Supplementary information.
Selective control of fear expression by optogenetic manipulation of infralimbic cortex after extinction
Hyung-Su Kim1,2, Hye-Yeon Cho1, George J. Augustine2-4, Jin-Hee Han1
Contents:
Supplementary Materials and Methods
Supplementary Results
Supplementary Figures
Supplementary References
Supplementary Materials and Methods
Electrophysiology
For electrophysiological measurement of eNpHR3.0 or ChR2 properties in slice, mice were sacrificed and brains were sliced using a vibratome (VT-1200S, Leica, Germany) in ice-cold sucrose solution containing (in mM): 87 NaCl, 2.5KCl, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 d(+)-glucose, and 75 sucrose, bubbled with 95% O2 and 5% CO2. Prepared brain slices were transferred to an incubation chamber filled with oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 d(+)-glucose, 3 Na-pyruvate, 1 ascorbic acid and 2 kynurenic acid, included to eliminate excitatory synaptic. The patch electrode was filled with an internal solution containing (in mM): 130K-gluconate, 2 NaCl, 4 MgCl2, 20 HEPES, 4 Na2ATP, 0.4 Na3GTP, 0.5 EGTA, and 10 Na2Phosphocreatine (pH 7.25; 290–295 mOsm). Recordings were amplified using a Multiclamp 700B amplifier (Molecular Devices, CA, USA) and digitized at 20 kHz using an A–D converter (Digidata 1440A, Molecular Devices). Electrical responses were detected with pClamp software (Molecular Devices) and analyzed using Clampfit software (Molecular Devices).A mercury arc lamp was used to activate ChR2 (470–495 nm) and eNpHR3.0 (530–550 nm). Light stimuli were delivered through a water-immersion objective (60×). Pulse duration was controlled by an electronic shutter (Uniblitz VS25,Vincent,NY, USA).
Immunohistochemistry
For immunohistochemistry, brain sections (40-µm thick) were washed with PBS and incubated with primary antibody against calcium/calmodulin-dependent protein kinase IICaMKIIα) protein (1:1,000, 05-532, Millipore, Germany) or glutamic acid decarboxylase67 (GAD67) protein (1:1,000, MAB5406, Millipore, Germany) for 72 h or 48 h at 4 °C. Alexa Flour 594-conjugated goat anti-mouse antibody (1:1,000, A-11005, Invitrogen, NY, USA) was used as a secondary antibody. Immunohistochemical signals were visualized and image obtained using a Zeiss LSM780 upright confocal laser-scanning microscope.
For cell counting analysis to determine the proportion of CaMKIIα- or GAD67-positive cells in AAV-hSyn-eNpHR3.0-EYFP injected mice, CaMKIIα- or GAD67-positive cells were quantified by manually counting in a blinded manner. Three brain sections corresponding to 1.94, 1.70, and 1.54 mm anterior from bregma from each animal were selected for the analysis (n = 7 mice). The percentage of eNpHR3.0 and CaMKIIα or GAD67 double labeled cells in a given IL section was obtained by calculating the ratio of the number of double positive cells to the total number of eNpHR3.0-EYFP expressing cells.
The number of excluded animals in each group
Animals with no or low virus infection, off-target infection or physical damage were excluded in the data analysis. The number of excluded animals in each group was reported in the following: : eNpHR3.0 (n=5) and EGFP (n=5), Figure 1h,i,j; eNpHR3.0 (n=1) and EGFP (n=3), Figure 1k; eNpHR (n=3) and EGFP (n=1), Figure 2c, d; ChR2 (n=1) and EGFP (n=0), Figure 3h, i; ChR2 (n=1) and EGFP (n=0), Figure 4b; ChR2 (n=3) and EGFP (n=1), Figure 4d; ChR2 (n=2) and EGFP (n=0), Figure 4e; ChR2 (n=1) and EGFP (n=0), Figure 4f; ChR2 (n=1) and EGFP (n=0), Figure 5.
The rationale for optogenetic manipulation protocols
Unilateral manipulation: The main reason we used unilateral manipulation was to minimize potential damages to the brain regions surrounding IL due to surgical processes such as cannula implantation, virus injection and optic fiber insertion etc. which would compromise data interpretation. Indeed, some previous studies also used unilateral manipulation due to the same reason (Milad and Quirk, 2002; Milad et al, 2004).
Expression of eNpHR3.0-EYFP using hSyn promoter: The in vivo recording or imaging of neuronal activity established that IL activity is increased during extinction retrieval (Knapska and Maren, 2009;Milad and Quirk, 2002;Phelps et al, 2004). However, to our knowledge, it has not been clearly specified exactly which types of neurons in the IL are activated and how different types of neurons in the IL contribute to the expression of fear extinction. In line with this, the possible contribution of interneurons in mPFC, although it is not specifically in the IL, in regulating fear expression and extinction has been suggested in the previous studies (Bissonette et al, 2014; Courtin et al, 2014). Thus, we reasoned that targeting neurons using a pan-neuronal promoter such as hSyn promoter should be the first step for optogenetic inhibition experiment to determine whether IL activity is necessary for the expression of fear extinction.
Stimulating during the ISI when activating eNpHR3.0 but not ChR2: In eNpHR3.0 manipulation, we attempted to silence IL activity as complete as possible during extinction retrieval process. Since it was unclear whether IL activity lasted even after tone offset in our condition, we continuously delivered light during extinction retrieval process including the ISI periods. We also confirmed that eNpHR3.0 stimulation alone in the absence of conditioned tone did not evoke freezing behavior as presented in Figure 1j. For IL activation scheme, previous studies using electrical stimulation method (Milad and Quirk, 2002; Milad et al, 2004) showed that activating IL during the tone is efficient to produce behavioral effects such as enhanced extinction. Thus, based on these previous reports, we tried to stimulate IL activity during the tone presentation in our optogenetic setup.
