After Impair Neurons Mice Conditioned Again

• Periodical Listing
• J Psychiatry Neurosci
• v.35(4); 2010 Jul
• PMC2895151

J Psychiatry Neurosci. 2010 Jul; 35(iv): 221–228.

Continuing the search for the engram: examining the mechanism of fearfulness memories

Received 2010 Jan 22; Revised 2010 April 7; Accepted 2010 Apr 12.

Abstract

The goal of my research is to gain insight using rodent models into the fundamental molecular, cellular and systems that make up the base of operations of memory formation. My work focuses on fear memories. Aberrant fear and/or feet may exist at the centre of many psychiatric disorders. In this commodity, I review the results of my enquiry group; these results bear witness that particular neurons in the lateral amygdala, a brain region important for fright, are specifically involved in particular fear memories. Nosotros started by showing that the transcription factor CREB (campsite/Ca²⁺ response element binding poly peptide) plays a central role in the formation of fear memories. Next, we used viral vectors to overexpress CREB in a subset of lateral amygdala neurons. This not just facilitated fright memory formation merely besides "drove" the memory into the neurons with relatively increased CREB function. Finally, we showed that selective ablation of the neurons overexpressing CREB in the lateral amygdala selectively erased the fear retentivity. These findings are the offset to evidence disruption of a specific memory past disrupting select neurons inside a distributed network.

A fundamental goal of neuroscience is to understand how memories are encoded and stored in the brain. Ensembles (or groups) of neurons are thought to serve as the physical representation of memory (the retention trace or "engram," a term first coined past Richard Semon in 1921^ane). Still, identifying the precise neurons that constitute a memory trace has been challenging neuroscientists since Karl Lashley began his famous search for the elusive engram in the 1920s. In his studies, Lashley trained rats in various tasks (such as traversing a maze to notice a food reward) and lesioned diverse portions of different cortical regions either before or after training. After 30 years of piece of work, he summarized his findings in a seminal newspaper entitled "In search of the engram."ⁱⁱ

Lashley observed that cortical lesions disrupted operation in the maze and that the degree of disruption was roughly proportional to the amount of cortex removed but not to the location of the lesion. From this, he concluded that all cortical areas can substitute for each other as far as learning is concerned (the principle of equipotentiality) and that the cortex tends to act as a whole in that the corporeality, rather than the location, of cortical tissue removed correlated with performance in the maze (the principle of mass action). These findings led Lashley to conclude that memories are not localized, just rather distributed, in the cortex. In his words,

"…this series of experiments has yielded a good bit of information about what and where the memory trace is not. Information technology has discovered zip directly of the real nature of the memory trace. I sometimes feel, in reviewing the evidence of the localization of the retentivity trace, that the necessary conclusion is that learning is but non possible. Information technology is difficult to excogitate of a mechanism that can satisfy the conditions set for information technology. Nevertheless, in spite of such prove against it, learning sometimes does occur."²

Lashley's failure to observe the engram may have been because of his choice of retention task (learning a maze is a circuitous job, and this sort of retention likely relies on many brain regions) and brain region examined (regions other than the cortex may be involved in retentivity).

Since Lashley's studies, though, at that place has been progress in "finding the engram." Both cellular imaging and electro-physioloic studies^{three – vii} accept detected neurons whose activity is correlated with memory encoding or expression or both, suggesting that these active neurons brand upwards the memory trace. For example, John Guzowski's laboratory used a technique they developed, referred to equally catFISH (compartmental analysis of temporal activeness past fluorescence in situ hybridization),^viii and institute that some of the aforementioned neurons in the dorsal hippocampus of rats were active when the rats were re-exposed to a unique environment. More recently, Mark Mayford'due south laboratory used a sophisticated transgenic mouse approach that immune them to tag active neurons.⁵ They showed that some of the aforementioned amygdala neurons that were active during fright learning were also active during the retrieval of that fear memory. Furthermore, the number of reactivated neurons in the amygdala was correlated with the forcefulness of the retrieved fear memory.

These findings advise that specific neurons are involved in a memory. Still, these correlative studies practice not address whether these active neurons are essential components of the retentiveness trace. A direct test of this hypothesis would require specifically disrupting only these activated neurons while leaving their neighbours intact and then determining whether subsequent memory expression is blocked. Establishing such a causal part for item neurons in a retentivity has been difficult because the neuronal ensembles that make up this memory trace are thought to exist sparsely distributed,^nine and the ability of electric current pharmacological, genetic and lesioning techniques to target specific subsets of neurons inside a brain region is limited.

