Effects of optogenetic silencing the anterior cingulate cortex in a delayed non-match to trajectory task.

Working memory is a fundamental cognitive ability, allowing us to keep information in memory for the time needed to perform a given task. A complex neural circuit fulfills these functions, among which is the anterior cingulate cortex (CG). Functionally and anatomically connected to the medial prefrontal, retrosplenial, midcingulate and hippocampus, as well as motor cortices, CG has been implicated in retrieving appropriate information when needed to select and control appropriate behavior. The role of cingulate cortex in working memory-guided behaviors remains unclear due to the lack of studies reversibly interfering with its activity during specific epochs of working memory. We used eNpHR3.0 to silence cingulate neurons while animals perform a standard delayed non-match to trajectory task, and found that, while not causing an absolute impairment in working memory, silencing cingulate neurons during retrieval decreases the mean performance if compared to silencing during encoding. Such retrieval-associated changes are accompanied by longer delays observed when light is delivered to control animals, when compared to eNpHR3.0+ ones, consistent with an adaptive recruitment of additional cognitive resources.

Easy to process, hard to control: Transient and sustained processing fluency impairs cognitive control adjustments to conflict.

Recent research suggests that the cognitive monitoring system of control could be using negative affective cues intrinsic to changes in information processing to initiate top-down regulatory mechanisms. Here, we propose that positive feelings of ease-of-processing could be picked up by the monitoring system as a cue indicating that control is not necessary, leading to maladaptive control adjustments. We simultaneously target control adjustments driven by task context and on a trial-by-trial level, macro-, and micro-adjustments. This hypothesis was tested using a Stroop-like task comprised trials varying in congruence and perceptual fluency. A pseudo randomisation procedure within different proportion of congruence conditions was used to maximise discrepancy and fluency effects. Results suggest that in a mostly congruent context participants committed more fast errors when incongruent trials were easy-to-read. Moreover, within the mostly incongruent condition, we also found more errors on incongruent trials after experiencing the facilitation effect of repeated congruent trials. These results suggest that transient and sustained feelings of processing fluency can downregulate control mechanisms, leading to failed adaptive adjustments to conflict.

Bad after bad is good: previous trial disfluency reduces interference promoted by incongruence.

Conflict and perceptual disfluency have been shown to lead to adaptive, sequential, control adjustments. Here, we propose that these effects can be additive, suggesting their integration into a general feeling of disfluent information processing. This hypothesis was tested using an interference task that dynamically mixed trials varying in legibility and/or congruence. Moreover, the manipulation of the proportion of congruent trials within the task allowed differentiating conditions in which these experiences of fluency may vary. Results showed that progressive increases in processing disfluency elicited a matching decrease in the interference of incongruent fluent trials. This linear effect was significant for all proportion of congruence conditions, although lower when incongruent trials were more frequent. These results highlight the role of feelings in the initiation of control and suggest that the monitoring system could be using changes in information processing fluency as a need-for-control signal.

Medial Entorhinal Cortex Excitatory Neurons Are Necessary for Accurate Timing.

The hippocampal region has long been considered critical for memory of time, and recent evidence shows that network operations and single-unit activity in the hippocampus and medial entorhinal cortex (MEC) correlate with elapsed time. However, whether MEC activity is necessary for timing remains largely unknown. Here we expressed DREADDs (designer receptors exclusively activated by designer drugs) under the CaMKIIa promoter to preferentially target MEC excitatory neurons for chemogenetic silencing, while freely moving male rats reproduced a memorized time interval by waiting inside a region of interest. We found that such silencing impaired the reproduction of the memorized interval and led to an overestimation of elapsed time. Trial history analyses under this condition revealed a reduced influence of previous trials on current waiting times, suggesting an impairment in maintaining temporal memories across trials. Moreover, using GLM (logistic regression), we show that decoding behavioral performance from preceding waiting times was significantly compromised when MEC was silenced. In addition to revealing an important role of MEC excitatory neurons for timing behavior, our results raise the possibility that these neurons contribute to such behavior by holding temporal information across trials.SIGNIFICANCE STATEMENT Medial temporal lobe (MTL) structures are implicated in processing temporal information. However, little is known about the role MTL structures, such as the hippocampus and the entorhinal cortex, play in perceiving or reproducing temporal intervals. By chemogenetically silencing medial entorhinal cortex (MEC) excitatory activity during a timing task, we show that this structure is necessary for the accurate reproduction of temporal intervals. Furthermore, trial history analyses suggest that silencing MEC disrupts memory mechanisms during timing. Together, these results suggest that MEC is necessary for timing behavior, possibly by representing the target interval in memory.

Transection of the Superior Sagittal Sinus Enables Bilateral Access to the Rodent Midline Brain Structures.

