels as a percentage of time of rats that had received either vehicle control (subcutaneously), ADX71743 (subcutaneously) or propranolol (intraperitoneally) on day 2 after fear memory recall. Conventions as for the other files.

Ciobanu_Fig3.xlsx
The Excel file contains two tabs, one marked Frequency and the other Amplitude. The frequency tab shows the frequency of spontaneous excitatory postsynaptic currents (EPSC; in pA) in the lateral amygdala of male rats before, during and after ADX71743 wash-in. ADX71743 (500 nM) started at 300 seconds and ended at 660 seconds. Frequencies were calculated in bins of 20 or 30 seconds. The amplitude tab shows the amplitudes of the EPSCs on the first tab.

Ciobanu_Fig4.xlsx
These data are electrically (first tab) optogenetically (second tab) evoked EPSCs in pA, by stimulating descending fibers from the thalamic Medial Geniculate Nucleus and recorded in lateral amygdala neurons, and in the presence of ADX71743 (500 nM). Data is raw data before, during and after ADX71743 application (first block), and normalized and aligned to onset of treatment or maximum response (blocks 2 – 4 to the right in the spreadsheet).

Ciobanu_Fig5.xlsx
Three examples are given for the induction of LTP in the presence or absence of ADX71743. The first few lines per cell are baseline recordings of excitatory postsynaptic potentials (EPSP) amplitude, expressed as the slope of their rising phase (percentage of baseline). The next series of empty lines reflects the time of the LTP induction protocol, and the final set of lines is EPSP amplitude after the induction protocol.

Ciobanu_Fig6.xlsx
Three examples of recordings of spontaneous EPSCs in neurons in human brain tissue are given during the period before, during and after ADX71743 (500 nM) bath application. The application starts at 310 seconds and lasts until 670 seconds. Frequency (in Hz) and amplitudes (in pA) are given on separate tabs. CTX are cortical cells, and AMY is an amygdala cell.

Cannula implants sites.pptx
This file gives five examples of cannula implantations. The first slide is a schematic taken from the Paxinos brain atlas to show where the lateral amygdala (LA), the target site, is situated. The next slides show an example of implantation sites of five rats. In four of these rats, the cannulas had been properly inserted into the LA, as visualized by an image and a schematic of the brain for each rat. The rat presented in example 2 shows a cannula placement outside the LA. This was the rat presented in Supplementary Figure 2, for which we have indicated that the cannula implantation sites were incorrect and left out of the statistical analysis.

Sous cut Veh.mp4 
This is a video of a rat that had received vehicle solution on Day 2, 10 min after fear memory recall. The video shows the readout of the test, which is the fear memory retention test on Day 5. The video starts with a recording of behavior during the Habituation period during which the animal is allowed to freely explore its unfamiliar environment. At time t = 18 seconds, the five CS presentations start, and the animal displays freezing behavior in response to each CS.

Sous cut ADX.mp4
This is an example video recording of a rat that received ADX71743 (100 mg/kg bodyweight, 1 ml) subcutaneously 10 min after fear memory recall on Day 2, and tested for fear memory retention on Day 5. After the Habituation phase, five CS are presented, starting at t = 23 seconds. The animal hardly freezes in response to the CS presentations.
