Systems analysis of cell-scale reconstructions has become a staple of modern biological discovery in the era of high -throughput data generation. Reconstructions of bacteria currently assimilate the existing wealth of biochemical knowledge for a given organism, accounting for metabolites and the stoichiometry of their transport and transformation via enzymes. However, the bulk of the material and energy consumed during cellular growth is devoted to the synthesis of the macromolecules involved in information transfer: RNA, protein, and the genome itself. In this dissertation, a framework for incorporating the fundamental processes of bacterial transcription and translation is described. This framework is used to integrate mRNA expression and half-life data towards assessing the global transcription state of the Escherichia coli genome under numerous experimental conditions. To address how the translation network would be constrained within this framework, a model is used to determine the theoretical limits in translational efficiency achievable by altering the synonymous codon usage of each gene given measured tRNA abundances. A sensitivity analysis of translational efficiency, which can be varied by an average 6.5-fold, demonstrates that wild-type synonymous codon usage and measured tRNA abundances in E. coli are highly synchronized. However, the results from these studies also expose the limitations of network reconstructions that neglect three-dimensional spatial information. The transcription state of a genome can be highly nonrandom with respect to chromosomal position. Furthermore, tRNA diffusion limitations must be taken into account to accurately model translational efficiency. This dissertation thus advances a method for characterizing the spatial organization of chromosome position-dependent data. A comprehensive assessment of the periodic pattern content contained within 163 prokaryotic chromosomes is performed using wavelet analysis. The degree of patterning in sequence-derived properties correlates with genome-size, overall GC-content, and the occurrence of motility and chromosomal-binding proteins. Given additional functional data for E. coli, long-range patterns in multiple heterogeneous properties are shown to be highly correlated and are consistent with experimentally detected chromosomal macrodomains. Taken together, the findings reported in this dissertation demonstrate that the field of cell-scale modeling will ultimately enter a phase in which network connectivity is viewed within the context of topobiological, spatial constraints

A critical feature of episodic memory is the ability to remember the order of events as they occurred in time, a capacity shared across species including humans, nonhuman primates, and rodents. Accumulating evidence suggests that this capacity depends on a network of structures including the hippocampus and the prefrontal cortex, but their respective contributions remain poorly understood. As addressing this important issue will require converging evidence from complementary investigative techniques, we developed a cross-species, nonspatial sequence memory task suitable for behavioral and neurophysiological studies in rodents and in humans. The task involves the repeated presentation of sequences of items (odors in rats and images in humans) and requires subjects to make a judgment as to whether each item is presented in sequence or out of sequence. To shed light on the cognitive processes and sequence representations supporting performance, different types of out of sequence probe trials were used including: (i) repeating an item from earlier in the sequence (Repeats; e.g., ABAD), (ii) skipping ahead in the sequence (Skips; e.g., ABD), and (iii) inserting an item from a different sequence into the same ordinal position (Ordinal Transfers; e.g., A2CD). We found a remarkable similarity in the performance of rats and humans, particularly in the pattern of results across probe trial types. Thus, the results suggest that rats and humans not only remember the sequences of events, but also use similar underlying cognitive processes and mnemonic representations. This strong cross-species correspondence validates this task for use in future basic and clinical interdisciplinary studies aimed at examining the neural mechanisms underlying episodic memory.

Considerable research in rodents and humans indicates the hippocampus and prefrontal cortex are essential for remembering temporal relationships among stimuli, and accumulating evidence suggests the perirhinal cortex may also be involved. However, experimental parameters differ substantially across studies, which limits our ability to fully understand the fundamental contributions of these structures. In fact, previous studies vary in the type of temporal memory they emphasize (e.g., order, sequence, or separation in time), the stimuli and responses they use (e.g., trial-unique or repeated sequences, and incidental or rewarded behavior), and the degree to which they control for potential confounding factors (e.g., primary and recency effects, or order memory deficits secondary to item memory impairments). To help integrate these findings, we developed a new paradigm testing incidental memory for trial-unique series of events, and concurrently assessed order and item memory in animals with damage to the hippocampus, prefrontal cortex, or perirhinal cortex. We found that this new approach led to robust order and item memory, and that hippocampal, prefrontal and perirhinal damage selectively impaired order memory. These findings suggest the hippocampus, prefrontal cortex and perirhinal cortex are part of a broad network of structures essential for incidentally learning the order of events in episodic memory.

