Granular retrosplenial cortex layer 2/3 generates high-frequency oscillations dynamically coupled with hippocampal rhythms across brain states.

The granular retrosplenial cortex (gRSC) exhibits high-frequency oscillations (HFOs; ∼150 Hz), which can be driven by a hippocampus-subiculum pathway. How the cellular-synaptic and laminar organization of gRSC facilitates HFOs is unknown. Here, we probe gRSC HFO generation and coupling with hippocampal rhythms using focal optogenetics and silicon-probe recordings in behaving mice. ChR2-mediated excitation of CaMKII-expressing cells in L2/3 or L5 induces HFOs, but spontaneous HFOs are found only in L2/3, where HFO power is highest. HFOs couple to CA1 sharp wave-ripples (SPW-Rs) during rest and the descending phase of theta. gRSC HFO current sources and sinks are the same for events during both SPW-Rs and theta oscillations. Independent component analysis shows that high gamma (50-100 Hz) in CA1 stratum lacunosum moleculare is comodulated with HFO power. HFOs may thus facilitate interregional communication of a multisynaptic loop between the gRSC, hippocampus, and medial entorhinal cortex during distinct brain and behavioral states.

Simultaneous electrophysiology and optogenetic perturbation of the same neurons in chronically implanted animals using µLED silicon probes.

Micro-light-emitting-diode (µLED) silicon probes feature independently controllable miniature light-emitting-diodes (LEDs) embedded at several positions in each shank of a multi-shank probe, enabling temporally and spatially precise optogenetic neural circuit interrogation. Here, we present a protocol for performing causal and reproducible neural circuit manipulations in chronically implanted, freely moving animals. We describe steps for introducing optogenetic constructs, preparing and implanting a µLED probe, performing simultaneous in vivo electrophysiology with focal optogenetic perturbation, and recovering a probe following termination of an experiment. For complete details on the use and execution of this protocol, please refer to Watkins de Jong et al. (2023).1.

Granular retrosplenial cortex layer 2/3 generates high-frequency oscillations coupled with hippocampal theta and gamma in online states or sharp-wave ripples in offline states.

Neuronal oscillations support information transfer by temporally aligning the activity of anatomically distributed 'writer' and 'reader' cell assemblies. High-frequency oscillations (HFOs) such as hippocampal CA1 sharp-wave ripples (SWRs; 100-250 Hz) are sufficiently fast to initiate synaptic plasticity between assemblies and are required for memory consolidation. HFOs are observed in parietal and midline cortices including granular retrosplenial cortex (gRSC). In 'offline' brain states (e.g. quiet wakefulness) gRSC HFOs co-occur with CA1 SWRs, while in 'online' states (e.g. ambulation) HFOs persist with the emergence of theta oscillations. The mechanisms of gRSC HFO oscillations, specifically whether the gRSC can intrinsically generate HFOs, and which layers support HFOs across states, remain unclear. We addressed these issues in behaving mice using optogenetic excitation in individual layers of the gRSC and high density silicon-probe recordings across gRSC layers and hippocampus CA1. Optogenetically induced HFOs (iHFOs) could be elicited by depolarizing excitatory neurons with 100 ms half-sine wave pulses in layer 2/3 (L2/3) or layer 5 (L5) though L5 iHFOs were of lower power than in L2/3. Critically, spontaneous HFOs were only observed in L2/3 and never in L5. Intra-laminar monosynaptic connectivity between excitatory and inhibitory neurons was similar across layers, suggesting other factors restrict HFOs to L2/3. To compare HFOs in online versus offline states we analyzed, separately, HFOs that did or did not co-occur with CA1 SWRs. Using current-source density analysis we found uniform synaptic inputs to L2/3 during all gRSC HFOs, suggesting layer-specific inputs may dictate the localization of HFOs to L2/3. HFOs occurring without SWRs were aligned with the descending phase of both gRSC and CA1 theta oscillations and were coherent with CA1 high frequency gamma oscillations (50-80 Hz). These results demonstrate that gRSC can internally generate HFOs without rhythmic inputs and that HFOs occur exclusively in L2/3, coupled to distinct hippocampal oscillations in online versus offline states.

