Mystery of the memory engram: History, current knowledge, and unanswered questions.

The quest to understand the memory engram has intrigued humans for centuries. Recent technological advances, including genetic labelling, imaging, optogenetic and chemogenetic techniques, have propelled the field of memory research forward. These tools have enabled researchers to create and erase memory components. While these innovative techniques have yielded invaluable insights, they often focus on specific elements of the memory trace. Genetic labelling may rely on a particular immediate early gene as a marker of activity, optogenetics may activate or inhibit one specific type of neuron, and imaging may capture activity snapshots in a given brain region at specific times. Yet, memories are multifaceted, involving diverse arrays of neuronal subpopulations, circuits, and regions that work in concert to create, store, and retrieve information. Consideration of contributions of both excitatory and inhibitory neurons, micro and macro circuits across brain regions, the dynamic nature of active ensembles, and representational drift is crucial for a comprehensive understanding of the complex nature of memory.

Pathway specific activation of ventral hippocampal cells projecting to the prelimbic cortex diminishes fear renewal.

The ability to learn that a stimulus no longer signals danger is known as extinction. A major characteristic of extinction is that it is context-dependent, which means that fear reduction only occurs in the same context as extinction training. In other contexts, there is re-emergence of fear, known as contextual renewal. The ability to properly extinguish fear memories and generalize safety associations to multiple contexts provides therapeutic potential, but little is known about the specific neural pathways that mediate fear renewal and extinction generalization. The ventral hippocampus (VH) is thought to provide a contextual gating mechanism that determines whether fear or safety is expressed in particular contexts through its projections to areas of the fear circuit, including the infralimbic (IL) and prelimbic (PL) cortices. Moreover, VH principal cells fire in large, overlapping regions of the environment, a characteristic that is ideal to support generalization; yet it is unclear how different projection cells mediate this process. Using a pathway-specific (intersectional) chemogenetic approach, we demonstrate that selective activation of VH cells projecting to PL attenuates fear renewal without affecting fear expression. These results have implications for anxiety disorders since they uncover a neural pathway associated with extinction generalization.

Receptor-stimulated phospholipase A(2) liberates arachidonic acid and regulates neuronal excitability through protein kinase C.

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca(2+) increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca(2+), co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA(2) stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 microM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 microM). Furthermore, direct stimulation of PLA(2) by melittin (10 microM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 microM) and light (to elevate intracellular Ca(2+)), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 microM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for > or =90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 microm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca(2+) to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca(2+) may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.

Synaptic efficacy is commonly regulated within a nervous system and predicts individual differences in learning.

The hypothesis that an individual's capacity for learning might be predicted or influenced by basal levels of synaptic efficacy has eluded empirical tests, owing in part to the inability to compare between animals single identified synaptic responses in the mammalian brain. To overcome this limitation, we have focused our analysis on the invertebrate Hermissenda, whose nervous system is composed of identifiable cells and synaptic interactions. Hermissenda were exposed to paired presentations of light and rotation such that the light came to elicit a learned defensive motor response. An animal's rate of learning was strongly correlated with the amplitude of the synaptic potential evoked in that animal's visual (light sensitive) receptors in response to stimulation of presynaptic vestibular (rotation sensitive) hair cells. In naive animals, strong correlations between the amplitude of both inhibitory and excitatory synaptic potentials were observed between synapses distributed throughout an animal's nervous system, and this conservation of synaptic efficacy was largely attributable to a common influence on transmitter release. These observations suggest that basal synaptic efficacy may be uniformly regulated throughout a nervous system, and provide direct evidence that the basal efficacy of synaptic transmission predicts, and possibly contributes to, individual differences between animals in their capacity to learn.

ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors.

MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

Neurophysiological substrates of context conditioning in Hermissenda suggest a temporally invariant form of activity-dependent neuronal facilitation.

