LC Paper Flashcards
What’s the overall research question?
What are the neuromodulatory neurons that promote the consolidation of hippocampal-dependent memory by firing after encoding?
What is the overarching motivation for the experiments described in this paper? What specific
question do the authors want to address?
Flashbulb memory
–> Initial retention when something novel or categorically relevant happens shortly before or after time of memory encoding
What neurons mediate the dopamine novelty effect?
Past studies demonstrated the dopamine-dependent novelty effect originating in TH+ neurons in the ventral tegmental area (VTA).
Studies have suggested from long term potentiation and depression support the concept of dopamine dependent enhancement of persistence.
TH+ neurons in the locus coeruleus shown to occasionally release dopamine and noradrenaline -> so researchers investigated these neurons as well.
VTA-TH+ neurons
Chosen because linkage between VTA and hippocampus. Dopaminergic.
LC-TH+
TH+ neurons in the locus coeruleus shown to occasionally release dopamine and noradrenaline -> so researchers investigated these neurons as well
Th-Cre mice –> methods
120 Th-Cre mice -> express Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter, allowing for specific labeling and manipulation of TH+ neurons.
TH+
TH+ is a marker for dopaminergic neurons - so this is how they can confirm that they are specifically looking at dopaminergic neurons (and they care because novelty effect is dopamine-dependent).
Experimental approach
‘Everyday’ or episodic-like memory task - placed mice in a small arena and allowed them to search for a food reward in sandwells that changed location across days
Changing of the reward’s daily location was encoded during ‘sample trials’ and displayed in ‘choice trials’ where the mice had to detect which sandwell(out of 5) had the reward
A control test ensured that there were no confounding factors related to the sense of smell
–> Mice were presented with a series of sample trials, where they were given either low or high reward
–> The effectiveness of their memory was then tested using occasional probe test, which sometimes followed the sample trials at different intervals
–> Results: even the smallest reward had a positive effect on memory at 1 hr after the sample trial, indicating that reward availability during memory encoding improves memory retention. Also, forgetting across 24 hours had an inverse relationship with reward magnitude - larger the reward, slower the rate of forgetting. So, higher rewards led to better long-term memory.
Novelty probe - exploration of a box with an unfamiliar floor surface placed inside the event arena 30 min after the encoding trial
What question(s) is the figure trying to answer?
–> Do mice experience enhanced memory encoding due to novelty (environmental)?
What methods are used in the experiments in the figure?
–> Using an arena with different configurations of sandwells, the experimenters put the mice through trials with one rewarded sandwell (sample trials), one rewarded and four unrewarded sandwells (choice trials) and then only unrewarded (initially) sandwells for memory probe tests to see if memory encoding in mice has occurred.
–> There were variations in size of rewards, variability in availability of rewards, and also the introduction of a novel floor surface to test the effect of novelty on post-encoding memory enhancement (which was tested with memory probe test configuration).
1A:
Experimental design, sample + choice trials.
Graph of 100 training sessions
Initial training builds up
Novelty wears off
Drop at non-encoding control session performance (59.6%)
1B:
No novelty 1 h vs 24 h and 24 h vs 24 h [novelty]
–> With novelty there is preservation, enhanced memory encoding
–> Without novelty there is lack of retention
Novelty is the pink floor
1C:
Two drugs administered
–> SCH - which blocks D1/D5 receptor in the hippocampus in rats => Dopamine antagonist
–> Prop - no effect on novelty induced enhancement (not a dopamine antagonist, it’s actually a b-adrenergic receptor antagonist, so norepinephrine and epinephrine antagonist).
–> This confirms that novelty-dependence memory encoding enhancement is dopamine-dependent, because blocking the dopamine receptor wipes out the novelty effect on enhanced memory encoding.
–> SCH blocked enhancement while Prop did not
What and why:
It’s a great way to run a lot of trials, determine base level of success in memory encoding based on how well/often mice choose the right sandwell, and then compare that control performance with the same experiments + new floor surface (novelty), and determine if there’s a change in memory encoding (which they can measure by running the memory probe test over the course of 24 hrs).
What question(s) is the figure trying to answer?
