Machine learning-based segmentation of the rodent hippocampal CA2 area from Nissl-stained sections.

The hippocampus is a center of learning, memory, and spatial navigation. This region is divided into the CA1, CA2, and CA3 areas, which are anatomically different from each other. Among these divisions, the CA2 area is unique in terms of functional relevance to sociality. The CA2 area is often manually detected based on the size, shape, and density of neurons in the hippocampal pyramidal cell layer, but this manual segmentation relying on cytoarchitecture is impractical to apply to a large number of samples and dependent on experimenters' proficiency. Moreover, the CA2 area has been defined based on expression pattern of molecular marker proteins, but it generally takes days to complete immunostaining for such proteins. Thus, we asked whether the CA2 area can be systematically segmented based on cytoarchitecture alone. Since the expression pattern of regulator of G-protein signaling 14 (RGS14) signifies the CA2 area, we visualized the CA2 area in the mouse hippocampus by RGS14-immunostaining and Nissl-counterstaining and manually delineated the CA2 area. We then established "CAseg," a machine learning-based automated algorithm to segment the CA2 area with the F1-score of approximately 0.8 solely from Nissl-counterstained images that visualized cytoarchitecture. CAseg was extended to the segmentation of the prairie vole CA2 area, which raises the possibility that the use of this algorithm can be expanded to other species. Thus, CAseg will be beneficial for investigating unique properties of the hippocampal CA2 area.

Postnatal Maturation of Membrane Potential Dynamics during in Vivo Hippocampal Ripples.

Sharp-wave ripples (SWRs) are transient high-frequency oscillations of local field potentials (LFPs) in the hippocampus and play a critical role in memory consolidation. During SWRs, CA1 pyramidal cells exhibit rapid spike sequences that often replay the sequential activity that occurred during behavior. This temporally organized firing activity gradually emerges during 2 weeks after the eye opening; however, it remains unclear how the organized spikes during SWRs mature at the intracellular membrane potential (Vm) level. Here, we recorded Vm of CA1 pyramidal cells simultaneously with hippocampal LFPs from anesthetized immature mice of either sex after the developmental emergence of SWRs. On postnatal days 16 and 17, Vm dynamics around SWRs were premature, characterized by prolonged depolarizations without either pre- or post-SWR hyperpolarizations. The biphasic hyperpolarizations, features typical of adult SWR-relevant Vm, formed by approximately postnatal day 30. This Vm maturation was associated with an increase in SWR-associated inhibitory inputs to pyramidal cells. Thus, the development of SWR-relevant inhibition restricts the temporal windows for spikes of pyramidal cells and allows CA1 pyramidal cells to organize their spike sequences during SWRs.SIGNIFICANCE STATEMENT Sharp-wave ripples (SWRs) are prominent hippocampal oscillations and play a critical role in memory consolidation. During SWRs, hippocampal neurons synchronously emit spikes with organized temporal patterns. This temporal structure of spikes during SWRs develops during the third and fourth postnatal weeks, but the underlying mechanisms are not well understood. Here, we recorded in vivo membrane potentials from hippocampal neurons in premature mice and suggest that the maturation of SWR-associated inhibition enables hippocampal neurons to produce precisely controlled spike times during SWRs.

Theta oscillations represent collective dynamics of multineuronal membrane potentials of murine hippocampal pyramidal cells.

Theta (θ) oscillations are one of the characteristic local field potentials (LFPs) in the hippocampus that emerge during spatial navigation, exploratory sniffing, and rapid eye movement sleep. LFPs are thought to summarize multineuronal events, including synaptic currents and action potentials. However, no in vivo study to date has directly interrelated θ oscillations with the membrane potentials (Vm) of multiple neurons, and it remains unclear whether LFPs can be predicted from multineuronal Vms. Here, we simultaneously patch-clamp up to three CA1 pyramidal neurons in awake or anesthetized mice and find that the temporal evolution of the power and frequency of θ oscillations in Vms (θVms) are weakly but significantly correlate with LFP θ oscillations (θLFP) such that a deep neural network could predict the θLFP waveforms based on the θVm traces of three neurons. Therefore, individual neurons are loosely interdependent to ensure freedom of activity, but they partially share information to collectively produce θLFP.

Inhibition allocates spikes during hippocampal ripples.

Sets of spikes emitted sequentially across neurons constitute fundamental pulse packets in neural information processing, including offline memory replay during hippocampal sharp-wave ripples (SWRs). The relative timing of neuronal spikes is fine-tuned in each spike sequence but can vary between different sequences. However, the microcircuitry mechanism that enables such flexible spike sequencing remains unexplored. We recorded the membrane potentials of multiple hippocampal CA1 pyramidal cells in mice and found that the neurons were transiently hyperpolarized prior to SWRs. The pre-SWR hyperpolarizations were spatiotemporally heterogeneous, and larger hyperpolarizations were associated with later spikes during SWRs. Intracellular blockade of Cl--mediated inhibition reduced pre-SWR hyperpolarizations and advanced spike times. Single-unit recordings also revealed that the pre-SWR firing rates of inhibitory interneurons predicted the SWR-relevant spike times of pyramidal cells. Thus, pre-SWR inhibitory activity determines the sequential spike times of pyramidal cells and diversifies the repertoire of sequence patterns.