Cannabidiol modulates excitatory-inhibitory ratio to counter hippocampal hyperactivity.

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsies, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking the pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G-protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3-CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABAARγ2 and gephyrin puncta. LPI effects at excitatory and inhibitory synapses were eliminated by CBD pre-treatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated GPR55 and LPI levels, and chronic lithium-pilocarpine-induced epileptogenesis potentiated LPI's pro-excitatory effects. We propose that CBD exerts potential anti-seizure effects by blocking LPI's synaptic effects and dampening hyperexcitability.

Simultaneous Electrophysiology and Optogenetic Perturbation of the Same Neurons in Chronically Implanted Animals using µLED Silicon Probes.

Optogenetics are a powerful tool for testing how a neural circuit influences neural activity, cognition, and behavior. Accordingly, the number of studies employing optogenetic perturbation has grown exponentially over the last decade. However, recent studies have highlighted that the impact of optogenetic stimulation/silencing can vary depending on the construct used, the local microcircuit connectivity, extent/power of illumination, and neuron types perturbed. Despite these caveats, the majority of studies employ optogenetics without simultaneously recording neural activity in the circuit that is being perturbed. This dearth of simultaneously recorded neural data is due in part to technical difficulties in combining optogenetics and extracellular electrophysiology. The recent introduction of µLED silicon probes, which feature independently controllable miniature LEDs embedded at several levels of each of multiple shanks of silicon probes, provides a tractable method for temporally and spatially precise interrogation of neural circuits. Here, we provide a protocol addressing how to perform chronic recordings using µLED probes. This protocol provides a schematic for performing causal and reproducible interrogations of neural circuits and addresses all phases of the recording process: introduction of optogenetic construct, implantation of the µLED probe, performing simultaneous optogenetics and electrophysiology in vivo , and post-processing of recorded data. SUMMARY: This method allows a researcher to simultaneously perturb neural activity and record electrophysiological signal from the same neurons with high spatial specificity using silicon probes with integrated µLEDs. We outline a procedure detailing all stages of the process for performing reliable µLED experiments in chronically implanted rodents.

Hippocampal area CA2 controls seizure dynamics, interictal EEG abnormalities and social comorbidity in mouse models of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, abnormal activity between seizures, and impaired behavior. CA2 pyramidal neurons (PNs) are potentially important because inhibiting them with a chemogenetic approach reduces seizure frequency in a mouse model of TLE. However, whether seizures could be stopped by timing inhibition just as a seizure begins is unclear. Furthermore, whether inhibition would reduce the cortical and motor manifestations of seizures are not clear. Finally, whether interictal EEG abnormalities and TLE comorbidities would be improved are unknown. Therefore, real-time optogenetic silencing of CA2 PNs during seizures, interictal activity and behavior were studied in 2 mouse models of TLE. CA2 silencing significantly reduced seizure duration and time spent in convulsive behavior. Interictal spikes and high frequency oscillations were significantly reduced, and social behavior was improved. Therefore, brief focal silencing of CA2 PNs reduces seizures, their propagation, and convulsive manifestations, improves interictal EEG, and ameliorates social comorbidities. HIGHLIGHTS: Real-time CA2 silencing at the onset of seizures reduces seizure durationWhen CA2 silencing reduces seizure activity in hippocampus it also reduces cortical seizure activity and convulsive manifestations of seizuresInterictal spikes and high frequency oscillations are reduced by real-time CA2 silencingReal-time CA2 silencing of high frequency oscillations (>250Hz) rescues social memory deficits of chronic epileptic mice.

Once is enough for hippocampal replay.

