Cell-type-specific optogenetic stimulation of the locus coeruleus induces slow-onset potentiation and enhances everyday memory in rats

Significance Other events happening around the time a memory is encoded can modulate how well such a memory is retained. The significance of this study is in revealing that environmental novelty either before or after memory encoding can enhance the retention of allocentric spatial memory, and this can be mimicked by optogenetic light activation of a brain area involved in neural activity in response to novelty. This intriguing perievent neural modulation is also associated with an optogenetically induced slow-onset potentiation of synaptic strength in the hippocampus which likely contributes to the enhancement of memory retention.


In vivo optrode recordings
Three to four weeks before the optrode recordings Th-Cre rats were injected bilaterally in the LC with the Cre-inducible AAV-DJ ChR + virus (ChR + , n = 3).Once the injection was completed and the syringe removed from the brain, cerebrospinal fluid (CSF) liquid and blood residue were absorbed with sterile paper and the holes in the skull were covered with a sterile sponge.On the day of the optrode recording the rat was anesthetized with urethane [ethyl carbamate (Sigma-Aldrich), 1.5 g/kg of body weight, intraperitoneal (IP) injection] and placed in a stereotaxic frame, the skull was exposed and the sponge was removed.A parylene-coated tungsten microelectrode (diameter = 127 µm, AC resistance = 1.0 MΩ) was glued to a custom SMA optical patch cable (optic fiber diameter = 400 µM, numerical aperture = 0.5; Thorlabs): the microelectrode protruded 100 µm from the end of the fiber, and the whole assembly is referred to as an 'optrode'.Before each experiment the integrity of the optic fiber was checked and the LED driver calibrated to evoke consistent power across experiments using a Slim Photodiode Power Sensor (Thorlabs).The optrode was then placed in a stereotaxic arm with 20-degree angle in the AP direction.From a starting position 3.1 mm posterior and 1.2 mm lateral to lambda at the dura surface, the optrode was positioned above the LC (DV, -2.8 mm), then gradually lowered in 50 μm increments till -6.6 mm.
Multi-unit spiking activity was observed in the continuous wide-band local field potential (LFP) sampled and recorded via a USB-6003 acquisition device (National Instruments) connected to a PC.Light stimulation at power levels ranging from 3 to 13 mW was delivered using a Fiber-Coupled LED (M625F2 -625 nm, 13.2 mW (min), 1000 mA, SMA; Thorlabs).
Optical stimulation and multi-unit recording were controlled by OG2, a custom-written LabVIEW program (National Instruments) developed by P.S.

In-vivo electrophysiology combined with optogenetic stimulation
Three to four weeks prior the electrophysiological recordings, Th-Cre rats were injected with either Cre-inducible AAV-DJ ChR + or ChR -virus bilaterally in the LC as described above.
Once the injection was completed and the syringe removed from the brain, cerebrospinal fluid (CSF) and blood residue were absorbed with sterile paper and the holes in the skull were covered with sterile sponge.After the surgery Rimadyl (carprofen, 0.08 ml/kg of body weight) was administered by subcutaneous injection.All rats were monitored for 7 days for them to regain their pre-surgery weights.
On the day of the electrophysiological recording rats were anaesthetized with urethane 1.5 g/kg (as described above) and placed in a stereotaxic frame with the skull horizontal.
Body temperature was monitored by a rectal probe and maintained at 36.2 °C using a homeothermic heating blanket.Depth of anesthesia was assessed throughout the experiment, and urethane top-ups of 0.2 ml were administered as required.Subcutaneous injections of a glucose/saline mixture were administered every 3 h to maintain hydration (1.5 ml of 0.9% saline + 0.5 ml of 5% glucose).A fiber-optic cannula (MFC_400/430-0.48NA_9mm_SM3_FLT Fiber-optic Cannula, Doric lenses) was screwed to a fiber-optic patch cord (MFP_400/460/ARMO-0.48_0.8m_SMA-CM3Mono Fiber-optic Patch Cord, Doric lenses) and all connected to a Fiber-Coupled LED (M625F2 -625 nm) controlled by an optic driver (LEDD1B).Prior the acute implantation of the fiber-optic cannulae a power test was performed to ensure the integrity of both the cannulae and the patchcord and to calibrate the optic driver to deliver 10 mW.The sponges previously placed in the skull during viral injection were removed to allow acute implantation of fiber-optic cannula above the LC (unilateral).A small burr-hole was made in the skull above the dorsal hippocampus, and a polytetrafluoroethylene-insulated monopolar platinum/iridium recording electrode (external diameter = 0.103 mm) was lowered into the stratum radiatum of area CA1 (coordinates from bregma: AP, -3.8 mm; ML, -2.5 mm; DV from the dura, ~-2.5 mm).A bipolar stimulating electrode comprising two twisted wires identical in composition to the recording electrode was lowered into CA3 (coordinates from bregma: AP, -3.5 mm; ML, -3.0 mm; DV from the dura, ~−3.0 mm) in order to activate the Schaffer collateral input to CA1.Recordings were referenced to a skull screw positioned on the left parietal bone.
Stimulation and recording were controlled by a PC running custom-written LabView software (Evoked Potential Sampler, P.S.) for the control of electrical stimulation and the time-locked recording and online analysis of evoked field excitatory postsynaptic potentials (fEPSPs).Stimulation comprised biphasic constant-current pulses delivered via a pair of stimulus isolators (NL800A, Digitimer).fEPSPs were amplified and filtered (high pass = 1 Hz, low pass = 5 kHz) using a differential AC amplifier (Model 1700, A-M Systems), and sampled at 20 kHz using a PC data acquisition card (PCIe-6321, National Instruments).The slope of the early rising phase of each fEPSP (measured by linear regression between two fixed time points) and the peak amplitude were recorded.
At the start of each experiment, electrodes were lowered into the hippocampus: correct placement was determined based on the characteristic depth profiles observed during implantation (10), and final depths were adjusted to maximize the amplitude of the negativegoing apical dendritic fEPSP elicited in CA1 by stimulation of CA3.Stimulation current was adjusted to evoke a fEPSP with a slope value of circa 50% of the maximum value obtained during an initial input-output curve.
After electrode placement, the fiber-optic cannula was lowered above the ipsilateral LC (identical coordinates to those described above).Baseline recording then began; single biphasic test pulses (50-μs pulse width per phase) were delivered at 2-min intervals.After recording a 1-h stable baseline, optical stimulation was delivered to LC-TH + neurons, comprising 20 pulses at 25 Hz delivered every 5 s for a total duration of 300 s, i.e. 60 trains in total.Recording continued for at least 3 h after optical stimulation.fEPSP slope data were normalized to the mean of the 1-h baseline period (assigned a value of 100%), and group means were calculated.