–> Having confirmed that mice have enhanced memory encoding due to novelty, the authors wanted to confirm that TH+ neurons are actually responding/activated due to novelty. So the question is: do TH+ neurons activate in response to novelty/are TH+ neurons activated by novelty?
What methods are used in the experiments in the figure?
–> They wanted to identify TH+ neurons so that they could measure these neurons’ activity in response to novelty.
–> Mice injected with AAV carrying ChR2-eYFP into the VTA or LC
–> After a few weeks, brief low-frequency blue light was used to identify the ChR2-eYFP positive neurons in awake
–> Recording continued, after TH+ neurons were identified, without light pulses, monitoring the mice as they explored familiar and novel environments
–> This experiment determined a neuronal bursting pattern - ‘within-burst spike frequency of 12.8-53.4 Hz’ - which informed the optogenetic stimulation protocol
–> A raster plot is a simple method to visually examine the trial-by-trial variability of the response.
2A:
Diagram of the viral injection of Cre-inducible AAV (ChR2 + eYFP) into the VTA or the LC. Shows the optetrode and blue light stimulation.
–> Optetrode = device that allows for both optical (light) stimulation and also electrophysiological recordings of activity.
Graphs of spontaneous vs light-evoked spikes from identified VTA-TH+ neurons and LC-TH+ neurons.
Sweep number = Number of times they administered the light stimulation.
Comparing activity that is light evoked vs spontaneous, and we see that the activity similar (in terms of spike amplitude).
2B:
Shows the behaviour protocol
Initially identify the VTA/LC-TH+ neurons expressing ChR2-eYFP using blue light.
Turn off blue light, place the mice in the familiar environment then novel, or novel then familiar (counterbalanced sequence). THEn they placed the mice back in home cage + stimulation with blue light.
All the while, making electrophysiological recordings via the optetrode.
2D:
Compares the firing rates of VTA-TH+ neurons in novel vs familiar, and LC-TH+ neurons in novel vs. familiar.
–> Conclusion: firing rates of VTA-TH+ neurons and LC-TH+ neurons are higher in novel environments.
—> All of the LC-TH+ neurons have this higher firing rate in novel environments, whereas most but not all of the VTA-TH+ neurons have the higher firing rate in novel environments.
2E:
Graph shows us the firing rate (in % of baseline) of the LC/VTA neurons in the familiar and novel condition.
–> The difference (relative to baseline firing rate) of LC familiar FR vs LC novel FR is greater than the difference between VTA familiar FR and VTA novel.
–> SO, LC-TH+ neurons experience stronger modulation of firing rate/activity due to novelty than VTA-TH+ neurons.
–> Also, LC-TH+ neurons display habituation (VTA-TH+ don’t)
What and why:
They injected Cre-induced AAV containing ChR2-eYFP as well as an optetrode into the mice. This allows them to insert light-activated ChR2, which allows them to conduct optogenetic experiments (via the optetrode), so they can trigger APs with light. eYFP allows them to visualise ChR2 in neurons, because it’s a fluorescent protein and stimulating ChR2 with light will result in both APs, which they can measure with the optetrode, and the fluorescence readout (eYFP). With this setup, they can place mice in familiar and novel environments and measure LC-TH+/VTA-TH+ neuronal activity, and determine whether or not these neurons are responsive to novelty according to any changes in their firing rates/spike frequency.
What question(s) is the figure trying to answer?
They wanted to determine how extensively the LC projects to the hippocampus in comparison to the VTA. The overarching goal is to identify the neurons that promote hippocampal-dependent memory consolidation via post-encoding firing. Since this memory consolidation is hippocampal dependent, it’s important to establish if there is indeed projection to the hippocampus from the LC or the VTA, and to determine whether one areas projects more onto the hippocampus than others, because more substantial projection would implication one area more than the other.
–> TH+ axons in the hippocampus originate from LC-TH+ neurons
–> They wanted to establish that co-localization of TH in eYFP+ axons in the dorsal hippocampus and the co-localization of eYFP, TH and the noradrenergic transporter for LC axons but not VTA axons
–> Quantified eYFP+ axons projecting to hippocampus, calculating both the area occupied by all eYFP-TH double-positive axons and the ratio of double-positive axons relative to all TH+ axons.