In this issue of Neuron, Berners-Lee et al. (2022) reveal how neural dynamics in the hippocampus change after a single experience, offering a candidate mechanism for how hippocampal plasticity supports episodic memory.

Neurophysiology of Remembering.

By linking the past with the future, our memories define our sense of identity. Because human memory engages the conscious realm, its examination has historically been approached from language and introspection and proceeded largely along separate parallel paths in humans and other animals. Here, we first highlight the achievements and limitations of this mind-based approach and make the case for a new brain-based understanding of declarative memory with a focus on hippocampal physiology. Next, we discuss the interleaved nature and common physiological mechanisms of navigation in real and mental spacetime. We suggest that a distinguishing feature of memory types is whether they subserve actions for single or multiple uses. Finally, in contrast to the persisting view of the mind as a highly plastic blank slate ready for the world to make its imprint, we hypothesize that neuronal networks are endowed with a reservoir of neural trajectories, and the challenge faced by the brain is how to select and match preexisting neuronal trajectories with events in the world.

Recruitment and inhibitory action of hippocampal axo-axonic cells during behavior.

The axon initial segment of hippocampal pyramidal cells is a key subcellular compartment for action potential generation, under GABAergic control by the "chandelier" or axo-axonic cells (AACs). Although AACs are the only cellular source of GABA targeting the initial segment, their in vivo activity patterns and influence over pyramidal cell dynamics are not well understood. We achieved cell-type-specific genetic access to AACs in mice and show that AACs in the hippocampal area CA1 are synchronously activated by episodes of locomotion or whisking during rest. Bidirectional intervention experiments in head-restrained mice performing a random foraging task revealed that AACs inhibit CA1 pyramidal cells, indicating that the effect of GABA on the initial segments in the hippocampus is inhibitory in vivo. Finally, optogenetic inhibition of AACs at specific track locations induced remapping of pyramidal cell place fields. These results demonstrate brain-state-specific dynamics of a critical inhibitory controller of cortical circuits.

Subiculum as a generator of sharp wave-ripples in the rodent hippocampus.

Sharp wave-ripples (SWRs) represent synchronous discharges of hippocampal neurons and are believed to play a major role in memory consolidation. A large body of evidence suggests that SWRs are exclusively generated in the CA3-CA2 network. In contrast, here, we provide several lines of evidence showing that the subiculum can function as a secondary SWRs generator. SWRs with subicular origin propagate forward into the entorhinal cortex as well as backward into the hippocampus proper. Our findings suggest that the output structures of the hippocampus are not only passively facilitating the transfer of SWRs to the cortex, but they also can actively contribute to the genesis of SWRs. We hypothesize that SWRs with a subicular origin may be important for the consolidation of information conveyed to the hippocampus via the temporoammonic pathway.

Gating of hippocampal rhythms and memory by synaptic plasticity in inhibitory interneurons.

Mental experiences can become long-term memories if the hippocampal activity patterns that encode them are broadcast during network oscillations. The activity of inhibitory neurons is essential for generating these neural oscillations, but molecular control of this dynamic process during learning remains unknown. Here, we show that hippocampal oscillatory strength positively correlates with excitatory monosynaptic drive onto inhibitory neurons (E→I) in freely behaving mice. To establish a causal relationship between them, we identified γCaMKII as the long-sought mediator of long-term potentiation for E→I synapses (LTPE→I), which enabled the genetic manipulation of experience-dependent E→I synaptic input/plasticity. Deleting γCaMKII in parvalbumin interneurons selectively eliminated LTPE→I and disrupted experience-driven strengthening in theta and gamma rhythmicity. Behaviorally, this manipulation impaired long-term memory, for which the kinase activity of γCaMKII was required. Taken together, our data suggest that E→I synaptic plasticity, exemplified by LTPE→I, plays a gatekeeping role in tuning experience-dependent brain rhythms and mnemonic function.

Preexisting hippocampal network dynamics constrain optogenetically induced place fields.