Drugs associated with in-vivo electrophysiology
All drugs were dissolved in sterile saline and stored in aliquots at -20 °C before use.

Histology
Rats were terminally anaesthetised with sodium pentobarbital (Euthatal, 100 mg/kg of body weight, IP) and perfused transcardially with 4% paraformaldehyde (PFA) dissolved in phosphate buffered saline (PBS), pH 7.2.Brains were removed and stored in 4% PFA overnight at 4 °C, transferred to 30% sucrose in PBS for 2 days and then kept at -80 °C.
Coronal sections (60 μm) were cut using a cryostat for histological analysis.
To check the position of each bilateral drug cannula in the dorsal hippocampus (n = 10), sections were then mounted on slides, stained with cresyl violet and coverslipped using mounting media DPX.The sections were examined using a light microscope under 20-fold magnification to verify the drug cannula placements.These locations were then marked on the appropriate coronal sections from the Paxinos and Watson (2007) brain atlas (72).
To check the viral expression and optic cannula position in the LC (n = 14), coronal sections were washed in PBS, permeabilised with 10% normal donkey serum (Sigma-Aldrich) and 0.5% Triton X-100 in PBS.They were then incubated overnight at room temperature with a monoclonal mouse anti-TH antibody (1:1000 dilution; 22941, Immunostar) and a polyclonal rabbit anti-RFP antibody (1:200 dilution; ab62341, Abcam).Sections were then washed and incubated with secondary antibodies (1:200 dilution; donkey anti-mouse 647 and donkey anti-rabbit 488).After washing, slices were mounted in Fluoroshield with DAPI (Sigma-Aldrich).The deepest optic cannula position in the LC on the stained sections was located under a fluorescent microscope with 20-fold magnification and measured using Image-Pro (Media Cybernetics).

Statistical analysis
Statistical analyses were performed using IMB SPSS Statistics 28 (IMB) and Prism (GraphPad).All data were analyzed by ANOVA followed, where appropriate, by post-hoc pairwise comparisons (Tukey's HSD), or by independent-sample, paired-sample, or onesample t tests as appropriate.Data are reported as mean ± SEM.

Figures S1 Total dig time across all probe trials (PTs).
The absolute total dig �me was c. 30 -40 s in early PTs (1-3).There was a trend towards slightly less digging in the correct loca�on on later probe trials (PT 4-5).However, there were no significant differences between condi�ons in any of the PTs (F < 1, n.s).

Figure S5
Slow-onset potentiation The data was averaged over 1-h �me periods rela�ve to the onset of light s�mula�on and normalized to the 1-h baseline period: supplementary analysis of the data presented in Fig. 3.Note that the baseline 0-1 h before light s�mula�on is always 100% by defini�on.(A) Slow-onset poten�a�on induced in the TH-Cre ChR+ light-ON group (n = 9) rela�ve to the three control groups (TH-Cre ChR-light-ON; n = 4, TH-Cre ChR+ light-OFF; n = 4, and WT with no virus and no cannula; n = 6).A repeated-measures ANOVA in which treatment group was entered as a between-subjects factor, and �me a�er op�cal s�mula�on (0-1 h, 1-2 h, and 2-3 h) was entered as a within-subjects factor, revealed a significant main effect of group [F = 15.5, df = 3/19, p < 0.001] and a significant group x �me interac�on [F = 6.72, df = 6/38, p < 0.001].Post-hoc pairwise comparisons with Bonferroni correc�on revealed significant differences between the TH-Cre ChR+ light-ON group and both other groups 1-2 h a�er light s�mula�on (***p < = 0.001 in all cases), and 2-3 h a�er s�mula�on (**p < 0.01; *** p < 0.001).
Figure S1 Total Dig time

Figures
Figure S2

Figure S3 .
Figure S3.LC projections to dorsal hippocampus.(A) Representa�ve image of TdTomato+ posi�ve fibers in the dorsal hippocampus (red).The image is representa�ve of 4 animals analysed.Scale bar, 100µm.(B) Representa�ve image of TH projec�ons to the dorsal hippocampus stained with the an�-TH Millipore an�body (see Methods) (green).Nuclei are stained with DAPI (blue).The image is representa�ve of 3 animals analysed.(C) immunostaining of hippocampal sliced from animals expressing TdTomato in LC neurons.From le� to right, in red an�-RFP (TdTomato) signal, in green an�-TH staining, in blue an�-NET staining, and merge.Scale bar, 50µm.On the right, quan�fica�on of the frac�on of double posi�ve TH/NET fibers out of the total TdTomato+ fibers, n=5 animals.