In my laboratory'south "search for the engram," nosotros used auditory fear conditioning as our retentivity chore. In this Pavlovian paradigm, a neutral tone is paired with a mildly aversive footshock. One tone-shock pairing is sufficient to produce a robust and long-lasting fear retention that tin be quantified by measuring the percentage of fourth dimension mice spend freezing (an adaptive defensive response) when the tone is subsequently replayed. Automated measures have been established to easily and reliably quantify freezing.¹⁰ We chose this task because genetic, biochemical, electrophysiological and behavioural studies from many laboratories, including those of Mike Davis, Michael Fanselow, Joe LeDoux and Steven Maren, established that lateral amygdala is required for auditory-conditioned fright memories^{xi – 15} (only see¹⁶). Now we knew where to look in the brain for a fear memory trace. The next step was to target lateral amygdala neurons that are active following fear training or testing.

To target neurons whose activity is correlated with memory, Jin-Hee Han, Adelaide Yiu and Hwai-Lin (Liz) Hsiang in my laboratory, together with our collaborators Steven Kushner, Bruno Bontempi and Paul Frankland, took reward of our contempo findings that increasing the level of the transcription factor CREB (cAMP/Ca²⁺ responsive element bounden poly peptide) in a small portion of lateral amygdala neurons enhances auditory fear memory nether certain grooming weather.¹⁷ Furthermore, we found that neurons with relatively increased CREB levels are more likely than their neighbours to be active post-obit fear memory preparation or testing. This suggested to us that these neurons with loftier CREB levels outcompete their neighbours for inclusion in the fright memory trace.

The idea that CREB is involved in memory is non new. Indeed, studies by Tim Tully'south and Eric Kandel's laboratories showed that CREB is necessary for retention germination in invertebrates.^{18 – 22} Parallel studies in rodents have as well showed that CREB is of import for memory formation^{23 – 34} (but see³⁵).

I first began examining the effects of increasing CREB function on memory when I was a postdoctoral fellow at Yale in Mike Davis's laboratory. Eric Nestler's laboratory was located across the hall at the Connecticut Center for Mental Health, and Bill Carlezon, a postdoctoral beau in his laboratory at the fourth dimension, was manipulating CREB function using replication-defective herpes simplex viral (HSV) vectors to determine the effects of CREB on cocaine-induced drug seeking.³⁶ Although at that place were many ways to decrease CREB part, in that location were relatively few ways to transiently raise its office in rodents. The HSV system was an platonic tool to written report the furnishings of enhancing CREB on memory. This system, which allows expression of a transgene in a particular encephalon region at a particular time, was largely pioneered by my colleague, Rachael Neve, now at the Massachusetts Establish of Engineering science.

Herpes simplex viral vectors offer many advantages over other viral vector systems.³⁷ First, HSV infects adult (nondividing) neurons (rather than glia) with relatively high efficiency.^{38 , 39} Second, the Dna from HSV remains episomal, thus avoiding potentially confounding furnishings of integration into the host Dna. Third, these vectors offer a large chapters for the insertion of foreign DNA. In our studies, we drove transgene expression with the HSV immediate–early on cistron IE4/v such that transgene expression peaked ii–5 days following infusion and declined within vii days.^{xl , 41} In this style, we could examine the effects of acutely increasing CREB function on fear retentivity in the lateral amygdala. In collaboration with Nib Carlezon and Eric Nestler, Mike Davis and I showed that increasing CREB levels in the lateral amygdala enhanced memory for fear-potentiated startle, another fear memory task,¹² in rats that had been given weak training (consisting of several grooming trials massed together without intervening remainder periods, which has been shown to produce weak memory).⁴² What intrigued us almost our finding was that nosotros observed robust memory enhancement despite the fact that we increased CREB levels in a relatively small-scale percentage of lateral amygdala neurons (our HSV vectors infected roughly 15% of lateral amygdala neurons). This disconnect between the pocket-sized number of neurons infected but big behavioural results continued to motivate my experiments.