The neurophysiological basis for context conditioning is conceptually problematic because neurophysiological descriptions of activity-dependent (associative) forms of neuronal plasticity uniformly assume that a specific temporal relationship between signals is necessary for memory induction. In the present experiments, this problem is addressed empirically by presenting, as a temporally diffuse contextual signal, a stimulus that results in known neural modifications following punctate (temporally contiguous) pairings with an aversive unconditioned stimulus. Hermissenda were trained to discriminate between adjoining contexts that were distinguished only in that one was lit and one was dark. Thirty unsignaled rotations were presented during each of three 15-min sessions in one of the two (lit or dark) contexts. Prior to training, animals displayed a slight preference for the lit context. After exposure to unsignaled rotation, animal's preferences shifted strongly to the dark context if unsignaled rotations were presented in the light, and tended (nonsignificantly) to the lit context if unsignaled rotations were presented in the dark. The B photoreceptors of the Hermissenda eye undergo several forms of activity-dependent facilitation (e.g., an increase in neuronal input resistance and evoked spike frequency) following pairings of punctate light (CS) and presynaptic vestibular stimulation (US). Similar facilitation in the B photoreceptor was observed following in vitro training that mimicked context conditioning in which presynaptic vestibular stimulation was presented repetitively during a continuous 7.5-min light. Subsequently, Ca(2+)-imaging experiments were conducted with Fura-2AM. It was determined that intracellular Ca(2+), the CS-induced second messenger critical for the induction of activity-dependent facilitation, was elevated in the B photoreceptor throughout the 7.5-min light presentation. These results indicate that activity-dependent facilitation within similar neural structures can underlie learning about both temporally diffuse contextual stimuli and temporally punctate CS-US pairings. These results suggest that a common mechanism may underlie learning about diffuse contextual stimuli as well as punctate-conditioned stimuli, provided that the stimuli are processed similarly in each type of conditioning arrangement. Consequently, the expression of different responses to contextual and discrete stimuli are likely to reflect a higher property of the neural network, and do not necessarily arise from unique underlying mechanisms.

Interactive contributions of intracellular calcium and protein phosphatases to massed-trials learning deficits in Hermissenda.

Using Hermissenda as subjects, massed-trials training deficits were examined. Associative pairings of light and rotation induced a progressively greater conditioned foot contraction in response to light as the intertrial interval (ITI) was extended (up to 8 min). In contrast, a short ITI (30 s) produced no evidence of learning. In a corresponding in vitro conditioning experiment that mimicked training of the intact animal, facilitation of neuronal excitability in the animal's B photoreceptors paralleled the results obtained in vivo. Imaging of intracellular Ca2+ using Fura-2 indicated that Ca2+ levels remained elevated during short ITIs. This Ca2+ accumulation appears to induce activation of protein phosphatases because normal facilitation of the B photoreceptors was induced with a short ITI if training occurred in the presence of a phosphatase inhibitor. These results suggest that intracellular Ca2+ and protein phosphatases contribute interactively to the kinetics of memory formation and provide evidence that an accumulation of intracellular Ca2+ across training trials may impede memory formation.

Ubiquitous molecular substrates for associative learning and activity-dependent neuronal facilitation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction.

Intracellular Ca2+ and adaptation of voltage responses to light in Hermissenda photoreceptors.

Fluorescent imaging of Ca2+ and intracellular recordings were used to assess Ca2+ increases and voltage responses during light presentations in Hermissenda B photoreceptors. Ca2+ levels increased and were sustained during a relatively long exposure to light. Repeated presentations of a brief light induced an elevation of intracellular Ca2+ that persisted throughout short interlight intervals, but which dissipated during long interlight intervals. In all instances, the magnitude of the intracellular Ca2+ signal was inversely related to the amplitude of the light-induced generator potential. Blocking of voltage-dependent Ca2+ channels did not significantly affect the magnitude of the Ca2+ signal, suggesting that the intracellular Ca2+ response arises primarily from release from intracellular stores. These results indicate that Ca2+ plays an important role in the modulation of the voltage responses to light, acting to suppress the response during repetitive or prolonged stimulation.

Protein synthesis-dependent memory and neuronal enhancement in Hermissenda are contingent on parameters of training and retention.

