Two important themes in Ivan Izquierdo's research each offered both answers and questions about the topic of memory formation and maintenance. The first theme provided evidence supporting the view that short-and long-term memory were distinct processes and could be selectively modulated by several treatments, with some affecting only short-term, others only affecting long-term memory, and still others affecting both. Over many years, Izquierdo's laboratory documented molecular responses across time after training obtaining results that showed differences as well as similarities in the biochemical changes during the first 1-2 h and the next 4-6 h after training, i.e., during the transition from short- to long-term memory. This work clarified the biological underpinnings of the memory processes. The second theme described waves of susceptibility of memory to enhancing and impairing treatments after time, a biphasic profile that contrasted with earlier monotonic decreases in the efficacy of memory modulating treatments as a function of time between training and treatment. Remarkably, these waves of susceptibility to modification were accompanied by biphasic changes in molecular measures at similar times after training. Remarkably, some of the molecular players exhibited persistent changes after training, with increases in levels lasting days following the training experience. These persistent molecular changes may reveal a biological basis for the dynamic nature of memories seen long after the initial memory is consolidated. This article is part of a Special Issue entitled: In memory of Ivan Izquierdo South American pioneer of the Neuroscience of Memory Temporal dynamics and molecular mechanisms. (C) 2022 IBRO. Published by Elsevier Ltd. All rights reserved.

Studies of age-related changes in learning and memory often focus on hippocampus-sensitive tasks and reveal age-associated impairments across numerous species and contexts. However, cognitive decline with advanced age is not all-encompassing; for example, forms of striatum-sensitive learning are conserved or enhanced with age. Under certain conditions, hippocampal and striatal memory systems function in opposition. In young adult rodents, disruption of one structure can enhance learning on tasks dependent on the other, suggesting that competitive interactions across memory systems contribute to learning and memory abilities. This report examines whether imbalances across memory systems might contribute to cognitive aging. We inactivated the striatum using central infusions of lidocaine (sodium channel blocker) prior to hippocampus-sensitive spatial (place) training in young (3-4-month-old) and old (24-25-month-old) F344 male rats. Consistent with prior work, vehicle-infused old rats exhibited place learning impairments relative to young rats. Additionally, striatal inactivation enhanced learning in old rats, but not young rats, abolishing the age-related impairment. These findings suggest that age-related declines in learning tasks thought to engage the hippocampus may stem from exaggerated interference from other memory systems and that interventions to target the striatum may reverse some age-related learning decrements.

Recent evidence suggests that astrocytes convert glucose to lactate, which is released from the astrocytes and supports learning and memory. This report takes a multiple memory perspective to test the role of astrocytes in cognition using real-time lactate measurements during learning and memory. Extracellular lactate levels in the hippocampus or striatum were determined with lactate biosensors while rats were learning place (hippocampus-sensitive) or response (striatum-sensitive) versions of T-mazes. In the first experiment, rats were trained on the place and response tasks to locate a food reward. Extracellular lactate levels in the hippocampus increased beyond those of feeding controls during place training but not during response training. However, striatal lactate levels did not increase beyond those of controls when rats were trained on either the place or the response version of the maze. Because food ingestion itself increased blood glucose and brain lactate levels, the contribution of feeding may have confounded the brain lactate measures. Therefore, we conducted a second similar experiment using water as the reward. A very different pattern of lactate responses to training emerged when water was used as the task reward. First, provision of water itself did not result in large increases in either brain or blood lactate levels. Moreover, extracellular lactate levels increased in the striatum during response but not place learning, whereas extracellular lactate levels in the hippocampus did not differ across tasks. The findings from the two experiments suggest that the relative engagement of the hippocampus and striatum dissociates not only by task but also by reward type. The divergent lactate responses of the hippocampus and striatum in place and response tasks under different reward conditions may reflect ethological constraints tied to foraging for food and water.