Stereotaxic access to brain areas underneath the superior sagittal sinus (SSS) is notoriously challenging. As a major drainage vessel, covering the whole extension of the sagittal fissure, the SSS impedes direct bilateral access to underlying regions for recording and stimulation probes, drug-delivery cannulas, and injection devices. We now describe a new method for transection and retraction of the SSS in rats, that allows the accurate placement of microinjection devices, or chronic electrode probes, while avoiding hemorrhage and the ensuing deleterious consequences for local structures, animal health, and behavior. To demonstrate the feasibility of this approach we evaluated its consequences acutely during surgery, and thereafter during surgical survival, recovery, behavioral testing, as well as postmortem analysis of histologic impact in the related brain structures of male rats. This method provides a new approach enabling direct access for manipulation and recording of activity in brain areas previously obstructed by the SSS.

A Gradient of Hippocampal Inputs to the Medial Mesocortex.

Memory-guided decisions depend on complex interactions between the hippocampus (HIPP) and medial mesocortical (MMC) regions, including the anterior cingulate (CG) and retrosplenial (RSC). The functional circuitry underlying these interactions is unclear. Using anatomy, electrophysiology, and optogenetics, we show that such circuitry is characterized by a functional-anatomical gradient. While the CG receives hippocampal excitatory projections originated in CA1 stratum pyramidale, the RSC additionally receives long-range inhibitory inputs from radiatum and lacunosum-moleculare. Such hippocampal projections establish bona fide synapses, with the RSC densely targeted on its superficial layers L1-L3 by a combination of inhibitory and excitatory synapses. We show that the MMC is targeted by dorsal-intermediate CA1 (diCA1) axons following a caudorostral gradient in which a dense, dual (excitatory/inhibitory), layer-specific projection is progressively converted in a sparse, excitatory, and diffuse projection. This gradient is reflected in higher oscillatory synchronicity between the HIPP and RSC in the awake-behaving animal, compatible with their known functional proximity and contrasting with that found in the CG.

Scalable, Lightweight, Integrated and Quick-to-Assemble (SLIQ) Hyperdrives for Functional Circuit Dissection.

Independently adjustable multielectrode arrays are routinely used to interrogate neuronal circuit function, enabling chronic in vivo monitoring of neuronal ensembles in freely behaving animals at a single-cell, single spike resolution. Despite the importance of this approach, its widespread use is limited by highly specialized design and fabrication methods. To address this, we have developed a Scalable, Lightweight, Integrated and Quick-to-assemble multielectrode array platform. This platform additionally integrates optical fibers with independently adjustable electrodes to allow simultaneous single unit recordings and circuit-specific optogenetic targeting and/or manipulation. In current designs, the fully assembled platforms are scalable from 2 to 32 microdrives, and yet range 1-3 g, light enough for small animals. Here, we describe the design process starting from intent in computer-aided design, parameter testing through finite element analysis and experimental means, and implementation of various applications across mice and rats. Combined, our methods may expand the utility of multielectrode recordings and their continued integration with other tools enabling functional dissection of intact neural circuits.

Slow-γ Rhythms Coordinate Cingulate Cortical Responses to Hippocampal Sharp-Wave Ripples during Wakefulness.

Behavioral changes in response to reward require monitoring past behavior relative to present outcomes. This is thought to involve a fine coordination between the hippocampus (HIPP), which stores and replays memories of past events, and cortical regions such as cingulate cortex, responsible for behavioral planning. Sharp-wave ripple (SWR)-mediated memory replay in the HIPP of awake rodents contributes to learning, but cortical responses to hippocampal SWR during wakefulness are not known. We now show that in rats, during quiet-wakefulness, cingulate neurons exhibit significant responses to SWR, as well as increased modulation by the accompanying hippocampal local field potential slow-γ oscillation, a rhythm associated with intra-hippocampal information processing. The magnitude of cingulate neurons' responses to SWR is significantly correlated with the degree of their modulation by HIPP slow-γ. We hypothesize that during pauses cingulate neurons transiently access episodic information concerning previous choices, replayed by HIPP SWR, to evaluate past trajectories in light of their outcome.

Phase organization of network computations.

Coupled oscillations are hypothesized to organize the processing of information across distributed brain circuits. This idea is supported by recent evidence, and newly developed techniques promise to put such theoretical framework to mechanistic testing. We review evidence suggesting that individual oscillatory cycles constitute a functional unit that organizes activity in neural networks, and that oscillatory phase (defined as the fraction of the wave cycle that has elapsed relative to the start of the cycle) is a key oscillatory parameter to implement the functions of oscillations in limbic networks. We highlight neural manipulation techniques that currently allow for causal testing of these hypotheses.