Memory models often emphasize the need to encode novel patterns of neural activity imposed by sensory drive. Prior learning and innate architecture likely restrict neural plasticity, however. Here, we test how the incorporation of synthetic hippocampal signals is constrained by preexisting circuit dynamics. We optogenetically stimulated small groups of CA1 neurons as mice traversed a chosen segment of a linear track, mimicking the emergence of place fields. Stimulation induced persistent place field remapping in stimulated and non-stimulated neurons. The emergence of place fields could be predicted from sporadic firing in the new place field location and the temporal relationship to peer neurons before the optogenetic perturbation. Circuit modification was reflected by altered spike transmission between connected pyramidal cells and inhibitory interneurons, which persisted during post-experience sleep. We hypothesize that optogenetic perturbation unmasked sub-threshold place fields. Plasticity in recurrent/lateral inhibition may drive learning through the rapid association of existing states.

Monosynaptic inference via finely-timed spikes.

Observations of finely-timed spike relationships in population recordings have been used to support partial reconstruction of neural microcircuit diagrams. In this approach, fine-timescale components of paired spike train interactions are isolated and subsequently attributed to synaptic parameters. Recent perturbation studies strengthen the case for such an inference, yet the complete set of measurements needed to calibrate statistical models is unavailable. To address this gap, we study features of pairwise spiking in a large-scale in vivo dataset where presynaptic neurons were explicitly decoupled from network activity by juxtacellular stimulation. We then construct biophysical models of paired spike trains to reproduce the observed phenomenology of in vivo monosynaptic interactions, including both fine-timescale spike-spike correlations and firing irregularity. A key characteristic of these models is that the paired neurons are coupled by rapidly-fluctuating background inputs. We quantify a monosynapse's causal effect by comparing the postsynaptic train with its counterfactual, when the monosynapse is removed. Subsequently, we develop statistical techniques for estimating this causal effect from the pre- and post-synaptic spike trains. A particular focus is the justification and application of a nonparametric separation of timescale principle to implement synaptic inference. Using simulated data generated from the biophysical models, we characterize the regimes in which the estimators accurately identify the monosynaptic effect. A secondary goal is to initiate a critical exploration of neurostatistical assumptions in terms of biophysical mechanisms, particularly with regards to the challenging but arguably fundamental issue of fast, unobservable nonstationarities in background dynamics.

Publisher Correction: Propagation of hippocampal ripples to the neocortex by way of a subiculum-retrosplenial pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Propagation of hippocampal ripples to the neocortex by way of a subiculum-retrosplenial pathway.

Bouts of high frequency activity known as sharp wave ripples (SPW-Rs) facilitate communication between the hippocampus and neocortex. However, the paths and mechanisms by which SPW-Rs broadcast their content are not well understood. Due to its anatomical positioning, the granular retrosplenial cortex (gRSC) may be a bridge for this hippocampo-cortical dialogue. Using silicon probe recordings in awake, head-fixed mice, we show the existence of SPW-R analogues in gRSC and demonstrate their coupling to hippocampal SPW-Rs. gRSC neurons reliably distinguished different subclasses of hippocampal SPW-Rs according to ensemble activity patterns in CA1. We demonstrate that this coupling is brain state-dependent, and delineate a topographically-organized anatomical pathway via VGlut2-expressing, bursty neurons in the subiculum. Optogenetic stimulation or inhibition of bursty subicular cells induced or reduced responses in superficial gRSC, respectively. These results identify a specific path and underlying mechanisms by which the hippocampus can convey neuronal content to the neocortex during SPW-Rs.

Mechanisms of neural organization and rhythmogenesis during hippocampal and cortical ripples.