What methods are used in the experiments in the figure?
–> They injected the anterograde AAV (Cre-inducible eYFP virus) into either the LC or the VTA. Anterograde tracing would move forward synaptically to determine the afferents (if any) that come from the LC or VTA and project into the hippocampus. They focused on the dorsal hippocampus (coronal sections) which they visualised with immunohistochemistry (immunostaining with fluorescent antibodies).
–> In this experiment, co-localization was established by determining the presence of both TH and eYFP in axons within the dorsal hippocampus as well as the co-localization of eYFP, TH, and the noradrenergic transporter(NET) in LC axons but not VTA axons. Co-localization of eYFP with TH and NET was used to confirm the presence of dopaminergic and noradrenergic projections in the dorsal hippocampus. By identifying the source of these projections, the researchers could determine which neuromodulatory neurons were involved in the consolidation of hippocampal-dependent memory.
–> Co-localization refers to the identification of two or more molecules or proteins present in the same cellular or subcellular region.
–> The purpose of establishing co-localization was to identify the location of axons that contain eYFP, which was induced by Cre-dependent expression.
–> TH and NET are specific markers for dopaminergic and noradrenergic neurons, respectively.
3A:
Coronal sections showing distribution of eYFP+ axons from VTA in the dorsal hippocampus. Anterograde tracing revealed very few/scarce projections from VTA-TH+ neurons to dorsal hippocampus.
3B:
eYFP (green), TH (red), NET (blue)
Establishes co-localization of TH in eYFP+ axons in the dorsal hippocampus (b,d bottom panels)
Co-localization of eYFP, TH and the noradrenergic transporter (NET) for LC axons but not VTA axons (Fig. 3b, d, bottom panels)
–> The fact that we don’t see the same amount of co-labelling in the (fewer) VTA afferents supports the previous suggestion (from retrograde labelling experiments and immunohistochemical work in rats) that few VTA afferents are dopaminergic.
3C:
Coronal sections showing distribution of eYFP+ axons from LC in the dorsal hippocampus. Anterograde tracing revealed substantial projections from LC-TH+ neurons to dorsal hippocampus.
So more likely the modulators of environmental novelty in the hippocampus.
3D:
By identifying the source of these projections, the researchers could determine which neuromodulatory neurons were involved in the consolidation of hippocampal-dependent memory.
Looks like noradrenergic neurons are involved (LC).
What question(s) is the figure trying to answer?
–> What is the pattern of projections from VTA-TH+ and LC-TH+ neurons to the dorsal hippocampus? Do these projections differ between the two neuron populations?
What methods are used in the experiments in the figure?
–> They used quantification methods to compare how much area the TH+-eYFP+ (so neuron population they are interested in) from the LC take up in the hippocampus vs the area these neurons from the VTA take up in the hippocampus. They also quantified the ratio of axons that are both TH+ and eYFP+ to just TH+, to determine how many of the TH+ neurons originate from the LC (vs. elsewhere) as opposed to VTA (vs. elsewhere).
3E:
The injection procedure into VTA or LC
Cre-inducible eYFP; these are Th-Cre mice, so neurons that have TH expression will also have Cre activity/expression. eYFP is induced by Cre, so in principle most axons that are eYFP+ should originate from the VTA or LC and be TH+ axons as well.
3F:
The greater the area covered by the LC vs VTA afferents, the more likely those populations of neurons are involved in the hippocampus novelty-dependent memory consolidation.
The greater the ratio of double positive axons to all TH+ axons, the greater the proportion of TH+ axons that originate from LC or VTA afferents (so the more likely those TH+ neurons are meditating environmental novelty in the hippocampus).
If eYFP+, should originate from LC or VTA; if TH+ too, looking at the TH+ neurons of interest from the LC or VTA.
There is significantly more area in the hippocampus taken up by LC afferents than by VTA afferents, across the three areas of the hippocampus they looked at.
