How development sculpts hippocampal circuits and function.

In mammals, the selective transformation of transient experience into stored memory occurs in the hippocampus, which develops representations of specific events in the context in which they occur. In this review, we focus on the development of hippocampal circuits and the self-organized dynamics embedded within them since the latter critically support the role of the hippocampus in learning and memory. We first discuss evidence that adult hippocampal cells and circuits are sculpted by development as early as during embryonic neurogenesis. We argue that these primary developmental programs provide a scaffold onto which later experience of the external world can be grafted. Next, we review the different sequences in the development of hippocampal cells and circuits at anatomical and functional levels. We cover a period extending from neurogenesis and migration to the appearance of phenotypic diversity within hippocampal cells and their wiring into functional networks. We describe the progressive emergence of network dynamics in the hippocampus, from sensorimotor-driven early sharp waves to sequences of place cells tracking relational information. We outline the critical turn points and discontinuities in that developmental journey, and close by formulating open questions. We propose that rewinding the process of hippocampal development helps understand the main organization principles of memory circuits.

GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. METHODS: We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. RESULTS: Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. INTERPRETATION: Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.

Molecular and Cellular Mechanisms of Epilepsy.

Despite the availability of a large number of antiepileptic drugs, about 30% of patients with epilepsy, especially temporal lobe epilepsy (TLE), continue to experience seizures [...].

Comparative Study of Terminal Cortical Potentials Using Iridium and Ag/AgCl Electrodes.

Brain ischemia induces slow voltage shifts in the cerebral cortex, including waves of spreading depolarization (SD) and negative ultraslow potentials (NUPs), which are considered as brain injury markers. However, different electrode materials and locations yield variable SD and NUP features. Here, we compared terminal cortical events during isoflurane or sevoflurane euthanasia using intracortical linear iridium electrode arrays and Ag/AgCl-based electrodes in the rat somatosensory cortex. Inhalation of anesthetics caused respiratory arrest, associated with hyperpolarization and followed by SD and NUP on both Ir and Ag electrodes. Ag-NUPs were bell shaped and waned within half an hour after death. Ir-NUPs were biphasic, with the early fast phase corresponding to Ag-NUP, and the late absent on Ag electrodes, phase of a progressive depolarizing voltage shift reaching -100 mV by two hours after death. In addition, late Ir-NUPs were more ample in the deep layers than at the cortical surface. Thus, intracortical Ag and Ir electrodes reliably assess early manifestations of terminal brain injury including hyperpolarization, SD and the early phase of NUP, while the late, giant amplitude phase of NUP, which is present only on Ir electrodes, is probably related to the sensitivity of Ir electrodes to a yet unidentified factor related to brain death.

Somatosensory-Evoked Early Sharp Waves in the Neonatal Rat Hippocampus.

The developing entorhinal-hippocampal system is embedded within a large-scale bottom-up network, where spontaneous myoclonic movements, presumably via somatosensory feedback, trigger hippocampal early sharp waves (eSPWs). The hypothesis, that somatosensory feedback links myoclonic movements with eSPWs, implies that direct somatosensory stimulation should also be capable of evoking eSPWs. In this study, we examined hippocampal responses to electrical stimulation of the somatosensory periphery in urethane-anesthetized, immobilized neonatal rat pups using silicone probe recordings. We found that somatosensory stimulation in ~33% of the trials evoked local field potential (LFP) and multiple unit activity (MUA) responses identical to spontaneous eSPWs. The somatosensory-evoked eSPWs were delayed from the stimulus, on average, by 188 ms. Both spontaneous and somatosensory-evoked eSPWs (i) had similar amplitude of ~0.5 mV and half-duration of ~40 ms, (ii) had similar current-source density (CSD) profiles, with current sinks in CA1 strata radiatum, lacunosum-moleculare and DG molecular layer and (iii) were associated with MUA increase in CA1 and DG. Our results indicate that eSPWs can be triggered by direct somatosensory stimulations and support the hypothesis that sensory feedback from movements is involved in the association of eSPWs with myoclonic movements in neonatal rats.

Early patterns of activity in the developing cortex: Focus on the sensorimotor system.

Early development of somatotopic cortical maps occurs during the fetal period in humans and during the postnatal period in rodents. During this period, the sensorimotor cortex expresses transient patterns of correlated neuronal activity including delta waves, gamma- and spindle-burst oscillations. These early activity patterns are largely driven by the thalamus and triggered, in a topographic manner, by sensory feedback resulting from spontaneous movements. Early cortical activities are instrumental for competitive interactions between sensory inputs for the cortical territories, they prevent cortical neurons from apoptosis and their alteration may lead to disturbances in cortical network development in a number of neurodevelopmental diseases.

Emergence of Coordinated Activity in the Developing Entorhinal-Hippocampal Network.

