Phase locking of hippocampal CA3 neurons to distal CA1 theta oscillations selectively predicts memory performance.

How the coordination of neuronal spiking and brain rhythms between hippocampal subregions supports memory function remains elusive. We studied the interregional coordination of CA3 neuronal spiking with CA1 theta oscillations by recording electrophysiological signals along the proximodistal axis of the hippocampus in rats that were performing a high-memory-demand recognition memory task adapted from humans. We found that CA3 population spiking occurs preferentially at the peak of distal CA1 theta oscillations when memory was tested but only when previously encountered stimuli were presented. In addition, decoding analyses revealed that only population cell firing of proximal CA3 together with that of distal CA1 can predict performance at test in the present non-spatial task. Overall, our work demonstrates an important role for the synchronization of CA3 neuronal activity with CA1 theta oscillations during memory testing.

Recalling gist memory depends on CA1 hippocampal neurons for lifetime retention and CA3 neurons for memory precision.

Why some of us remember events more clearly than others and why memory loses precision over time is a major focus in memory research. Here, we show that the recruitment of specific neuroanatomical pathways within the medial temporal lobe (MTL) of the brain defines the precision of the memory recalled over the lifespan. Using optogenetics, neuronal activity mapping, and studying recent to very remote memories, we report that the hippocampal subfield CA1 is necessary for retrieving the gist of events and receives maximal support from MTL cortical areas (MEC, LEC, PER, and POR) for recalling the most remote memories. In contrast, reduction of CA3's activity alone coincides with the loss of memory precision over time. We propose that a shift between specific MTL subnetworks over time might be a fundamental mechanism of memory consolidation.

Lesion of the hippocampus selectively enhances LEC's activity during recognition memory based on familiarity.

The sense of familiarity for events is crucial for successful recognition memory. However, the neural substrate and mechanisms supporting familiarity remain unclear. A major controversy in memory research is whether the parahippocampal areas, especially the lateral entorhinal (LEC) and the perirhinal (PER) cortices, support familiarity or whether the hippocampus (HIP) does. In addition, it is unclear if LEC, PER and HIP interact within this frame. Here, we especially investigate if LEC and PER's contribution to familiarity depends on hippocampal integrity. To do so, we compare LEC and PER neural activity between rats with intact hippocampus performing on a human to rat translational task relying on both recollection and familiarity and rats with hippocampal lesions that have been shown to then rely on familiarity to perform the same task. Using high resolution Immediate Early Gene imaging, we report that hippocampal lesions enhance activity in LEC during familiarity judgments but not PER's. These findings suggest that different mechanisms support familiarity in LEC and PER and led to the hypothesis that HIP might exert a tonic inhibition on LEC during recognition memory that is released when HIP is compromised, possibly constituting a compensatory mechanism in aging and amnesic patients.

Dendritic Kv4.2 potassium channels selectively mediate spatial pattern separation in the dentate gyrus.

The capacity to distinguish comparable experiences is fundamental for the recall of similar memories and has been proposed to require pattern separation in the dentate gyrus (DG). However, the cellular mechanisms by which mature granule cells (GCs) of the DG accomplish this function are poorly characterized. Here, we show that Kv4.2 channels selectively modulate the excitability of medial dendrites of dentate GCs. These dendrites are targeted by the medial entorhinal cortex, the main source of spatial inputs to the DG. Accordingly, we found that the spatial pattern separation capability of animals lacking the Kv4.2 channel is significantly impaired. This points to the role of intrinsic excitability in supporting the mnemonic function of the dentate and to the Kv4.2 channel as a candidate substrate promoting spatial pattern separation.

Single-cell memory trace imaging with immediate-early genes.

For the past decades, an increasing number of studies has taken advantage of molecular imaging methods involving the detection of immediate-early genes' (IEGs) expression for investigating neural substrates underlying plasticity processes and memory function. The detection of IEGs RNA by Fluorescent In-Situ Hybridization (FISH) yields single-cell as well as high temporal resolution and has recently enabled the mapping of medial temporal lobe subareas/subnetworks activity induced by single or multiple behavioural events in the same animal. After briefly reviewing the function and the ties of the typical IEGs (Fos, Zif268, Arc, Homer1a) used for mapping plasticity, we focus on discussing technical considerations vital for the successful detection of IEGs with FISH with emphasis on the design of RNA probes, the optimization of experimental conditions and the necessity for controls. Finally, we discuss recent developments in brain clearing methods that in combination with FISH detection of IEGs' expression allow for 3D imaging with single cell resolution as well as whole brain analyses. This, in parallel with the recent development of fMRI cognitive tasks in awake rats and the use of high resolution fMRI in humans, holds great promises for bridging further memory in humans and animals.

Spatial information is preferentially processed by the distal part of CA3: Implication for memory retrieval.