Following contiguous pairings of light and rotation, light alone elicits a conditioned contraction of Hermissenda's foot, indicative of an associative memory. After a 5-min retention interval, this conditioned response was evident following two or nine (but not one) conditioning trials but persisted for 90 min only after nine trials. In vivo incubation of animals in the protein synthesis inhibitor anisomycin (ANI; 1 microM) did not affect the conditioned response at the 5-min retention interval but significantly attenuated conditioned responding at the 90-min interval even following nine training trials. Deacetylanisomycin (DANI; 1 microM; an inactive form of anisomycin) had no effect on either 5- or 90-min retention. In a companion procedure, groups of isolated nervous systems were exposed to comparable light and rotation pairings, and the B photoreceptors (considered a site of storage for the associative memory) underwent electrophysiological analysis. An increase in neuronal excitability (indexed by depolarizing voltage responses to injected current) in the B photoreceptors paralleled the expression of conditioned responding in intact animals, that is, two training trials produced a short-term increase in excitability that dissipated within 45 min, whereas nine trials produced a persistent (at least 90-min) increase in excitability. In a fmal experiment, isolated nervous systems were exposed to nine training trials, and ANI or DANI was either present in the bathing medium before and during training or was introduced 5 min after training. Following training in ANI, a short-term (5- to 45-min) but not persistent (90-min) increase in excitability in the B photoreceptors was observed. ANI had no effect on either the short-term or persistent increase in excitability if the drug was applied 5 min after the last (ninth) training trial, and DANI had no effect on training-induced increases in excitability at any retention intervals. These results suggest that short-term retention in Hermissenda is protein synthesis independent but that new protein synthesis initiated during or shortly after the training event is necessary for even 90-min retention. Moreover, these results indicate that under some conditions, a critical threshold of training must be exceeded to initiate protein synthesis-dependent retention.

Incremental redistribution of protein kinase C underlies the acquisition curve during in vitro associative conditioning in Hermissenda.

An incremental increase in the excitability (i.e., input resistance, evoked spike frequency) of B photoreceptors in Hermissenda accompanied successive pairings of light and presynaptic stimulation of vestibular hair cells (simulating light-rotation pairings in an intact animal). Analysis of protein kinase C (PKC) in the Hermissenda's photoreceptors indicated a training-induced incremental reduction of PKC in cytosolic compartments, a tendency toward an increase in membrane compartments, and a small decrease in total enzyme activity (possibly owing to a downregulation or conversion of PKC to a calcium-independent state). Neither the biophysical or biochemical effects were observed in Hermissenda exposed to unpaired light and rotation or in those trained in the presence of the selective PKC inhibitor NPC-15437 (which had no effect on synaptic interactions or light-induced generator potentials). These results suggest that the intracellular redistribution of a protein kinase contributes critically to the kinetics of new learning.

Phospholipases and arachidonic acid contribute independently to sensory transduction and associative neuronal facilitation in Hermissenda type B photoreceptors.

During contiguous pairings of light and rotation, B photoreceptors in the Hermissenda eye undergo an increase in excitability that contributes to a modification of several light-elicited behaviors. This excitability increase requires a light-induced rise in intracellular Ca2+ in the photoreceptor concomitant with transmitter binding to G protein-coupled receptors as a result of presynaptic vestibular hair cell stimulation. Phospholipases and arachidonic acid (ArA) are here reported to be involved in independent signal transduction pathways that underlie both receptor function and activity-dependent facilitation of the B photoreceptor. 4-Bromophenacyl bromide (BPB), an inhibitor of phospholipases A2 (PLA2) and C (PLC), blocked the generation of light-induced depolarizing generator potentials, but had no affect on the inhibitory postsynaptic potential (IPSP) in the B cell that results from hair cell stimulation. Quinacrine, which predominantly blocks the activity of PLA2 in neurons, had no affect on either the light response or the IPSP, but did block increases in excitability (i.e. increased input resistance and elicited spike rate) of the B cell that results from pairings of light and presynaptic vestibular stimulation (i.e., in vitro associative conditioning). Neither nordihydroquararetic acid (NDGA), which inhibits metabolism of ArA by cyclooxygenase, nor indomethacin, which inhibits lipoxygenase metabolism of ArA, affected the light response or IPSP, but both blocked the increases in excitability in the B cell that accompanied in vitro conditioning. In combination with earlier results, these data suggest that ArA activates PKC in a synergistic fashion with Ca2+ and diacylglycerol in the B cell, and suggest that PLA2-induced ArA release, though not necessary for transduction of light or the hair cell-induced IPSP in the B cell, is a critical component of the convergence of signals that precipitates associative facilitation in this system.

Timing effects of postevent information on infant memory.