Molecular Characterization of Superficial Layers of the Presubiculum During Development.

The presubiculum, a subarea of the parahippocampal region, plays a critical role in spatial navigation and spatial representation. An outstanding aspect of presubicular spatial codes is head-direction selectivity of the firing of excitatory neurons, called head-direction cells. Head-direction selectivity emerges before eye-opening in rodents and is maintained in adulthood through neurophysiological interactions between excitatory and inhibitory neurons. Although the presubiculum has been physiologically profiled in terms of spatial representation during development, the histological characteristics of the developing presubiculum are poorly understood. We found that the expression of vesicular glutamate transporter 2 (VGluT2) could be used to delimit the superficial layers of the presubiculum, which was identified using an anterograde tracer injected into the anterior thalamic nucleus (ATN). Thus, we immunostained slices from mice ranging in age from neonates to adults using an antibody against VGluT2 to evaluate the VGluT2-positive area, which was identified as the superficial layers of the presubiculum, during development. We also immunostained the slices using antibodies against parvalbumin (PV) and somatostatin (SOM) and found that in the presubicular superficial layers, PV-positive neurons progressively increased in number during development, whereas SOM-positive neurons exhibited no increasing trend. In addition, we observed repeating patch structures in presubicular layer III from postnatal days 12. The abundant expression of VGluT2 suggests that the presubicular superficial layers are regulated primarily by VGluT2-mediated excitatory neurotransmission. Moreover, developmental changes in the densities of PV- and SOM-positive interneurons and the emergence of the VGluT2-positive patch structures during adolescence may be associated with the functional development of spatial codes in the superficial layers of the presubiculum.

The subiculum sensitizes retrosplenial cortex layer 2/3 pyramidal neurons.

KEY POINTS: Neurons in the retrosplenial cortex (RSC), a cerebral region that connects synaptically with various brain regions, are known to increase neuronal activity in accordance with hippocampal sharp wave-ripples. Pyramidal cells in granular RSC (gRSC) layer 2/3, but not layer 5, exhibit slowly ramping depolarization and considerably delayed spikes in response to a step-pulse current injection. The latencies of delayed spikes in RSC layer 2/3 pyramidal neurons were shortened by a preceding current injection. This effect was mimicked by activation of axonal afferents from the subiculum, but not of neocortical afferents. The subiculum is likely to facilitate information processing and flow in the RSC. ABSTRACT: The retrosplenial cortex (RSC), a cerebral region involved in diverse cognitive functions, is an anatomical hub that forms monosynaptic connections with various brain areas. Here, we report a unique form of short-term intrinsic plasticity in mouse granular RSC layer 2/3 pyramidal cells. These cells exhibited delayed spikes in response to somatic current injection, but the spike latencies were shortened by a preceding brief depolarization (priming). This priming-induced sensitization is distinct from desensitization, which is commonly observed in other cortical neurons. The facilitatory priming effect lasted for more than 3 s, providing a time window for increased sensitivity to RSC inputs. Based on in vitro and in vivo patch-clamp recordings following optogenetic stimulation of axonal fibres, we found that preactivation of subicular afferents replicated the facilitatory priming effect. The results suggest that subicular inputs to RSC layer 2/3 neurons may modulate subsequent information integration in the RSC layer 2/3 circuits.

In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives.

Brain functions are fundamental for the survival of organisms, and they are supported by neural circuits consisting of a variety of neurons. To investigate the function of neurons at the single-cell level, researchers often use whole-cell patch-clamp recording techniques. These techniques enable us to record membrane potentials (including action potentials) of individual neurons of not only anesthetized but also actively behaving animals. This whole-cell recording method enables us to reveal how neuronal activities support brain function at the single-cell level. In this review, we introduce previous studies using in vivo patch-clamp recording techniques and recent findings primarily regarding neuronal activities in the hippocampus for behavioral function. We further discuss how we can bridge the gap between electrophysiology and biochemistry.

Urethane anesthesia suppresses hippocampal subthreshold activity and neuronal synchronization.

Urethane, an anesthetic utilized for animal experiments, induces neocortical slow oscillations in which a large number of neurons emit rhythmic synchronized activity. However, it remains unclear how urethane affects neuronal activity in the hippocampus. In this study, we obtained in vivo patch-clamp recordings from dorsal hippocampal CA1 neurons in mice and found a reduction in the fluctuation of subthreshold membrane potentials during urethane anesthesia, implying reduced synaptic activity in the hippocampus. We then performed spike unit recordings from dorsal hippocampal CA1 neuronal ensembles in rats and found prominent reductions in the spike rates of the majority of hippocampal units, especially spatially selective units, during urethane anesthesia, whereas a subset of nonspatial units exhibited increased spike rates. The overall reductions in neuronal spike rates induced by urethane led to prominent decreases in spike synchronization across neuronal units. Consistently, the magnitude of hippocampal sharp wave ripples was also reduced by urethane. The suppression of hippocampal neuronal synchronization by urethane may lead to the disruption of offline memory reactivation mechanisms.