Correlated activity in the entorhinal-hippocampal neuronal networks, supported by oscillatory and intermittent population activity patterns is critical for learning and memory. However, when and how correlated activity emerges in these networks during development remains largely unknown. Here, we found that during the first postnatal week in non-anaesthetized head-restrained rats, activity in the superficial layers of the medial entorhinal cortex (MEC) and hippocampus was highly correlated, with intermittent population bursts in the MEC followed by early sharp waves (eSPWs) in the hippocampus. Neurons in the superficial MEC layers fired before neurons in the dentate gyrus, CA3 and CA1. eSPW current-source density profiles indicated that perforant/temporoammonic entorhinal inputs and intrinsic hippocampal connections are co-activated during entorhinal-hippocampal activity bursts. Finally, a majority of the entorhinal-hippocampal bursts were triggered by spontaneous myoclonic body movements, characteristic of the neonatal period. Thus, during the neonatal period, activity in the entorhinal cortex (EC) and hippocampus is highly synchronous, with the EC leading hippocampal activation. We propose that such correlated activity is embedded into a large-scale bottom-up circuit that processes somatosensory feedback resulting from neonatal movements, and that it is likely to instruct the development of connections between neocortex and hippocampus.

Segregation of seizures and spreading depolarization across cortical layers.

OBJECTIVE: Cortical spreading depolarization (SD) and seizures are often co-occurring electrophysiological phenomena. However, the cross-layer dynamics of SD during seizures and the effect of SD on epileptic activity across cortical layers remain largely unknown. METHODS: We explored the spatial-temporal dynamics of SD and epileptic activity across layers of the rat barrel cortex using direct current silicone probe recordings during flurothyl-induced seizures. RESULTS: SD occurred in half of the flurothyl-evoked seizures. SD always started from the superficial layers and spread downward either through all cortical layers or stopping at the L4/L5 border. In cases without SD, seizures were characterized by synchronized population firing across all cortical layers throughout the entire seizure. However, when SD occurred, epileptic activity was transiently silenced in layers involved with SD but persisted in deeper layers. During partial SD, epileptiform activity persisted in deep layers throughout the entire seizure, with positive signals at the cortical surface reflecting passive sources of population spikes generated in deeper cortical layers. During full SD, the initial phase of SD propagation through the superficial layers was similar to partial SD, with suppression of activity at the superficial layers and segregation of seizures to deep layers. Further propagation of SD to deep layers resulted in a wave of transient suppression of epileptic activity through the entire cortical column. Thus, vertical propagation of SD through the cortical column creates dynamic network states during which epileptiform activity is restricted to layers without SD. SIGNIFICANCE: Our results point to the importance of vertical SD spread in the SD-related depression of epileptiform activity across cortical layers.

Inhibition of Cortical Activity and Apoptosis Caused by Ethanol in Neonatal Rats In Vivo.

Alcohol consumption during pregnancy causes fetal alcohol spectrum disorder, which includes neuroapoptosis and neurobehavioral deficits. The neuroapoptotic effects of alcohol have been hypothesized to involve suppression of brain activity. However, in vitro studies suggest that ethanol acts as a potent stimulant of cortical activity. We explored the effects of alcohol (1-6 g/kg) on electrical activity in the rat somatosensory cortex in vivo at postnatal days P1-23 and compared them with its apoptotic actions. At P4-7, when the peak of alcohol-induced apoptosis was observed, alcohol strongly suppressed spontaneous gamma and spindle-bursts and almost completely silenced neurons in a dose-dependent manner. The dose-dependence of suppression of neuronal activity strongly correlated with the alcohol-induced neuroapoptosis. Alcohol also profoundly inhibited sensory-evoked bursts and suppressed motor activity, a physiological trigger of cortical activity bursts in newborns. The suppressive effects of ethanol on neuronal activity waned during the second and third postnatal weeks, when instead of silencing the cortex, alcohol evoked delta-wave electrographic activity. Thus, the effects of alcohol on brain activity are strongly age-dependent, and during the first postnatal week alcohol profoundly inhibits brain activity. Our findings suggest that the adverse effects of alcohol in the developing brain involve suppression of neuronal activity.

The Nature of the Sensory Input to the Neonatal Rat Barrel Cortex.

UNLABELLED: Sensory input plays critical roles in the development of the somatosensory cortex during the neonatal period. This early sensory input may involve: (1) stimulation arising from passive interactions with the mother and littermates and (2) sensory feedback arising from spontaneous infant movements. The relative contributions of these mechanisms under natural conditions remain largely unknown, however. Here, we show that, in the whisker-related barrel cortex of neonatal rats, spontaneous whisker movements and passive stimulation by the littermates cooperate, with comparable efficiency, in driving cortical activity. Both tactile signals arising from the littermate's movements under conditions simulating the littermates' position in the litter, and spontaneous whisker movements efficiently triggered bursts of activity in barrel cortex. Yet, whisker movements with touch were more efficient than free movements. Comparison of the various experimental conditions mimicking the natural environment showed that tactile signals arising from the whisker movements with touch and stimulation by the littermates, support: (1) a twofold higher level of cortical activity than in the isolated animal, and (2) a threefold higher level of activity than in the deafferented animal after the infraorbital nerve cut. Together, these results indicate that endogenous (self-generated movements) and exogenous (stimulation by the littermates) mechanisms cooperate in driving cortical activity in newborn rats and point to the importance of the environment in shaping cortical activity during the neonatal period. SIGNIFICANCE STATEMENT: Sensory input plays critical roles in the development of the somatosensory cortex during the neonatal period. However, the origins of sensory input to the neonatal somatosensory cortex in the natural environment remain largely unknown. Here, we show that in the whisker-related barrel cortex of neonatal rats, spontaneous whisker movements and passive stimulation by the littermates cooperate, with comparable efficiency, in driving cortical activity during the critical developmental period.