When administered near the time of training, protein synthesis inhibitors such as anisomycin impair later memory. A common interpretation of these findings is that memory consolidation requires new protein synthesis initiated by training. However, recent findings support an alternative interpretation that abnormally large increases in neurotransmitter release after injections of anisomycin may be responsible for producing amnesia. In the present study, a local anesthetic was administered prior to anisomycin injections in an attempt to mitigate neurotransmitter actions and thereby attenuate the resulting amnesia. Rats received lidocaine and anisomycin injections into the amygdala 130 and 120 min, respectively, prior to inhibitory avoidance training. Memory tests 48 h later revealed that lidocaine attenuated anisomycin-induced amnesia. In other rats, in vivo microdialysis was performed at the site of amygdala infusion of lidocaine and anisomycin. As seen previously, anisomycin injections produced large increases in release of norepinephrine in the amygdala. Lidocaine attenuated the anisomycin-induced increase in release of norepinephrine but did not reverse anisomycin inhibition of protein synthesis, as assessed by c-Fos immunohistochemistry. These findings are consistent with past evidence suggesting that anisomycin causes amnesia by initiating abnormal release of neurotransmitters in response to the inhibition of protein synthesis.

These experiments examined the effects of prior stress, corticosterone, or epinephrine on learning in mazes that can be solved efficiently using either place or response strategies. In a repeated stress condition, rats received restraint stress for 6 h/day for 21 days, ending 24 h before food-motivated maze training. In two single stress conditions, rats received a 1-h episode of restraint stress ending 30 min or 24 h prior to training. Single stress ending 30 min prior to training resulted in a significant interaction of stress and learning on the two tasks, with significant enhancement of learning in the response task and non-significant impairment in the place task. Neither acute nor chronic stress significantly altered learning in either task when the stress ended 24 h before training. Thus, the anterograde effects of stress on maze learning ended within a single day. Two stress-related hormones, corticosterone and epinephrine, were tested for effects on learning parallel to those of acute stress. When administered 30 min prior to training, a corticosterone dose (40 mg/kg) that enhanced memory on a spontaneous alternation task did not significantly enhance or impair learning in either task. Two doses of epinephrine that modulate memory in other settings were used to test the effects of epinephrine on learning. Pre-training injections of 0.03 mg/kg epinephrine impaired place learning, while 0.1 mg/kg epinephrine impaired response learning. The epinephrine results mimicked those seen with acute stress on the place task, but were opposite those seen after acute stress on the response task. Thus, corticosterone does not appear to be a major factor mediating the effects of acute stress on place and response learning and epinephrine is, at most, a partial contributor to these effects.

Spatial working memory and the ability of a cholinesterase inhibitor to enhance memory were assessed at 4, 10, and 16 months of ages in control and Ts65Dn mice, a partial trisomy model of Down syndrome, with possibly significant relationships to Alzheimer’s disease as well. In addition, ACh release during memory testing was measured in samples collected from the hippocampus using in vivo microdialysis at 4, 10, and 22–25 months of age. When tested on a four-arm spontaneous alternation task, the Ts65Dn mice exhibited impaired memory scores at both 4 and 10 months. At 16 months, control performance had declined toward that of the Ts65Dn mice and the difference in scores across genotypes was not significant. Physostigmine (50μg/kg) fully reversed memory deficits in the Ts65Dn mice in the 4-month-old group but not in older mice. Ts65Dn and control mice exhibited comparable baseline levels of ACh release at all ages tested; these levels did not decline significantly across age in either genotype. ACh release increased significantly during alternation testing only in the young Ts65Dn and control mice. However, the increase in ACh release during alternation testing was significantly greater in control than Ts65Dn mice at this age. The controls exhibited a significant age-related decline in the testing-related increase in ACh release. With only a small increase during testing in young Ts65Dn mice, the age-related decline in responsiveness of ACh release to testing was not significant in these mice. Overall, these results suggest that diminished responsiveness of ACh release in the hippocampus to behavioral testing may contribute memory impairments in Ts65Dn mice.

This chapter reviews a range of pharmacological treatments that enhance memory formation. Notably, many drugs that enhance memory do so by acting at receptors of hormones or neurotransmitters and are effective when administered near the time of training. Of particular interest are the demonstrations that the drugs act when administered soon after training, in experiments using a posttraining design that distinguishes effects on memory from effects on non-mnemonic factors such as motivation or sensory-motor functions. Much of the attention given to understanding the molecular bases of memory has focused on interfering with signaling cascades downstream from neurotransmitter receptor activation, attributing memory impairments to substrate mechanisms by which memories are made. Additionally, consideration of the molecular biology that underlies enhancement of memory may prove to be a fruitful area of research.