Later, when I was a postdoctoral fellow in Alcino Silva'south laboratory, Steven Kushner, Alcino and I had long conversations nigh how we might make sense of these findings. We definitely believed the data. Mike Davis taught me to always follow the data (to borrow from Shakira, data, like hips, don't lie). Plus, our basic effect was replicated by ii additional groups using the same HSV vectors.^{43 , 44} We reasoned that one way to explicate the information was that the low number of neurons with increased levels of CREB were somehow outcompeting their neighbours for inclusion in the memory trace. When I began my laboratory in Toronto, Jin-Hee Han, a talented postdoctoral boyfriend in my laboratory, connected these studies.

First, we revamped our viral vectors to allow infected neurons to be easily visualized past fusing the transgenes with green fluorescent protein (GFP). Tagging the Northward-terminus of CREB with GFP does not interfere with the functional activeness of CREB.^{37 , forty , 45 , 46} In our outset studies, nosotros used 3 primary vectors: HSV-CREB expressing wild-type CREB (CREB^WT); HSV-mCREB (the mutant CREB vector) expressing a mutated version of CREB that cannot be phosphorylated at the key Ser133 residue (CREB^S133A); and HSV-GFP (control, Cntrl vector) expressing GFP or LacZ.

Our overall aim was to decide whether neurons infected with the CREB^WT vector were preferentially recruited to the lateral amygdala fear retentivity trace. We reasoned that if competition between neurons for inclusion in the retentivity trace occurred during learning, the easiest way to see this would be to "stack" the experimental deck by maximizing the difference between the neurons infected with the CREB^WT vector and their noninfected neighbours. Thus, we commencement infused our CREB^WT vector into the lateral amygdala of mice with a targeted disruption of the 2 primary isoforms of CREB, CREB^αδ−/− mice.⁴⁷ The CRE–DNA bounden is virtually abolished (past > 90%),^{48 , 49} and the levels of CREB protein are dramatically reduced (roughly 85%–90% reduction compared with controls) in the brains of CREB^αδ−/− mice.^{48 , 50} These CREB-deficient mice take previously been shown to be impaired in auditory fear conditioning.^{23 , 28} Chiefly, we replicated this retentivity arrears and showed that infusing our Cntrl vector into the lateral amygdala did non alter this. Yet, infusing the CREB^WT vector into the lateral amygdala of these mice completely rescued the auditory fear deficit; the CREB-deficient mice now froze at the same levels as their wild-type littermate control mice. Furthermore, under weak preparation conditions, we institute that increasing CREB role in a similar small fraction of lateral amygdala neurons enhanced memory in wild-type mice (like to my initial findings using fear-potentiated startle in rats⁴²). Together these findings suggested that these neurons with increased CREB function were preferentially recruited to the trace supporting the fear retentivity.

We were inspired to explore this possibility further by John Guzowski'south elegant studies using the catFISH technique. Specifically, Jin-Hee Han, Adelaide Yiu and Christina (Christy) Cole in my laboratory, together with our collaborators, Steven Kushner, John Guzowski and Alcino Silva, attempted to visualize the fearfulness retention trace by taking reward of the unique transcriptional time course of the activity-dependent gene Arc (activity-regulated cytoskeleton-associated poly peptide; Arg3.1).^{6 , 8} Under basal conditions, neurons contain very low levels of Arc RNA. However, neuronal activation (the type of activation that is associated with learning or long-term potentiation) produces a rapid but transient burst in Arc RNA synthesis (within almost 3–v min of activation).⁸ This RNA is and then delivered to dendrites within roughly xx minutes of activation. Therefore, the localization of Arc RNA (either in the nucleus, cytoplasm or both) can serve as an activity marker for that detail neuron, with Arc RNA localized to the nucleus beingness a molecular marker of a neuron that was active v minutes ago. We used this technique to identify neurons that were activated by fear retentivity training or testing and asked whether these neurons were as well the ones we infected with the CREB^WT vector.

Nosotros observed that neurons infected with the CREB^WT vector were 3 times more likely than their noninfected neighbours to be Arc positive in wild-type mice and 10 times more likely in CREB-deficient mice. In contrast, neurons infected with the dominant-negative CREB vector were 12 times less likely than their neighbours to exist Arc positive. This was consistent with our behavioural data showing that disrupting CREB function (using the CREB^S133A vector) in roughly 20% of lateral amygdala neurons had no outcome on memory in wild-type mice, perhaps considering the remaining neurons (over 80%) with normal levels of CREB were sufficient for normal retentivity.¹⁷

Together with many control studies, these findings suggest that neurons with relatively college CREB function are preferentially recruited to the memory trace. Would this mean that increasing CREB levels in all lateral amygdala neurons would enhance fearfulness memory? We hypothesized that it would non, because nosotros remember that contest betwixt eligible neurons is critical in memory enhancement. We reasoned that raising CREB levels in all neurons would not produce a stronger memory because all of the neurons would again be equal (and the signal-to-noise ratio would not be enhanced). As Gore Vidal put information technology, information technology is not enough to succeed; others must fail. The same may employ to neuronal competition underlying memory formation.