Episodic memory includes information about how long ago specific events occurred. Since most of our experiences have overlapping elements, remembering this temporal context is crucial for distinguishing individual episodes. The discovery of timing signals in hippocampal neurons, including evidence of "time cells" and of gradual changes in ensemble activity over long timescales, strongly suggests that the hippocampus is important for this capacity. However, behavioral evidence that the hippocampus is critical for the memory of elapsed time is lacking. This is possibly because previous studies have used time durations in the range of seconds when assessing hippocampal dependence, a timescale known to require corticostriatal circuits. Here we developed a nonspatial paradigm to test the hypothesis that the hippocampus is critical for keeping track of elapsed time over several minutes. We report that rats have a robust ability to remember durations at this timescale. We then determined the role of the hippocampus using infusions of fluorophore-conjugated muscimol, a GABAA agonist. We found that the hippocampus was essential for discriminating smaller, but not larger, temporal differences (measured in log units), consistent with a role in temporal pattern separation. Importantly, this effect was observed at long (minutes) but not short (seconds) timescales, suggesting an interplay of temporal resolution and timescale in determining hippocampal dependence. These results offer compelling evidence that the hippocampus plays a critical role in remembering how long ago events occurred.

The hippocampus is critical to the memory for sequences of events, a defining feature of episodic memory. However, the fundamental neuronal mechanisms underlying this capacity remain elusive. While considerable research indicates hippocampal neurons can represent sequences of locations, direct evidence of coding for the memory of sequential relationships among nonspatial events remains lacking. To address this important issue, we recorded neural activity in CA1 as rats performed a hippocampus-dependent sequence-memory task. Briefly, the task involves the presentation of repeated sequences of odors at a single port and requires rats to identify each item as "in sequence" or "out of sequence". We report that, while the animals' location and behavior remained constant, hippocampal activity differed depending on the temporal context of items-in this case, whether they were presented in or out of sequence. Some neurons showed this effect across items or sequence positions (general sequence cells), while others exhibited selectivity for specific conjunctions of item and sequence position information (conjunctive sequence cells) or for specific probe types (probe-specific sequence cells). We also found that the temporal context of individual trials could be accurately decoded from the activity of neuronal ensembles, that sequence coding at the single-cell and ensemble level was linked to sequence memory performance, and that slow-gamma oscillations (20-40 Hz) were more strongly modulated by temporal context and performance than theta oscillations (4-12 Hz). These findings provide compelling evidence that sequence coding extends beyond the domain of spatial trajectories and is thus a fundamental function of the hippocampus.

Significance statement

The ability to remember the order of life events depends on the hippocampus, but the underlying neural mechanisms remain poorly understood. Here we addressed this issue by recording neural activity in hippocampal region CA1 while rats performed a nonspatial sequence memory task. We found that hippocampal neurons code for the temporal context of items (whether odors were presented in the correct or incorrect sequential position) and that this activity is linked with memory performance. The discovery of this novel form of temporal coding in hippocampal neurons advances our fundamental understanding of the neurobiology of episodic memory and will serve as a foundation for our cross-species, multitechnique approach aimed at elucidating the neural mechanisms underlying memory impairments in aging and dementia.

We remember our lives as sequences of events, but it is unclear how these memories are controlled during retrieval. In rats, the medial prefrontal cortex (mPFC) is positioned to influence sequence memory through extensive top-down inputs to regions heavily interconnected with the hippocampus, notably the nucleus reuniens of the thalamus (RE) and perirhinal cortex (PER). Here, we used an hM4Di synaptic-silencing approach to test our hypothesis that specific mPFC→RE and mPFC→PER projections regulate sequence memory retrieval. First, we found non-overlapping populations of mPFC cells project to RE and PER. Second, suppressing mPFC activity impaired sequence memory. Third, inhibiting mPFC→RE and mPFC→PER pathways effectively abolished sequence memory. Finally, a sequential lag analysis showed that the mPFC→RE pathway contributes to a working memory retrieval strategy, whereas the mPFC→PER pathway supports a temporal context memory retrieval strategy. These findings demonstrate that mPFC→RE and mPFC→PER pathways serve as top-down mechanisms that control distinct sequence memory retrieval strategies.