They also quantified and compared the ratio of double positive axons to TH+ axons (so TH+ axons that are not originating from the VTA or LC), and found that this ratio is significantly higher in LC-TH+ neurons than in VTA.
Bars showing how much more prevalent TH+ projections are in the LC over the VTA
What and why together:
TH+ projections are stronger from LC than the VTA in CA1, CA3, and DG area
In visualising the prevalence of yellow fluorescence in the hippocampus in VTA vs LC, and the double positive prevalence and proportion to TH+ neurons, they conclude that the LC-TH+ neurons are most likely the mediators of environmental novelty.
Fluorescent retrobeads were injected into the dorsal hippocampus to confirm the pattern of projections from VTA-TH+ and LC-TH+ neurons.
Overall, the study used a combination of optogenetic and anatomical methods to identify and characterize the role of neuromodulatory neurons in hippocampal-dependent memory consolidation.
Why? What question(s) is the figure trying to answer?
–> Could optogenetic stimulation mimic the beneficial effects of environmental novelty on memory retention?
–> Now that they’ve decided to focus on LC-TH+ neurons, they want to determine if activation of the LC-TH+ neurons alone (via optogenetic stimulation) is sufficient to induce the novelty effect observed during arena activity.
–> So they know that this novelty effect happens in mice, and they know that this population of neurons (LC-TH+ neurons) are activated by/responsive to environmental novelty. They also know that the hippocampus (which the novelty process is dependent on) has many afferents from the LC. Now, they want to see if LC-TH+ neurons are responsible for the novelty effect, so they want to see if their activity alone (without a novel environment) is sufficient to result in increased memory consolidation similar to that observed due to novelty.
What methods are used in the experiments in the figure?
–> They injected the VTA or the LC with the Cre-inducible ChR2-eYFP virus. They also implanted optic cannulae into the LC or VTA (for optogenetic stimulation) and drug cannulae into the hippocampus (to inject with different antagonists). They then repeat the earlier encoding + probe tests in the arenas with sandwells, this time without placing a novel box into the arena. Instead, following the same timeline (30 mins post-encoding), they provide optogenetic stimulation to the VTA or the LC, and measure the effect of this stimulation on memory encoding at 24 hours.
–> Optogenetic burst stimulation frequency of 25 Hz was chosen, and both LC-TH+ and VTA-TH+ neurons could follow this frequency in awake mice
–> After training to a performance index of circa 75%, mice were tested in a 10 min probe
–> They used a control population of mice that was administered the eYFP virus but without ChR2, so VTA or LC neurons should not be responsive to optogenetic stimulation.
4A:
Shows the viral administration to the VTA or LC, implantation of drug cannulae into the dorsal hippocampus and optic cannulae into the VTA or LC.
4B:
Shows the optogenetic burst protocol. They selected the 25 Hz frequency, which is the optogenetic frequency they’ll administer to the VTA or LC.
This was chosen based on data regarding LC/VTA neurons’ in-burst firing. In principle, both VTA and LC neurons should be able to respond to this frequency.
The optogenetic bursts are administered for 5 mins (replacing the 5 min exploration of novel environment during previous experiments). 20 pulses are administered for 1 second every 5 seconds, until the end of the 5 mins.
–> The bursts of activity at this pulse frequency should mimic (in theory) the activity in these neurons during exploration of novel environments.
4C:
Showing the experimental setup of the arena activity. Mice are given the encoding task, and once they’ve reached sufficient performance of the task, they are given the probe test. Then this encoding task was repeated, but with 5 mins of optogenetic stimulation to the LC or the VTA 30 mins post-encoding. Then, the probe test was administered 23.5 hours later to determine memory encoding.
4D:
Quantification of memory encoding in the control group and experimental group during control task vs. task + optogenetic stimulation. The greater the dig time in the correct sandwell, the better the memory encoding.
–> LC-TH+
==> In the ChR2+ group (experimental, have LC or VTA neurons that are light-activated), there was no difference in dig accuracy (so not much memory encoding) when no optogenetic bursts were given. When optogenetic stimulation was given, there is a significantly greater performance in the probe test (memory consolidation) - greater difference in dig time in correct vs. incorrect sandwell.