Our findings suggested that neurons with college levels of CREB "win" the contest between neurons for inclusion in a retention trace. My laboratory then attempted to use this property to decide if selectively ablating just these neurons subsequently training disrupts the expression of an established fright retention. If so, this would indicate that these neurons are disquisitional components of the elusive retentivity trace. We used several techniques to do this but had no luck. Information technology turns out that these neurons, , similar Steven Segal, are hard to kill, perhaps because CREB is a survival cistron.⁵¹ Then, Steven Kushner, an avid reader of scientific journals, came across a paper in Nature Methods by Ari Waisman and colleagues⁵² describing a novel transgenic mouse used for prison cell lineage ablation studies based on diphtheria toxin (DT). We decided to endeavour to kill neurons overexpressing CREB using this arroyo.

Showtime, a bit of groundwork on DT. Corynebacterium diphtheriae produces DT, a stiff toxin that, in one case it binds to its receptor (diphtheria toxin receptor; DTR) and is internalized into a mammalian jail cell, efficiently blocks protein synthesis to cause rapid apoptotic jail cell death.^{53 – 55} For cell death to occur using the DT-based system, both fundamental components (DT and DTR) are required. Interestingly, mice do not normally limited a functional DTR.^{56 – 58} So, Waisman and colleagues⁵² engineered a mouse that expresses a simian DTR transgene (driven past the ubiquitous Rosa promoter; Fig. 1). Still, expression of DTR is dependent on the cre recombinase-mediated removal of a transcriptional End cassette (upstream of the DTR transgene they placed a floxed STOP cassette that silences DTR expression until the Finish cassette is removed by cre recombinase-mediated recombination). Systemic injection of DT anytime thereafter induced apoptosis, but just in the cells that have undergone cre-mediated recombination and limited DTR. A single internalized catalytically active fragment of DT (DT-A) is sufficient to kill a jail cell, indicating the sensitivity of DT-induced cell ablation.⁵⁹ Importantly, neither high doses of DT in wild-type mice nor the expression of DTR lonely (without DT) induces apoptosis, indicating the specificity of the organisation.^{threescore , 61} Because DT readily crosses the blood–encephalon barrier,⁶² these inducible DTR (iDTR) mice accept been used to induce cell expiry in the brain.^{52 , lx}

Schematic diagram depicting the strategy for selectively ablating neurons using inducible diphtheria toxin receptor (iDTR) transgenic mice. The mice express a simian DTR transgene. Expression of the DTR transgene is suppressed by a STOP cassette, which is floxed and removed when a neuron is infected with canker simplex virus expressing cre recombinase (cAMP/Ca²⁺ response element binding protein [CREB]–cre vector or control-cre vector). Only neurons that have undergone cre-induced recombination volition constitutively express DTR on the cell surface. At whatsoever point thereafter, systemic injection of diphtheria toxin (DT) induces apoptosis only in cells expressing DTR. In this manner, only neurons infected with CREB-cre vector or Cntrl-cre vector will be ablated.

Many strategies can be used to delete defined cells, including the expression of cytotoxic proteins,⁶³ antibodies⁶⁴ or chemicals.⁶⁵ Even so, these systems, and indeed before versions of the DT arrangement, were not suited for our studies because they lacked temporal control over cell expiry and/or are prone to "leakiness" (in that cell expiry is non temporally or spatially limited to the targeted cell population).⁶³ The iDTR mice were designed to accost these drawbacks. Considering DT induces cell decease past apoptosis, it is thought to produce less inflammation than necrosis-induced cell death.⁶⁶ In this way, the iDTR mice were designed to minimize the "bystander upshot," in which the death of a targeted jail cell direct or indirectly affects neighbouring neurons.