There has not been a major change in how neuroscientists approach stereotaxic methods in decades. Here, we present a new stereotaxic method that provides an alternative approach to a traditional u-frame stereotaxic device and reduces costs, surgical time, and aids repeatability. The RatHat brain implantation system is a 3D-printable stereotaxic device for rats that is fabricated prior to surgery and fits to the shape of the skull. RatHat builds are directly implanted into the brain without the need for head-leveling or coordinate-mapping during surgery. The RatHat can be used in conjunction with the traditional u-frame stereotaxic device, but does not require the use of a micromanipulator for successful implantations. Each RatHat contains several primary components including the implant for mounting intracranial components, the surgical stencil for targeting drill sites, and the protective cap for preventing damage from impacts and debris. Each component serves a unique function and can be used together or separately. We demonstrate the feasibility of the RatHat in four different proof-of-principle experiments: (1) a three-pole cannula apparatus, (2) an optrode-electrode assembly, (3) a fixed-electrode array, and (4) a tetrode hyperdrive. Implants were successful, durable, and long-lasting (up to nine months). RatHat print files are easily created, can be modified in computer aided design (CAD) software for a variety of applications, and are easily shared, contributing to open science goals and replications. The RatHat has been adapted to multiple experimental paradigms in our lab and should be a useful new way to conduct stereotaxic implant surgeries in rodents.

The hippocampus plays a critical role in the memory for sequences of events, a defining feature of episodic memory. To shed light on the fundamental mechanisms supporting this capacity, we recently recorded neural activity in CA1 as rats performed a nonspatial odor sequence memory task. Our main finding was that, while the animals' location and behavior remained constant, a proportion of CA1 neurons fired differentially to odors depending on whether they were presented in or out of sequence (sequence cells). Here, we further examined if such sequence coding varied along the distal-to-proximal axis of the dorsal CA1 region (distal: toward subiculum; proximal: toward CA3). Differences in information processing along this axis have been suggested by recent anatomical and electrophysiological evidence that odor information may be more strongly represented in the distal segment, whereas spatial information may be more strongly represented in the proximal segment. Recorded neurons were grouped into four arbitrary sections of dorsal CA1, ranging from distal to proximal. We found that, although sequence cell coding was observed across the distal-to-proximal extent of CA1 from which we recorded, it was significantly higher in intermediate CA1, a region with more balanced anatomical input from lateral and medial entorhinal regions. More specifically, in that particular segment of CA1, we observed a significant increase in the magnitude of sequence coding of all cells, as well as in the sequential information content of sequence cells. Importantly, a different pattern was observed when examining the distribution of spatial coding from the same electrodes. Consistent with previous reports, our results suggest that spatial information was more strongly represented in the proximal section of CA1 (higher proportion of cells with place fields). These findings indicate that nonspatial sequence memory coding is not uniformly distributed along the transverse axis of CA1, and that this distribution does not simply follow the expected gradient based on the stimulus modality or the degree of spatial selectivity. Instead, the observed distribution suggests this form of sequence coding may be associated with convergent input from lateral and medial entorhinal regions, which is present throughout the proximodistal axis but greater in intermediate CA1.

Typical aging is associated with diminished episodic memory performance. To improve our understanding of the fundamental mechanisms underlying this age-related memory deficit, we previously developed an integrated, cross-species approach to link converging evidence from human and animal research. This novel approach focuses on the ability to remember sequences of events, an important feature of episodic memory. Unlike existing paradigms, this task is nonspatial, nonverbal, and can be used to isolate different cognitive processes that may be differentially affected in aging. Here, we used this task to make a comprehensive comparison of sequence memory performance between younger (18-22 yr) and older adults (62-86 yr). Specifically, participants viewed repeated sequences of six colored, fractal images and indicated whether each item was presented "in sequence" or "out of sequence." Several out of sequence probe trials were used to provide a detailed assessment of sequence memory, including: (i) repeating an item from earlier in the sequence ("Repeats"; e.g., AB A: DEF), (ii) skipping ahead in the sequence ("Skips"; e.g., AB D: DEF), and (iii) inserting an item from a different sequence into the same ordinal position ("Ordinal Transfers"; e.g., AB 3: DEF). We found that older adults performed as well as younger controls when tested on well-known and predictable sequences, but were severely impaired when tested using novel sequences. Importantly, overall sequence memory performance in older adults steadily declined with age, a decline not detected with other measures (RAVLT or BPS-O). We further characterized this deficit by showing that performance of older adults was severely impaired on specific probe trials that required detailed knowledge of the sequence (Skips and Ordinal Transfers), and was associated with a shift in their underlying mnemonic representation of the sequences. Collectively, these findings provide unambiguous evidence that the capacity to remember sequences of events is fundamentally affected by typical aging.