Cingulate-hippocampus coherence and trajectory coding in a sequential choice task.

Interactions between cortex and hippocampus are believed to play a role in the acquisition and maintenance of memories. Distinct types of coordinated oscillatory activity, namely at theta frequency, are hypothesized to regulate information processing in these structures. We investigated how information processing in cingulate cortex and hippocampus relates to cingulate-hippocampus coordination in a behavioral task in which rats choose from four possible trajectories according to a sequence. We found that the accuracy with which cingulate and hippocampal populations encode individual trajectories changes with the pattern of cingulate-hippocampal theta coherence over the course of a trial. Initial theta coherence at ~8 Hz during trial onsets lowers by ~1 Hz as animals enter decision stages. At these stages, hippocampus precedes cingulate in processing increased amounts of task-relevant information. We hypothesize that lower theta frequency coordinates the integration of hippocampal contextual information by cingulate neuronal populations, to inform choices in a task-phase-dependent manner.

Role for a cortical input to hippocampal area CA1 in the consolidation of a long-term memory.

A dialogue between the hippocampus and the neocortex is thought to underlie the formation, consolidation and retrieval of episodic memories, although the nature of this cortico-hippocampal communication is poorly understood. Using selective electrolytic lesions in rats, here we examined the role of the direct entorhinal projection (temporoammonic, TA) to the hippocampal area CA1 in short-term (24 hours) and long-term (four weeks) spatial memory in the Morris water maze. When short-term memory was examined, both sham- and TA-lesioned animals showed a significant preference for the target quadrant. When re-tested four weeks later, sham-lesioned animals exhibited long-term memory; in contrast, the TA-lesioned animals no longer showed target quadrant preference. Many long-lasting memories require a process called consolidation, which involves the exchange of information between the cortex and hippocampus. The disruption of long-term memory by the TA lesion could reflect a requirement for TA input during either the acquisition or consolidation of long-term memory. To distinguish between these possibilities, we trained animals, verified their spatial memory 24 hours later, and then subjected trained animals to TA lesions. TA-lesioned animals still exhibited a deficit in long-term memory, indicating a disruption of consolidation. Animals in which the TA lesion was delayed by three weeks, however, showed a significant preference for the target quadrant, indicating that the memory had already been adequately consolidated at the time of the delayed lesion. These results indicate that, after learning, ongoing cortical input conveyed by the TA path is required to consolidate long-term spatial memory.

Molecular mechanisms contributing to long-lasting synaptic plasticity at the temporoammonic-CA1 synapse.

The hippocampus and the nearby medial temporal lobe structures are required for the formation, consolidation, and retrieval of episodic memories. Sensory information enters the hippocampus via two inputs from entorhinal cortex (EC): One input (perforant path) makes synapses on the dendrites of dentate granule cells as the first set of synapses in the trisynaptic circuit, the other (temporoammonic; TA) makes synapses on the distal dendrites of CA1 neurons. Here we demonstrate that TA-CA1 synapses undergo both early- and late-phase long-term potentiation (LTP) in rat hippocampal slices. LTP at TA-CA1 synapses requires both NMDA receptor and voltage-gated Ca2+ channel activity. Furthermore, TA-CA1 LTP is insensitive to the blockade of fast inhibitory transmission (GABAA-mediated) and, interestingly, is dependent on GABAB-dependent slow inhibitory transmission. These findings indicate that the TA-CA1 synapses may rely on a refined modulation of inhibition to exhibit LTP.

Direct cortical input modulates plasticity and spiking in CA1 pyramidal neurons.

The hippocampus is necessary for the acquisition and retrieval of declarative memories. The best-characterized sensory input to the hippocampus is the perforant path projection from layer II of entorhinal cortex (EC) to the dentate gyrus. Signals are then processed sequentially in the hippocampal CA fields before returning to the cortex via CA1 pyramidal neuron spikes. There is another EC input-the temporoammonic (TA) pathway-consisting of axons from layer III EC neurons that make synaptic contacts on the distal dendrites of CA1 neurons. Here we show that this pathway modulates both the plasticity and the output of the rat hippocampal formation. Bursts of TA activity can, depending on their timing, either increase or decrease the probability of Schaffer-collateral (SC)-evoked CA1 spikes. TA bursts can also significantly reduce the magnitude of synaptic potentiation at SC-CA1 synapses. The TA-CA1 synapse itself exhibits both long-term depression (LTD) and long-term potentiation (LTP). This capacity for bi-directional plasticity can, in turn, regulate the TA modulation of CA1 activity: LTP or LTD of the TA pathway either enhances or diminishes the gating of CA1 spikes and plasticity inhibition, respectively.