Jin-Hee Han, together with other members of my laboratory and our collaborators Steven Kushner and Paul Frankland, took advantage of the iDTR transgenic mice to selectively induce cell death in the neurons that we hypothesized were preferentially involved in the memory trace (neurons infected with the CREB^WT vector). To this end, we re-engineered our viral vectors to include cDNA for cre recombinase. Our first vector expressed both CREB and cre recombinase (CREB-cre vector). When injected into the lateral amygdala of the iDTR mouse, we expected that a pocket-sized portion of neurons would have increased levels of CREB and that a subsequent injection of DT would ablate but these neurons. Recombination and DTR expression can only occur in neurons expressing cre recombinase, allowing us to persistently tag infected neurons for subsequent ablation. This tagging is critical considering of the relatively brief time course of transgene expression using HSV.⁴⁰ Considering this technique produces a small lesion of the lateral amygdala that may affect memory on its ain, we needed a control vector. Then we added cre recombinase to our Cntrl vector. Our thinking was that this Cntrl-cre vector would ablate a similar number of lateral amygdala neurons but that these neurons would be randomly located in the lateral amygdala and non involved in the memory trace (because they did not overexpress CREB). As a first footstep in this study, Jin-Hee confirmed that this iDTR/vector organisation selectively induced cell decease past examining 2 markers of apoptosis (activated caspase 3 and concluding deoxynucleotidyl transferase dUTP nick stop labeling [TUNEL]). We quantified the number of cells with activated caspase 3 and those that were positive for TUNEL and found that significant apoptotic cell expiry was only observed in the experimental groups (iDTR mice infused with either the CREB-cre or Cntrl-cre vectors). Importantly, nosotros found that the CREB-cre and Cntrl-cre vectors produced a similar level of cell death. Nosotros were excited because we thought that this system, which allowed the states to temporally ablate tagged neurons, would bring us 1 step closer to finding the engram.

To examine the effects of mail service-preparation ablation of neurons overexpressing CREB, we performed a series of behavioural tests. First, nosotros found that increasing CREB levels in a population of lateral amygdala neurons (by microinjecting the CREB-cre vector) enhanced retentivity following weak grooming in iDTR mice. This finding replicated our earlier finding using the CREB^WT vector (without cre recombinase) in wild-type mice.¹⁷ This retentiveness enhancement, notwithstanding, was completely reversed later on systemic administration of DT (to delete just those neurons infected with the CREB-cre vector; Fig. 2). Importantly, the reversal of this retention enhancement was not observed in the control groups that lacked a cardinal component of the DT-killing system (either cre recombinase or DT), consequent with the lack of cell death in these control groups. This was important because it showed that the memory reversal was not due to fear retentiveness extinction caused by the 2nd memory examination.

Neurons with relatively increased CREB (military camp/Ca^two+ response element binding poly peptide) function are essential for memory remember. (A) Schematic diagram of an experiment designed to exam whether neurons with increased CREB function at the time of training are required for subsequent retentivity remember. (i) A subset of lateral amygdale (LA) neurons are infected with a CREB-cre vector (labelled light-green via dark-green fluorescent poly peptide [GFP]), which increases CREB in these neurons and induces them to undergo cre-induced recombination to express diphtheria toxin receptor (DTR). (2) Mice are trained. A subset of LA neurons have high CREB levels, but because diphtheria toxin (DT) has not been administered, there is no cell death. (iii) Mice are tested. Neurons involved in the memory trace are depicted in ruby-red. Neurons with increased CREB out-compete their neighbours for inclusion in the fear memory trace (labelled yellow [green+red]). (four) Mice receive systemic injections of DT to induce jail cell death only in the cells that express DTR (have undergone cre-induced recombination and, therefore, must accept been infected with the CREB-cre vector). Mice are tested once more. Neurons that previously had high CREB (infected) are at present ablated. (B) Results from this experiment indicate that selectively ablating neurons with increased CREB at the fourth dimension of training impairs subsequent retentivity recall. The CREB-cre group shows that increasing CREB function in a portion of neurons at the fourth dimension of preparation enhances memory (Examination 1) and subsequent ablation of these item neurons completely reverses this enhancement (Test 2). The Cntrl-cre grouping show that GFP does not raise memory and that selectively ablating these cells does not alter this. The off-target group shows that increasing CREB office in neurons outside the LA does not enhance memory. The CREB alone (no cre) grouping shows that increasing CREB in the LA enhances memory (Test 1) and that DT injection (with no recombination) does non contrary this enhancement Test 2). The mice that received phosphate-buffered saline just no DT (PBS, no DT) show that CREB enhances memory in inducible DTR mice, and a systemic injection of PBS (rather than DT) does non alter this on the second test day.