==> In the ChR2- group, there was no difference in dig accuracy when no optogenetic simulation was given (little memory encoding). When optogenetic stimulation was provided, there was not a great difference in dig time in the correct vs. incorrect sandwell (a little but not much memory encoding).
–> VTA-TH+
==> In ChR2+ group, there was no difference in dig time in correct vs incorrect sandwell when no optogenetic stimulation was given. There was some difference in dig time in correct vs incorrect sandwell when optogenetic stimulation was given, but similar to the difference observed in the ChR2- group.
==> So non-significant difference, and also observed in the negative control, so this result is likely not related to optogenetic stimulation.
SO: we see increased memory consolidation at 24hrs during the probe task when ChR2+ mice receive optogenetic stimulation to the LC. We see a non-significant increased memory consolidation at 24hrs when the same ChR2+ mice received optogenetic stimulation to the VTA.
Did they compare this to the performance during the task with the novel box?
They were confused by this, because the LC-TH+ neurons are not known for sure to be dopaminergic, and they know that blocking dopamine receptors during novelty inhibits this novelty-depending memory consolidation. They want to confirm/repeat this with drug infusion during optogenetic stimulation.
What methods are used in the experiments in the figure?
They also conducted the optogenetic stimulation experiment alongside drug administration. They administered a D1/D5 receptor (dopamine receptor) antagonist during light activation of the LC. They also administered Prop (norepinephrine and epinephrine antagonist) during light activation of the LC.
–> Th-Cre mice were stereotaxically prepared with bilateral injections fo the ChR2-eYFP+ virus or a no opsin control virus, accompanied by implantation of bilateral optic cannulae into both LC and VTA and bilateral drug cannulae targeting dorsal hippocampus
4E:
Shows us the microinfusion set up to the dorsal hippocampus during the encoding-probe task with and without LC optogenetic stimulation.
Schematic design for optogenetic LC activation with pharmacological interventions to reconcile memory enhancement paradox between 1c and 4d (Why does a dopamine inhibitor wipe out the novelty effect of LC neurons but LC neurons are noradrenergic?).
4F:
Showing dig time (proxy for memory consolidation) with and without optogenetic stimulation, and with varying conditions during optogenetic stimulation (with non-drug administration, with Prop administration, and with SCH administration). Did this in the ChR2+ (light responsive) and ChR2- (not light responsive) mice.
Again, optogenetic administration in the ChR2+ mice enhanced their memory for the probe test (measured according to dig time in correct vs incorrect sandwell). When these mice were administered Prop to their hippocampus during optogenetic stimulation of the LC, this memory enhancement was preserved. When these mice were administered SCH to their hippocampus during optogenetic stimulation of the LC, this memory enhancement disappeared.
Prop, which is a norepinephrine and epinephrine antagonist, does not interfere with memory enhancement due to optogenetic stimulation
SCH, which is a dopamine receptor antagonist, does block memory enhancement due to optogenetic stimulation.
SO WHAT:
Suggests that LC-TH+ terminals, which are known to release noradrenaline, might also co-release dopamine in the hippocampus. Or maybe there is heterodimerization of noradrenaline and dopamine receptors.
Using optogenetic stimulation experiments during the encoding and probe tasks, the authors found that activating the LC optogenetically mimics the novelty effect (increases memory 24hrs post-encoding).
→ Optogenetic activation of LC-TH+ neurons enhances memory persistence.
Key finding: The striking persistence of memory over 24h when 5 min of post-encoding (30 min) intermittent burst stimulation of LC-TH+ neurons with blue light was given to the ChR2+ mice in their home cages.
What question(s) is the figure trying to answer?
–> What is a possible mechanism of memory enhancement?
The idea that LC activation enhances synaptic efficacy
Explored one possible mechanism of the enhancement of memory retention, ex vivo electrophysiological experiments examined the response of CA1 pyramidal neurons to CA3 Schaffer collateral synaptic input
What methods are used in the experiments in the figure?