Neural activity during ripples has attracted great theoretical and experimental attention over the last three decades. Perhaps one reason for such interest is that ripples occur during quiet waking moments and during sleep, times when we reflect and dream about what has just occurred and what we expect to happen next. The hope is that understanding such 'offline' activity may yield insights into reflection, planning, and the purposes of sleep. This review focuses on the mechanisms by which neurons organize during these high-frequency events. In studying ripples, broader principles have emerged that relate intrinsic neural properties, network topology and synaptic plasticity in controlling neural activity. Ripples, therefore, serve as an excellent model for studying how properties of a neural network relate to neural dynamics. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Inferring and validating mechanistic models of neural microcircuits based on spike-train data.

The interpretation of neuronal spike train recordings often relies on abstract statistical models that allow for principled parameter estimation and model selection but provide only limited insights into underlying microcircuits. In contrast, mechanistic models are useful to interpret microcircuit dynamics, but are rarely quantitatively matched to experimental data due to methodological challenges. Here we present analytical methods to efficiently fit spiking circuit models to single-trial spike trains. Using derived likelihood functions, we statistically infer the mean and variance of hidden inputs, neuronal adaptation properties and connectivity for coupled integrate-and-fire neurons. Comprehensive evaluations on synthetic data, validations using ground truth in-vitro and in-vivo recordings, and comparisons with existing techniques demonstrate that parameter estimation is very accurate and efficient, even for highly subsampled networks. Our methods bridge statistical, data-driven and theoretical, model-based neurosciences at the level of spiking circuits, for the purpose of a quantitative, mechanistic interpretation of recorded neuronal population activity.

A High-Resolution Opto-Electrophysiology System With a Miniature Integrated Headstage.

This work presents a fully integrated neural interface system in a small form factor (1.9 g), consisting of a µLED silicon optoelectrode (12 µLEDs and 32 recording sites in a 4-shank configuration), an Intan 32-channel recording chip, and a custom optical stimulation chip for controlling 12 µLEDs. High-resolution optical stimulation with approximately 68.5 nW radiant flux resolution is achieved by a custom LED driver ASIC, which enables individual control of up to 48 channels with a current precision of 1 µA, a maximum current of 1.024 mA, and an update rate of >10 kHz. Recording is performed by an off-the-shelf 32-channel digitizing front-end ASIC from Intan. Two compact custom interface printed circuit boards were designed to link the headstage with a PC. The prototype system demonstrates precise current generation, sufficient optical radiant flux generation , and fast turn-on of µLEDs . Single animal in vivo experiments validated the headstage's capability to precisely modulate single neuronal activity and independently modulate activities of separate neuronal populations near neighboring optoelectrode shanks.

Dual color optogenetic control of neural populations using low-noise, multishank optoelectrodes.

Optogenetics allows for optical manipulation of neuronal activity and has been increasingly combined with intra- and extra-cellular electrophysiological recordings. Genetically-identified classes of neurons are optically manipulated, though the versatility of optogenetics would be increased if independent control of distinct neural populations could be achieved on a sufficient spatial and temporal resolution. We report a scalable multi-site optoelectrode design that allows simultaneous optogenetic control of two spatially intermingled neuronal populations in vivo. We describe the design, fabrication, and assembly of low-noise, multi-site/multi-color optoelectrodes. Each shank of the four-shank assembly is monolithically integrated with 8 recording sites and a dual-color waveguide mixer with a 7 × 30 µm cross-section, coupled to 405 nm and 635 nm injection laser diodes (ILDs) via gradient-index (GRIN) lenses to meet optical and thermal design requirements. To better understand noise on the recording channels generated during diode-based activation, we developed a lumped-circuit modeling approach for EMI coupling mechanisms and used it to limit artifacts to amplitudes under 100 µV upto an optical output power of 450 µW. We implanted the packaged devices into the CA1 pyramidal layer of awake mice, expressing Channelrhodopsin-2 in pyramidal cells and ChrimsonR in paravalbumin-expressing interneurons, and achieved optical excitation of each cell type using sub-mW illumination. We highlight the potential use of this technology for functional dissection of neural circuits.