Next, we showed that deleting neurons infected with the CREB-cre vector (but not a like portion of random neurons infected with the Cntrl-cre vector) also impaired the expression of a strong memory. Therefore, even though increasing CREB function in a portion of lateral amygdala neurons did not further enhance retention in mice that were trained using a potent protocol, these neurons are yet important in the fright retentivity. In both of these experiments, still, we administered DT after a fearfulness retentivity examination. This retention test, however, may reactivate the fright retentiveness and trigger a second moving ridge of consolidation (referred to as reconsolidation).⁶⁷ Karim Nader, Joe LeDoux and several other groups have shown that, similar to initial consolidation, reconsolidation requires protein synthesis.⁶⁷ Because DT disrupts protein synthesis, we wanted to examine whether the memory disruption that nosotros observed in the previous experiments was due to a disruption in reconsolidation. Therefore, we examined additional groups of mice but did not reactivate the retentiveness (we did non give the mice a memory test) earlier DT assistants. Instead, we administered DT at the same time simply in the homecage. Again, we observed that mice microinjected with the CREB-cre vector and administered DT showed impaired memory whereas similarly treated Cntrl-cre mice did not.

If neurons overexpressing CREB during training (neurons with the CREB-cre vector) are critically involved in the memory trace, deleting this subpopulation should permanently block retentivity expression. To examine the persistence of memory loss, we trained mice using a potent protocol, administered DT in the homecage and assessed memory 2, 5 and 12 days later. The retentivity loss observed in the CREB-cre vector mice was long lasting; mice showed low freezing during the tone over these repeated tests. In contrast, retentivity remained robust over repeated tests in similarly treated Cntrl-cre mice. Therefore, nosotros constitute no prove of memory recovery in mice in which neurons overexpressing CREB were deleted, indicating that the retentivity was not just transiently suppressed.

To determine whether the retentiveness loss could be due to a nonspecific impairment of lateral amygdala office (the mice did have a small lesion of the lateral amygdala), we retrained these mice. Following retraining, both groups of mice (CREB-cre and Cntrl-cre mice) showed as high levels of freezing during the tone. That is, the mail service-grooming ablation of neurons that were overexpressing CREB (and cre recombinase) at the time of training produced a memory deficit, merely these mice were capable of learning a tone-shock clan (and freezing). Similarly, nosotros also plant that ablating neurons with the CREB-cre vector before fear workout did non impair subsequent memory formation. In this experiment, we infused the CREB-cre vector and systemically administered DT (to kill the neurons overexpressing CREB) earlier strong training and tested fear memory daily for 3 days. We found that even though a subpopulation of neurons was ablated earlier grooming, the mice acquired retention normally. Furthermore, this memory was neither more prone to extinction nor more fragile than a retention caused without this killing (iDTR mice microinjected with the CREB-cre vector but systemically administered phosphate-buffered saline instead of DT). Together, these findings point that ablation of neurons that were overexpressing CREB at the fourth dimension of retention-encoding blocks the retentiveness of that detail learning event while leaving subsequent learning intact. Much like the data from wild-type mice infused with the mCREB vector (which showed normal memory), the loftier portion of remaining (noninfected) neurons seemed sufficient to encode a new retention. Finally, nosotros examined the effects of ablating neurons that were infected with the CREB-cre vector after preparation. In this experiment, nosotros reasoned that there would exist no memory disruption because the CREB-infected neurons would not be part of the memory trace (considering training had already taken place). This is exactly what we observed. Therefore, the retention loss induced by ablating neurons overexpressing CREB is robust, persistent and specific.

Our results bear witness that the neurons with increased CREB levels at the time of fright learning are critical to the stability of that memory because selectively ablating simply these neurons after training blocks this fear memory. This indicates that these neurons themselves are essential for later on memory expression; they are not simply creating a local environment that promotes memory formation (such as releasing trophic factors). Fright learning may generate a broad memory trace that encompasses more lateral amygdala neurons than affected past our treatment or multiple retentiveness traces throughout the brain. Still, deleting just the neurons over-expressing CREB at the time of grooming produces amnesia, which suggests that these neurons play an essential role in what is likely a broader fright neuronal network.