Supplementary Results
Specificity of optogenetic manipulation of IL
To determine the specificity of viral expression, we also measured the percentage area in PL that showed fluorescence signal of eNpHR3.0-EYFP in Figure 1h, i. For this purpose, we analyzed three brain sections from each animal (total 9 animals) where strong NpHR signal was detected in the IL. Imaging analysis showed that fluorescence signal was detected in approximately 2% of PL areas on average (2.22±0.97%, n=9) and in general the signals in the PL were relatively very weak compared to that in the main injection site in the IL. Fluorescence could reflect either opsin-expressing cell bodies of PL neurons or the presence of neuronal processes in the IL that project or extend into PL (distinct from viral leak causing inhibition of PL neurons) so we tried to count the number of eNpHR3.0-EYFP-expressing cell bodies in the PL to distinguish these two cases, although it was technically difficult to clearly distinguish signal in the projections fromthat in the cell body membranes because the fluorescence signals in the PL was generally too weak for this purpose. We barely detected fluorescence signal patterns that reflect opsin expression in the cell body membrane of PL neurons, suggesting that probably most fluorescence signals detected in the PL may reflect the presence of neuronal processes in IL that project or extend into PL. Therefore, these results further support that our manipulation was specific to the IL.
Supplementary Figures
Supplementary figure 1The proportion of glutamatergic or GABAergic neurons expressing eNpHR3.0-EYFP under hSyn promoter in the IL.(a) Representative confocal microscopic images showing expression of eNpHR3.0-EYFP under hSyn promoter in CaMKII-positive neurons in the IL. (b) Representative confocal microscopic images showing expression of eNpHR3.0-EYFP under hSyn promoter in GAD67-positive neurons in the IL. (c) Quantification of the percentage numbers of double positive neurons in eNpHR3.0 expressing neurons. Data in c is expressed as means ± s.e.m.
Supplementary figure 2 Extinction procedure successfully reduced conditioned freezing during extinction retrieval. (a) The percentage of freezing to the tone CS during auditory fear conditioning, extinction training, and extinction memory expression. A single trial block in extinction training consisted of four sessions of tone presentation (30 s each). Freezing level for each trial block was determined by averaging freezing levels from these four sessions. Mice injected with EGFP alone were randomly divided into two groups after fear conditioning: Extinction group (Ext; n = 9) and No-extinction group (No-ext; n = 6). *p<0.05 (Student’s t test). (b) The percentage of freezing during the first extinction trial block on day 2 and test session on day 4. Mice in the Ext group, but not those in the No-ext group, showed substantially reduced freezing during extinction memory tests compared with the first trial block during extinction training. ** p<0.01 (Student’s t test). Data in a,b are expressed as means ± s.e.m.n.s. = not statistically significant.x, baseline (pre-CS) freezing levels.
Supplementary figure 3Photoinactivation of CaMKIIα-positive neurons in the IL had no effect on expression of fear extinction. (a) Representative confocal microscopic images showing selective expression of eNpHR3.0 in CaMKII-positive neurons in the IL. (b)Experimental procedure. Mice expressing either eNpHR3.0 or control EGFP under CaMKIIα promoterin the IL were trained for auditory fear conditioning and subsequently administered extinction training. Green light (561 nm) was delivered to inactivate IL activity. (c) The percentage of freezing during auditory fear conditioning (FC) and extinction training procedure (Ext). (d) The percentage of freezing during extinction retrieval. Mice from CaMKIIα-eNpHR3.0 and control CaMKIIα-EGFP groups displayed no significant differences in freezing throughout tone presentations, even with light illumination (CaMKIIα-EGFP, n = 9; CaMKIIα-eNpHR3.0, n = 8). Data in c, d are expressed as means ± s.e.m.x, baseline (pre-CS) freezing levels.
Supplementary figure 4Light stimulation conditions used for behavior experimentsevoked reliable action potential firing in ChR2 expressing neurons in IL. (a) Representative recording traces (black line) from slice patch recording of ChR2 expressing IL neurons. Light stimulation patterns (top, 10 Hz/20 ms pulse duration; bottom, 20 Hz/10 ms pulse duration) are also shown (blue line). (b) Mean probability of evoked action potentials by two light stimulation conditions (n = 15 for each condition). Data in b are expressed as mean ± s.e.m.
Supplementary References
Bissonette GB, Bae MH, Suresh T, Jaffe DE, Powell EM (2014). Prefrontal cognitive deficits in mice with altered cerebral cortical GABAergic interneurons.Behav Brain Res259: 143–151.
Courtin J, Chaudun F, Rozeske RR, Karalis N, Gonzalez-Campo C, Wurtz Het al(2014). Prefrontal parvalbumin interneurons shape neuronal activity to drive fear expression.Nature505: 92–96.
Knapska E, Maren S (2009). Reciprocal patterns of c-Fos expression in the medial prefrontal cortex and amygdala after extinction and renewal of conditioned fear.Learn Mem16: 486–493.
Milad MR, Quirk GJ (2002). Neurons in medial prefrontal cortex signal memory for fear extinction. Nature420: 70–74.
Milad MR, Vidal-Gonzalez I, Quirk GJ (2004). Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally specific manner. Behav Neurosci118: 389–394.
Phelps EA, Delgado MR, Nearing KI, LeDoux JE (2004). Extinction learning in humans: role of the amygdala and vmPFC. Neuron43: 897–905.