This special issue of Neurobiology of Learning and Memory is devoted to the question of the role of protein synthesis in learning and memory. Prospective authors with very different views on this subject were invited to contribute to the special issue. Happily, most of those invited quickly agreed to participate. All papers were peer-reviewed and most were revised prior to publication. The result is a collection of papers representing significant diversity of opinion regarding the role of protein synthesis in memory formation. The authors took different approaches to represent their views of the role of protein synthesis in memory, with formats including broad reviews, targeted reviews, and experimental papers. The goal of collecting these papers was not to find a single clear view, laying to rest one alternative view or another—a rather delusional goal at best. Instead, the attempt was to provide a venue through which different perspectives could appear together, with the understanding that all contributors are interested in a common purpose, to identify the ways in which brains make and hold new memories. When approached with a description of the theme for this special issue, the authors were also provided with some general questions that they might use to guide the preparation of their papers. What current data provide the best evidence for and against the view that memory formation passes through protein synthesis dependent and independent stages? What is the significance of observations of different rates for onset of amnesia, as well as for decay of LTP, within and across laboratories and procedures? What future data are needed to provide the best evidence for and against the view that memory formation passes through protein synthesis dependent and independent stages? Across these papers, there is agreement on the basic findings. All authors agree that proteins and protein synthesis are important to memory formation, but disagree on the question of whether new protein synthesis specifically triggered by an event is important for the formation of memory for that event. Some of the alternatives suggested include protein synthesis needed to maintain cell integrity, to replenish proteins ‘consumed’ by plasticity mechanisms, and to provide particular proteins that might be modified by experience, with long-lasting modification perhaps themselves representing cellular memory. The authors also agree that protein synthesis inhibitors impair memory but disagree about whether such findings reveal a core mechanism of memory formation. There is related disagreement regarding whether the loss of protein synthesis following inhibition is responsible for the amnesia. In part, the different views stem from the idea that the mechanisms of amnesia need not be the inverse of the mechanisms of memory. Some of the opposing views are based on the distinction between mechanisms of amnesia and mechanisms of memory. A second issue is whether inhibition of protein synthesis is a primary cause of amnesia produced by protein synthesis inhibitors or a side effect of these drugs. An extension of the latter view is that inhibition of protein synthesis induces neural responses to the challenge, and these responses then produce amnesia, i.e., that the mechanisms of amnesia are initiated by protein synthesis inhibition but the inhibition, per se, does not cause amnesia. Embedded in the various perspectives offered here are very different ideas about the significance of timing. Some writers see relatively fixed times for retrograde and anterograde amnesia after administration of protein synthesis inhibitors and other treatments, taking this evidence to imply two-process—e.g., early/late, short-term/long-term—theories of memory formation. Other writers see widely variable times for retrograde and anterograde amnesia, taking this as evidence for high-order multiple processes of memory formation. Still others compare the slow times of onset of amnesia and decay of LTP to the fast times of synaptic physiological and anatomical plasticity and suggest that the molecular changes occur after the plasticity is complete, and therefore attempt to assign other functional roles to the molecular changes triggered by experiences. The diversity of opinion collected in this special issue, and briefly summarized here, offers an opportunity for readers to examine how different researchers, each sharing a common goal of understanding how memories are made, can view the same data set and come away with disparate opinions. In this way, the readers may find this discourse useful in identifying the important questions, if not the answers, surrounding the roles of protein synthesis in memory.