Our results established a causal link between the activity of a defined subpopulation of neurons and expression of a fear retentivity, thereby identifying a central component of the memory trace. They also bespeak that neurons with relatively loftier CREB levels are selectively recruited to a fright memory trace. But what is the machinery underlying this preferential selection? Why are the neurons that overexpress CREB so special? 1 possibility is that increasing CREB alters the intrinsic excitability of a neuron. Intrinsic excitability (the propensity of a neuron to burn down action potentials in response to an input) is determined past the distribution and backdrop of ion channels (e.g., Na⁺, K⁺, Ca²⁺) in the plasma membrane. Indeed, increasing CREB function stimulates transcription of a voltage-dependent Na⁺ subunit (1β subunit) and inhibits transcription of a voltage-dependent Grand⁺ channel subunit (Kv1.4).⁶⁸ Hebb proposed that links between 2 cells are strengthened if both cells are active simultaneously.⁶⁹ That is, coincident firing of the presynaptic neuron and depolarization of the postsynaptic neuron is necessary for Hebbian plasticity. A postsynaptic neuron that is more excitable than its neighbour might therefore be more probable to be depolarized and subsequently "burn down together" and "wire together" with the presynaptic neuron. In this mode, neurons with increased intrinsic excitability may be "primed" for learning and more probable to outcompete their neighbours for inclusion in a fear retentiveness trace. Unlike synaptic plasticity, which involves changes at the level of the private synapse, intrinsic excitability or plasticity involves changes at the level of the entire neuron,⁷⁰ thus making it an attractive mechanism for the effects of CREB on neuronal contest during memory formation.

To investigate this, my laboratory collaborated with Mike Salter's laboratory at The Hospital for Sick Children. We examined the electrophysiological characteristics of primary neurons in the lateral amygdala infected with CREB^WT and Cntrl vectors in astute slices from wild-type mice. We were inspired by work from Eric Nestler'southward and Robert Malenka's laboratories⁷¹ showing that medium aspiny neurons in the nucleus accumbens infected with a similar CREB vector showed increased excitability (increased evoked action potential firing, decreased rheobase), whereas disrupting CREB (using the ascendant-negative CREB^S133A vector) decreased intrinsic excitability without affecting passive membrane backdrop.

We found a similar effect in lateral amygdala neurons. Our initial results showed that neurons with the CREB^WT vector were more excitable than both their noninfected neighbours and those infected with the Cntrl vector.⁷² Specifically, neurons with the CREB^WT vector fired more than action potentials in response to the same input stimulus than neurons with the Cntrl vector or the noninfected neighbouring neurons. Importantly, nosotros observed no modify in passive membrane properties (average resting membrane potential, input conductance) or action potential waveforms between the groups. This increase in neuronal excitability is in agreement with findings using viral vectors^{73 , 74} and transgenic mice^{75 – 78} to increase CREB levels. Recently, the effects of increasing CREB function on the electrophysiological backdrop of lateral amygdala neurons has been replicated and extended past Alcino Silva's group.⁷⁹

Francis Crick wrote that a crucial footstep in agreement the mechanisms underlying memory is to interfere with divers neuronal populations in intact neural circuits.⁸⁰ He emphasized that it was necessary that such manipulations target specific neurons in time and space. With the appearance of modern genetic, molecular and imaging tools, this is becoming possible.⁸¹ Our studies take shown that interfering (retrograde deletion) with a defined neuronal population (lateral amygdala neurons overexpressing CREB) in intact circuits disrupts memory expression. This may be the first example of the disruption of a specific retentiveness within a distributed network. We believe that this takes us one step closer to finding and even manipulating the elusive engram.

Acknowledgments

In this review, I outlined my scientific journey. Of course, I did non take this journeying alone, equally all the piece of work described here has been a squad effort. I mentioned many of the important people along the way. I would too like to specifically acknowledge certain individuals whose contributions were vital. First, my postdoctoral mentors, Mike Davis and Alcino Silva. Next, the members of my laboratories (both past and present), specially Jin-Hee Han, Adelaide Yiu, Christy Cole and Hwai-Lin (Liz) Hsiang. Finally, I thank my collaborators, Eric Nestler, Bill Carlezon, Rachael Neve and John Guzowski, and peculiarly Steven Kushner and Paul Frankland. I would like to give thanks the people in my lab and my many collaborators for making this work possible. I would also similar to thank Paul West. Frank-country for suggestions on this manuscript. This piece of work was supported by Canadian Institutes of Health Research (MOP74650), EJLB Foundation and grants from the National Sciences and Applied science Research Quango of Canada.

Footnotes

Competing interests: None declared.

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