Heterogeneous expression patterns of fibronectin in the mouse subiculum.

The subiculum displays as much anatomical and physiological heterogeneity as the hippocampus. Recent studies suggest that the subiculum is also diverse in terms of gene expression. However, few studies have investigated the heterogeneity of the entire subiculum. To address this issue, we focused on fibronectin because its mRNA (FN1 mRNA) is expressed in the dorsal and ventral subiculum. We immunohistochemically characterized the intracellular expression of fibronectin in the entire subiculum along three axes (i.e., the dorsoventral, proximodistal, and superficial-deep axes). We first confirmed that FN1 mRNA is translated into protein inside cells. Moreover, we found that fibronectin was expressed evenly in the pyramidal cell layer of the dorsal subiculum, whereas in the ventral subicular pyramidal field, fibronectin was most concentrated in the superficial, distal corner. These results suggest that excitatory neurons labeled by fibronectin are more localized in the ventral subiculum than in the dorsal subiculum. Therefore, fibronectin may be useful as an indicator for studying the heterogeneity of principal cells in the subiculum.

Quick visualization of neurons in brain tissues using an optical clearing technique.

Neurons are classified into several morphological types according to the locations of their somata and the branching patterns of their axons and dendrites. Recent studies suggest that these morphological features are related to their physiological properties, including firing characteristics, responses to neuromodulators, and wiring patterns. Therefore, rapid morphological identification of electrophysiologically recorded neurons promises to advance our understanding of neuronal circuits. One of the most common anatomical cell identification methods is neuronal reconstruction with biocytin delivered through whole-cell patch-clamp pipettes. However, conventional reconstruction methods usually take longer than 24 h and limit the throughput of electrophysiological experiments. Here, we developed a quick, simple cell reconstruction method by optimizing the tissue clearing protocol ScaleSQ. We found that adding 200 mM NaCl almost entirely prevented tissue swelling without compromising optical clearing ability. This solution, termed IsoScaleSQ, allowed us to increase the transparency of the gray matter of 500-µm-thick slices within 30 min, meaning that the total time required to reconstruct whole-cell recorded neurons was reduced to 1 h. This novel method will improve the efficacy and effectiveness of electrophysiological experiments linked to cell morphology.

Whisker electromyograms signify awake and anesthetized states in mice.

The behavioral state of animals is essential information for functional recordings of neuronal activity; practically, the exact timing when animals recover from anesthesia is important information. Recordings of cortical local field potentials and dorsal neck electromyograms (EMGs), a widely used method to identify behavioral states, requires at least two recording electrodes, one of which also requires a craniotomy procedure. In the present study, recordings of whisker EMGs alone are sufficient to detect the state switch from anesthesia to awakening in head-fixed mice. This method uses a single electrode and thus is technically simple and demands a less physical burden to animals. Moreover, whisker EMGs recorded under anesthesia reflect respiratory rhythms.

Simultaneous monitoring of mouse respiratory and cardiac rates through a single precordial electrode.

Normal respiratory and circulatory functions are crucial for survival. However, conventional methods of monitoring respiration, some of which use sensors inserted into the nasal cavity, may interfere with naïve respiratory rates. In this study, we conducted a single-point measurement of electrocardiograms (ECGs) from the pectoral muscles of anesthetized and waking mice and found low-frequency oscillations in the ECG baseline. Using the fast Fourier transform of simultaneously recorded respiratory signals, we demonstrated that the low-frequency oscillations corresponded to respiratory rhythms. Moreover, the baseline oscillations changed in parallel with the respiratory rhythm when the latter was altered by pharmacological manipulation. We also demonstrated that this method could be combined with in vivo whole-cell patch-clamp recordings from the hippocampus. Thus, we developed a non-invasive form of respirometry in mice. Our recording method using a simple derivation algorithm is applicable to a variety of physiological and pharmacological experiments, providing an experimental platform in studying the mechanisms underlying the interaction of the central nervous system and the peripheral functions.

Juvenile Hippocampal CA2 Region Expresses Aggrecan.

Perineuronal nets (PNNs) are distributed primarily around inhibitory interneurons in the hippocampus, such as parvalbumin-positive interneurons. PNNs are also present around excitatory neurons in some brain regions and prevent plasticity in these neurons. A recent study demonstrated that PNNs also exist around mouse hippocampal pyramidal cells, which are the principle type of excitatory neurons, in the CA2 subregion and modulate the excitability and plasticity of these neurons. However, the development of PNNs in the CA2 region during postnatal maturation was not fully investigated. This study found that a main component of PNNs, aggrecan, existed in the pyramidal cell layer of the putative CA2 subarea prior to the appearance of the CA2 region, which was defined by the CA2 marker protein regulator of G protein signaling 14 (RGS14). We also found that aggrecan immunoreactivity was more evident in the anterior sections of the CA2 area than the posterior sections, which suggests that the function of CA2 PNNs varies along the anterior-posterior axis.