For the past decades, CA3 was considered as a single functional entity. However, strong differences between the proximal (close to the dentate gyrus) and the distal (close to CA2) parts of CA3 in terms of connectivity patterns, gene expression and electrophysiological properties suggest that it is not the case. We recently showed that proximal CA3 (together with distal CA1) preferentially deals with non-spatial information [1]. In contrast to proximal CA3, distal CA3 mainly receives and predominantly projects to spatially tuned areas. Here, we tested if distal CA3 preferentially processes spatial information, which would suggest a segregation of the spatial information along the proximodistal axis of CA3. We used a high-resolution imaging technique based on the detection of the expression of the immediate-early gene Arc, commonly used to map activity in the medial temporal lobe. We showed that distal CA3 is strongly recruited in a newly designed delayed nonmatching-to-location task with high memory demands in rats, while proximal CA3 is not. These results indicate a functional segregation of CA3 that mirrors the one reported in CA1, and suggest the existence of a distal CA3- proximal CA1 spatial subnetwork. These findings bring further evidence for the existence of 'specialized' spatial and non-spatial subnetworks segregated along the proximodistal axis of the hippocampus and put forward the 'segregated' view of information processing in the hippocampus as a reasonable alternative to the well-accepted 'integrated' view, according to which spatial and non-spatial information are systematically integrated in the hippocampus to form episodic memory.

The memory for time and space differentially engages the proximal and distal parts of the hippocampal subfields CA1 and CA3.

A well-accepted model of episodic memory involves the processing of spatial and non-spatial information by segregated pathways and their association within the hippocampus. However, these pathways project to distinct proximodistal levels of the hippocampus. Moreover, spatial and non-spatial subnetworks segregated along this axis have been recently described using memory tasks with either a spatial or a non-spatial salient dimension. Here, we tested whether the concept of segregated subnetworks and the traditional model are reconcilable by studying whether activity within CA1 and CA3 remains segregated when both dimensions are salient, as is the case for episodes. Simultaneously, we investigated whether temporal or spatial information bound to objects recruits similar subnetworks as items or locations per se, respectively. To do so, we studied the correlations between brain activity and spatial and/or temporal discrimination ratios in proximal and distal CA1 and CA3 by detecting Arc RNA in mice. We report a robust proximodistal segregation in CA1 for temporal information processing and in both CA1 and CA3 for spatial information processing. Our results suggest that the traditional model of episodic memory and the concept of segregated networks are reconcilable, to a large extent and put forward distal CA1 as a possible "home" location for time cells.

Spatial information is preferentially processed by the distal part of CA3: implication for memory retrieval.

For the past decades, CA3 was considered as a single functional entity. However, strong differences between the proximal (close to the dentate gyrus) and the distal (close to CA2) parts of CA3 in terms of connectivity patterns, gene expression and electrophysiological properties suggest that it is not the case. We recently showed that proximal CA3 (together with distal CA1) preferentially deals with non-spatial information [1]. In contrast to proximal CA3, distal CA3 mainly receives and predominantly projects to spatially tuned areas. Here, we tested if distal CA3 preferentially processes spatial information, which would suggest a segregation of the spatial information along the proximodistal axis of CA3. We used a high-resolution imaging technique based on the detection of the expression of the immediate-early gene Arc, commonly used to map activity in the medial temporal lobe. We showed that distal CA3 is strongly recruited in a newly designed delayed nonmatching-to-location task with high memory demands in rats, while proximal CA3 is not. These results indicate a functional segregation of CA3 that mirrors the one reported in CA1, and suggest the existence of a distal CA3- proximal CA1 spatial subnetwork. These findings bring further evidence for the existence of 'specialized' spatial and non-spatial subnetworks segregated along the proximodistal axis of the hippocampus and put forward the 'segregated' view of information processing in the hippocampus as a reasonable alternative to the well-accepted 'integrated' view, according to which spatial and non-spatial information are systematically integrated in the hippocampus to form episodic memory.

Noradrenergic activation of the basolateral amygdala maintains hippocampus-dependent accuracy of remote memory.