These experiments examined the release of acetylcholine in the hippocampus and striatum when rats were trained, within single sessions, on place or response versions of food-rewarded mazes. Microdialysis samples of extra-cellular fluid were collected from the hippocampus and striatum at 5-min increments before, during, and after training. These samples were later analyzed for ACh content using HPLC methods. In Experiment 1, ACh release in both the hippocampus and striatum increased during training on both the place and response tasks. The magnitude of increase of training-related ACh release in the striatum was greater in rats trained on the response task than in rats trained on the place task, while the magnitude of ACh release in the hippocampus was comparable in the two tasks. Experiment 2 tested the possibility that the hippocampus was engaged and participated in learning the response task, as well as the place task, because of the availability of extra-maze cues. Rats were trained on a response version of a maze under either cue-rich or cue-poor conditions. The findings indicate that ACh release in the hippocampus increased similarly under both cue conditions, but declined during training on the cue-poor condition, when spatial processing by the hippocampus would not be suitable for solving the maze. In addition, high baseline levels of ACh release in the hippocampus predicted rapid learning in the cue-rich condition and slow learning in the cue-poor condition. These findings suggest that ACh release in the hippocampus augments response learning when extra-maze cues can be used to solve the maze but impairs response learning when extra-maze cues are not available for use in solving the maze.

The present experiments examined the effects of injecting glucose into the dorsal hippocampus or dorsolateral striatum on learning rates and on strategy selection in rats trained on a T-maze that can be solved by using either a hippocampus-sensitive place or striatum-sensitive response strategy. Percentage strategy selection on a probe trial (P(crit)) administered after rats achieved criterion (nine of 10 correct choices) varied by group. All groups predominately exhibited a response strategy on a probe trial administered after overtraining, i.e., after 90 trials. In experiment 1, rats that received intrahippocampal glucose injections showed enhanced acquisition of the T-maze and showed increased use of response solutions at P(crit) compared with that of unimplanted and artificial cerebral spinal fluid (aCSF)-treated groups. These findings suggest that glucose enhanced hippocampal functions to accelerate the rate of learning and the early adoption of a response strategy. In experiment 2, rats that received intrastriatal glucose injections exhibited place solutions early in training and reached criterion more slowly than did aCSF controls, with learning rates comparable to those of unoperated and operated-uninjected controls. Relative to unoperated, operated-uninjected and glucose-injected rats, rats that received intrastriatal aCSF injections showed enhanced acquisition of the T-maze and increased use of response solutions at P(crit). The unexpected enhanced acquisition seen after striatal aCSF injections suggests at least two possible interpretations: (1) aCSF impaired striatal function, thereby releasing competition with the hippocampus and ceding control over learning to the hippocampus during early training trials; and (2) aCSF enhanced striatal functioning to facilitate striatal-sensitive learning. With either interpretation, the results indicate that intrastriatal glucose injections compensated for the aCSF-induced effect. Finally, enhanced acquisition regardless of treatment was accompanied by rapid adoption of a response solution for the T-maze.

Although baseline blood glucose levels in aged Fischer-344 rats are comparable to those of young rats, the rise in blood glucose in response to training-related stress is substantially attenuated. The diminished response may contribute to increased depletion of extracellular brain glucose levels during training in aged rats; the depletion is blocked and memory is enhanced by systemic injections of glucose. The present experiment examined the role of glucose in regulating memory for reward reduction training. Blood glucose levels exhibited a significant rise after reward reduction trials in young adult but not 2-year-old rats. Although young and aged rats exhibited comparable learning during the day of reward reduction training, aged rats exhibited more rapid forgetting of the learning response. Post-training glucose injections (200 mg/kg, i.p.) facilitated memory formation and slowed the rate of forgetting in young and old rats, consistent with the view that deficiencies in circulating glucose responses to training may contribute to the rapid forgetting evident in aged Fischer-344 rats.

Extensive evidence supports the view that cholinergic mechanisms modulate learning and memory formation. This paper reviews evidence for cholinergic regulation of multiple memory systems, noting that manipulations of cholinergic functions in many neural systems can enhance or impair memory for tasks generally associated with those neural systems. While parallel memory systems can be identified by combining lesions with carefully crafted tasks, most—if not all—tasks require the combinatorial participation of multiple neural systems. This paper offers the hypothesis that the magnitude of acetylcholine (ACh) release in different neural systems may regulate the relative contributions of these systems to learning. Recent studies of ACh release, obtained with in vivo microdialysis samples during training, together with direct injections of cholinergic drugs into different neural systems, provide evidence that release of ACh is important in engaging these systems during learning, and that the extent to which the systems are engaged is associated with individual differences in learning and memory.