–> Ex vivo electrophysiological experiments to exam the response of CA1 pyramidal neurons to CA3 Schaffer collateral synaptic input. 3 weeks before obtaining slices, bilateral injections of Coe-inducible ChR2-eYFP virus were made into the LC of Th-Cre mice.
–> Administration of Optogenetics stimulation to the hippocampal axons and measuring excitatory post-synaptic currents (EPSCs).
–> Examining LTP at CA3-CA1 synapses using theta-burst stimulation to induce LTP.
–> Hippocampal LC-TH+ axons were selectively activated with protocol closely mimicking LC-TH+ firing patterns recorded during novelty exploration.
5A:
Schematic of setup. How they decided to slice the hippocampal section and the site where optogenetic activation takes places.
5B:
Schaffer collateral (SC) - Axon collaterals given off by CA3 pyramidal cells in the hippocampus. These collaterals project to area CA1 of the hippocampus and are an integral part of memory formation and the emotional network.
–> Following three trains of burst optogenetic stimulation of hippocampal LC-TH+ axons (Extended Data Fig. 7b), the CA3–CA1 excitatory postsynaptic currents (EPSCs) gradually increased by 55% over ~30 min
An increase unaffected by the presence of the α- and β-adrenoceptors antagonists prazosin (Praz) and Prop.
In contrast, there was no increase following optogenetic LC-TH+ activation in the presence of SCH, revealing a pattern of EPSC potentiation consistent with mediation by a dopaminergic mechanism.
5C:
LTP differed in magnitude across four conditions.
–> Thetaburst stimulation alone induced LTP by 29% at 45min (light off + LTP) relative to a no-LTP baseline; by 59% when combined with optogenetic LC-TH+ activation (light on + LTP);
but by was blocked by SCH (light on + LTP with SCH). Taken together, these findings indicate that depolarization of hippocampal LC-TH+ axons by optogenetic stimulation can enhance synaptic transmission, and that a physiologically realistic pattern potentiates LTP at CA3–CA1 synapses in a manner consistent with release of dopamine from hippocampal LC-TH+ terminals.
What question(s) is the figure trying to answer?
–> Does blocking VTA-TH+ neurons during novelty affect memory enhancement?
What methods are used in the experiments in the figure?
They injected mice with drug cannulae into the VTA. They measured VTA neuronal firing during lidocaine administration to verify that blocking sodium channel activity inhibits VTA activity.
Then they had the mice do the encoding-probe task and administered lidocaine into the VTA during novelty exploration and measured memory consolidation based on dig time in correct vs incorrect sandwells.
They also had the mice perform the encoding-probe task and administered clonidine (alpha-2-adrenoceptor agonist, an inhibitor of LC neurons) and measured memory consolidation based on dig time in correct vs incorrect sandwells.
6A:
But shows that after administration of lidocaine during the novelty period (meaning equivalent time of novelty period), measured neuronal activity in the VTA drops sharply to almost nothing, then gradually recovers. We see this in an example trace + population data.
Small figure top right of A shows comparison of neuronal activity pre-lidocaine injection, during lidocaine injection, and post-lidocaine.
Lidocaine, which blocks sodium channels, inhibits VTA activity/firing.
6B:
Administration of lidocaine to the VTA during novelty exploration (in the encoding-probe test task). Compared memory consolidation (measure by dig time in correct vs. incorrect sandwells) when mice were administered lidocaine during novelty exploration or administered vehicle (control).
Found that there was no difference in memory consolidation 24hrs after encoding when VTA activity was blocked.
If VTA had been involved in the novelty effect, inhibiting its activity should’ve resulted in reduced dig time in correct sandwells related to the control, which we don’t see.
6C:
Administration of clonidine during novelty exploration (encoding-probe test task). Compared memory consolidation (dig time in correct vs. incorrect sandwells) when mice were administered vehicle or clonidine during novelty, or when mice were given vehicle and were not exposed to environmental novelty.
Clonidine administration (LC neuron inhibitor) during novelty wipes out the increased memory consolidation observed with exposure to environmental novelty (w/ vehicle - no inhibition).
What and why together:
Inhibiting VTA activity has no impact on the novelty effect.
Inhibiting LC neurons wipes out the novelty effect.