Emotional enhancement of memory by noradrenergic mechanisms is well-described, but the long-term consequences of such enhancement are poorly understood. Over time, memory traces are thought to undergo a neural reorganization, that is, a systems consolidation, during which they are, at least partly, transferred from the hippocampus to neocortical networks. This transfer is accompanied by a decrease in episodic detailedness. Here we investigated whether norepinephrine (NE) administration into the basolateral amygdala after training on an inhibitory avoidance discrimination task, comprising two distinct training contexts, alters systems consolidation dynamics to maintain episodic-like accuracy and hippocampus dependency of remote memory. At a 2-d retention test, both saline- and NE-treated rats accurately discriminated the training context in which they had received footshock. Hippocampal inactivation with muscimol before retention testing disrupted discrimination of the shock context in both treatment groups. At 28 d, saline-treated rats showed hippocampus-independent retrieval and lack of discrimination. In contrast, NE-treated rats continued to display accurate memory of the shock-context association. Hippocampal inactivation at this remote retention test blocked episodic-like accuracy and induced a general memory impairment. These findings suggest that the NE treatment altered systems consolidation dynamics by maintaining hippocampal involvement in the memory. This shift in systems consolidation was paralleled by time-regulated DNA methylation and transcriptional changes of memory-related genes, namely Reln and Pkmζ, in the hippocampus and neocortex. The findings provide evidence suggesting that consolidation of emotional memories by noradrenergic mechanisms alters systems consolidation dynamics and, as a consequence, influences the maintenance of long-term episodic-like accuracy of memory.

Recognition memory: Cellular evidence of a massive contribution of the LEC to familiarity and a lack of involvement of the hippocampal subfields CA1 and CA3.

A highly debated issue in memory research is whether familiarity is supported by the parahippocampal region, especially the lateral (LEC) and the perirhinal (PER) cortices, or whether it is supported by the same brain structure as recollection: the hippocampus. One reason for this is that conflicting results have emerged regarding the contribution of the hippocampus to familiarity. This might stem from the lack of dissociation between hippocampal subfields CA1 and CA3 as these areas are involved to a different extent in processes which are pertinent to familiarity. Another reason is that empirical evidence for a contribution of the LEC is still missing. Furthermore, it is unclear whether the superficial and the deep layers of the LEC would equally contribute to this process as these layers are differentially recruited during memory retrieval which partly relies on familiarity. To identify the specific contribution of the LEC, CA1, and CA3, we imaged with cellular resolution activity in the brain of rats performing a version of a standard human memory task adapted to rats that yields judgments based on familiarity. Using this translational approach, we report that in striking contrast to CA1 and CA3, the LEC is recruited for familiarity-judgments and that its contribution is comparable to that of the PER. These results show for the first time that the LEC, specifically its deep layers, contributes to familiarity and constitute the first cellular evidence that the hippocampus does not, thus establishing that familiarity does not share the same neural substrate as recollection.

Imaging a memory trace over half a life-time in the medial temporal lobe reveals a time-limited role of CA3 neurons in retrieval.

Whether retrieval still depends on the hippocampus as memories age or relies then on cortical areas remains a major controversy. Despite evidence for a functional segregation between CA1, CA3 and parahippocampal areas, their specific role within this frame is unclear. Especially, the contribution of CA3 is questionable as very remote memories might be too degraded to be used for pattern completion. To identify the specific role of these areas, we imaged brain activity in mice during retrieval of recent, early remote and very remote fear memories by detecting the immediate-early gene Arc. Investigating correlates of the memory trace over an extended period allowed us to report that, in contrast to CA1, CA3 is no longer recruited in very remote retrieval. Conversely, we showed that parahippocampal areas are then maximally engaged. These results suggest a shift from a greater contribution of the trisynaptic loop to the temporoammonic pathway for retrieval.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

The inhibitory avoidance discrimination task to investigate accuracy of memory.

The present study was aimed at developing a new inhibitory avoidance task, based on training and/or testing rats in multiple contexts, to investigate accuracy of memory. In the first experiment, male Sprague-Dawley rats were given footshock in an inhibitory avoidance apparatus and, 48 h later, retention latencies of each rat were assessed in the training apparatus (Shock box) as well as in a novel, contextually modified, apparatus. Retention latencies in the Shock box were significantly longer than those in the Novel box, indicating accurate memory of the training context. When the noradrenergic stimulant yohimbine (0.3 mg/kg, sc) was administered after the training, 48-h retention latencies in the Shock box, but not Novel box, were increased, indicating that the noradrenergic activation enhanced memory of the training experience without reducing memory accuracy. In the second experiment, rats were trained on an inhibitory avoidance discrimination task: They were first trained in an inhibitory avoidance apparatus without footshock (Non-Shock box), followed 1 min later by footshock training in a contextually modified apparatus (Shock box). Forty-eight-hour retention latencies in the Shock and Non-Shock boxes did not differ from each other but were both significantly longer than those in a Novel box, indicating that rats remembered the two training contexts but did not have episodic-like memory of the association of footshock with the correct training context. When the interval between the two training episodes was increased to 2 min, rats showed accurate memory of the association of footshock with the training context. Yohimbine administered after the training also enhanced rats' ability to remember in which training context they had received actual footshock. These findings indicate that the inhibitory avoidance discrimination task is a novel variant of the well-established inhibitory avoidance task suitable to investigate accuracy of memory.

Epigenetic modification of the glucocorticoid receptor gene is linked to traumatic memory and post-traumatic stress disorder risk in genocide survivors.