The role of the timing of postevent information on retention was assessed with 124 6-month-olds. In four experiments, infants learned to kick to move a particular crib mobile (the original target), were passively exposed to a discriminably different mobile (the postevent information), and were tested for recognition of the original mobile, the postevent-exposure mobile, or a completely novel mobile 24 h later. All interpolated exposures occurred after delays when infants' retention of the training mobile is excellent. In Experiment 1, the interpolated information precluded recognition of the training mobile (memory impairment) after all exposure delays. In Experiment 2, when the interpolated information was exposed within a week of training, infants treated the exposure mobile as if they had actually been trained with it (memory facilitation). Despite the recognition failures in Experiments 1-2, both original and exposure mobiles later reactivated the training memory, but only if the interpolated exposure was early in the retention interval; if it was later, only the exposure mobile was an effective reminder (Experiments 3A-3B). In Experiment 4, exposing postevent information after longer delays led infants to respond to a completely novel mobile (categorization). These findings demonstrate that postevent information has different qualitative effects depending on its timing and provides a basis for understanding discrepant reports of postevent-information effects with children and adults.

Variations in learning reflect individual differences in sensory function and synaptic integration.

With the invertebrate Hermissenda as subjects, variability in acquisition of a learned association between light and rotation was correlated with the magnitude of the unconditioned responses elicited by these stimuli. Moreover, learning was facilitated by increasing stimulus intensity. In the isolated nervous system, pairings of light and mechanical stimulation of the animal's vestibular hair cells resulted in an increase in the excitability of B photoreceptors (an in vitro index of learning) that was strongly correlated with the strength of the synaptic interaction between the hair cells and the photoreceptors and weakly correlated with the magnitude of the light response in the photoreceptors. Because these in vitro results are not attributable to motor or motivational variables, they suggest that the efficacy of synaptic integration between sensory systems and sensory transduction is the primary determinant of the variability in learning.

Diverse current and voltage responses to baclofen in an identified molluscan photoreceptor.

1. gamma-Aminobuturic acid-B (GABAB) receptors play a role in the mediation of slow inhibitory postsynaptic potentials in mammalian as well as some nonmammalian species. In identified photoreceptors from the marine mollusc Hermissenda, recent evidence has suggested that GABA, as well as the GABAB receptor agonist baclofen, might simultaneously modulate multiple conductances on the postsynaptic membrane. Here, using intracellular current-clamp and single-electrode voltage-clamp techniques, we have characterized responses to baclofen in the B photoreceptors of the Hermissenda eye. 2. Microapplication of baclofen (12.5-62.5 microM) to the terminal branches of the B photoreceptors induced a slow, concentration-dependent hyperpolarization (approximately 3-8 mV) that was accompanied by a cessation of spontaneous action potentials and a positive shift in firing threshold. Both the hyperpolarization and the shift in spike threshold in response to baclofen were attenuated largely by the K+ channel blocker tetraethylammonium chloride (TEA; 50 mM). 3. Bath application of baclofen (100 microM) decreased the amplitude, duration, and the afterhyperpolarization (AHP) of evoked action potentials. Although baclofen's effect on spike duration and amplitude persisted in the absence of extracellular Ca2+, the reduction of the AHP by baclofen was eliminated, suggesting that multiple conductances mediated the baclofen-induced modification of the action potential. 4. Using a single-electrode voltage-clamp technique, microapplication of baclofen to the terminal branches of the B photoreceptor produced a slow, net outward current (< 0.5 nA) that reversed near the equilibrium potential for K+ and shifted to more positive potentials when extracellular K+ was increased, in approximate agreement with the Nernst equation for K+. 5. Baclofen induced an increase in amplitude of the nonvoltage dependent leak conductance (IL), and the increase was blocked by TEA. The baclofen-induced increase of IL was accompanied by an increase in amplitude and a negative shift in the voltage dependence of a slow, steeply voltage-dependent K+ current (IK), which displays selective sensitivity to TEA but does not normally contribute to leak conductance. The amplitude and steady-state inactivation of a fast, transient K+ current, as well as the amplitude of an inwardly rectifying K+ current were unaffected by baclofen. 6. Both the rate of activation as well as the amplitude of a voltage-dependent Ca2+ current (ICa) were reduced by baclofen. The reduction of ICa resulted in a concomitant suppression of a Ca(2+)-dependent K+ current (IK-Ca) that was sufficient to account for the reduction of the AHP after evoked action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)