Recent evidence suggests that altered expression and epigenetic modification of the glucocorticoid receptor gene (NR3C1) are related to the risk of post-traumatic stress disorder (PTSD). The underlying mechanisms, however, remain unknown. Because glucocorticoid receptor signaling is known to regulate emotional memory processes, particularly in men, epigenetic modifications of NR3C1 might affect the strength of traumatic memories. Here, we found that increased DNA methylation at the NGFI-A (nerve growth factor-induced protein A) binding site of the NR3C1 promoter was associated with less intrusive memory of the traumatic event and reduced PTSD risk in male, but not female survivors of the Rwandan genocide. NR3C1 methylation was not significantly related to hyperarousal or avoidance symptoms. We further investigated the relationship between NR3C1 methylation and memory functions in a neuroimaging study in healthy subjects. Increased NR3C1 methylation-which was associated with lower NR3C1 expression-was related to reduced picture recognition in male, but not female subjects. Furthermore, we found methylation-dependent differences in recognition memory-related brain activity in men. Together, these findings indicate that an epigenetic modification of the glucocorticoid receptor gene promoter is linked to interindividual and gender-specific differences in memory functions and PTSD risk.

Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator.

Glucocorticoid receptor (GR) antagonism may be of considerable therapeutic value in stress-related psychopathology such as depression. However, blockade of all GR-dependent processes in the brain will lead to unnecessary and even counteractive effects, such as elevated endogenous cortisol levels. Selective GR modulators are ligands that can act both as agonist and as antagonist and may be used to separate beneficial from harmful treatment effects. We have discovered that the high-affinity GR ligand C108297 is a selective modulator in the rat brain. We first demonstrate that C108297 induces a unique interaction profile between GR and its downstream effector molecules, the nuclear receptor coregulators, compared with the full agonist dexamethasone and the antagonist RU486 (mifepristone). C108297 displays partial agonistic activity for the suppression of hypothalamic corticotropin-releasing hormone (CRH) gene expression and potently enhances GR-dependent memory consolidation of training on an inhibitory avoidance task. In contrast, it lacks agonistic effects on the expression of CRH in the central amygdala and antagonizes GR-mediated reduction in hippocampal neurogenesis after chronic corticosterone exposure. Importantly, the compound does not lead to disinhibition of the hypothalamus-pituitary-adrenal axis. Thus, C108297 represents a class of ligands that has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain, while retaining beneficial aspects of GR signaling.

Involvement of the insular cortex in regulating glucocorticoid effects on memory consolidation of inhibitory avoidance training.

Glucocorticoids are known to enhance the consolidation of memory of emotionally arousing experiences by acting upon a network of interconnected brain regions. Although animal studies typically do not consider the insular cortex (IC) to be part of this network, the present findings indicate that the IC is importantly involved in regulating glucocorticoid effects on memory consolidation of emotionally arousing inhibitory avoidance training. The specific glucocorticoid receptor (GR) agonist RU 28362 (3 or 10 ng in 0.5 µl) infused bilaterally into the IC of male Sprague-Dawley rats immediately after one-trial inhibitory avoidance training dose-dependently enhanced 48 h retention performance. Moreover, training on the inhibitory avoidance task increased neuronal activity of the IC, as assessed by an increased number of cells expressing immunoreactivity for phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2). However, systemic administration of a memory-enhancing dose of corticosterone (1 mg/kg) after inhibitory avoidance training rapidly reduced the number of pERK1/2-positive cells in the IC, suggesting that glucocorticoid administration reduces overall neuronal activity of the IC. To investigate which components of the inhibitory avoidance training experience were influenced by the intra-IC glucocorticoid administration, in the last experiment rats were trained on a modified inhibitory avoidance task in which context exposure and footshock training occur on two sequential days. RU 28362 administration into the IC enhanced later retention when infused immediately after either the context or footshock training. Thus, these findings indicate that the IC mediates glucocorticoid effects on the consolidation of memory of different components of inhibitory avoidance training and suggest that the IC might be an important element of the rodent brain network involved in emotional regulation of learning and memory.

Rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience.

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents(1-4), public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals(5)) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus(6).

Structure-activity relationship of etomidate derivatives at the GABA(A) receptor: Comparison with binding to 11beta-hydroxylase.

At the GABA(A) receptor, low concentrations of etomidate potentiate the inhibitory effect of GABA on specific binding of the closed channel ligand [(3)H]ethynylpropylbicycloorthobenzoate ([(3)H]EBOB). Here, we present SARs for etomidate and structurally related compounds inducing this effect. In the absence of GABA, similar SARs, but 14-20 times weaker potencies were observed. We discuss these SARs in comparison to the much higher potencies of these compounds as inhibitors of 11beta-hydroxylase.