Dynamic Changes from Depolarizing to Hyperpolarizing GABAergic Actions during Giant Depolarizing Potentials in the Neonatal Rat Hippocampus.

During development, GABA exerts depolarizing action on immature neurons and, acting in synergy with glutamate, drives giant depolarizing potentials (GDPs) in the hippocampal network. Yet, blockade of the GABA(A) receptors transforms GDPs to epileptiform discharges suggesting dual, both excitatory and inhibitory, actions of GABA in the immature hippocampal network. However, the nature of this dualism in early GABA actions is poorly understood. Here we characterized the dynamics of synaptic currents mediated by GABA(A) and glutamate receptors through an estimation of the changes in their conductance and driving forces in neonatal rat CA3 pyramidal cells during GDPs. We found that depolarizing GABAergic and glutamatergic currents act in synergy at the GDPs' onset. However, during the peak of the population discharge, the inward synaptic current was essentially mediated by glutamate receptors whereas GABA currents transiently switched their direction from depolarizing to hyperpolarizing as a result of neuronal depolarization above the GABA(A) reversal potential. Thus, the action of GABA on CA3 pyramidal cells dynamically changes during GDPs from excitatory at the GDPs' onset to inhibitory at the GDPs' peak. We propose that the dynamic changes in GABA actions occurring during GDPs enable GABAergic interneurons not only to initiate the discharge of pyramidal cells but also to control excitation in the recurrent CA3 network preventing epileptiform synchronization. SIGNIFICANCE STATEMENT: During development GABA exerts a depolarizing action on immature neurons. However, at the network level the effects of GABA are complex involving both excitatory and inhibitory actions. Here we show that GABA actions critically depend on the network state. Although GABA depolarizes neurons at rest and at the onset of population bursts, it transiently becomes hyperpolarizing at the peak of the population bursts. These dynamic changes in GABA actions enable GABAergic interneurons not only to initiate the network discharge but also to control excitation to prevent epileptiform synchronization.

Imprecise Whisker Map in the Neonatal Rat Barrel Cortex.

The somatosensory barrel cortex in rodents contains a topographic map of the facial whiskers where each cortical barrel is tuned to a corresponding whisker. However, exactly when this correspondence is established during development and how precise the functional topography of the whisker protomap is at birth, before the anatomical formation of barrels, are questions that remain unresolved. Here, using extracellular and whole-cell recordings from the barrel cortex of 0- to 7-day-old (P0-7; P0 = day of birth) rat pups in vivo, we report a low level of tuning to the principal whisker at P0-1, with multiple adjacent whiskers evoking large multi- and single-unit responses and excitatory postsynaptic currents in cortical neurons. Additionally, we found broad and largely overlapping projection fields (PFs) for neighboring whiskers in the barrel cortex at P0-1. Starting from P2-3, a segregated whisker map emerged, characterized by preferential single whisker tuning and segregated whisker PFs. These results indicate that the functional whisker protomap in the somatosensory cortex is imprecise at birth, that for 2-3 days after birth, whiskers compete for the cortical target territories, and that formation of a segregated functional whisker map coincides with emergence of the anatomical barrel map.

Synchronous excitation in the superficial and deep layers of the medial entorhinal cortex precedes early sharp waves in the neonatal rat hippocampus.

Early Sharp Waves (eSPWs) are the earliest pattern of network activity in the developing hippocampus of neonatal rodents. eSPWs were originally considered to be an immature prototype of adult SPWs, which are spontaneous top-down hippocampal events that are self-generated in the hippocampal circuitry. However, recent studies have shifted this paradigm to a bottom-up model of eSPW genesis, in which eSPWs are primarily driven by the inputs from the layers 2/3 of the medial entorhinal cortex (MEC). A hallmark of the adult SPWs is the relay of information from the CA1 hippocampus to target structures, including deep layers of the EC. Whether and how deep layers of the MEC are activated during eSPWs in the neonates remains elusive. In this study, we investigated activity in layer 5 of the MEC of neonatal rat pups during eSPWs using silicone probe recordings from the MEC and CA1 hippocampus. We found that neurons in deep and superficial layers of the MEC fire synchronously during MEC sharp potentials, and that neuronal firing in both superficial and deep layers of the MEC precedes the activation of CA1 neurons during eSPWs. Thus, the sequence of activation of CA1 hippocampal neurons and deep EC neurons during sharp waves reverses during development, from a lead of deep EC neurons during eSPWs in neonates to a lead of CA1 neurons during adult SPWs. These findings suggest another important difference in the generative mechanisms and possible functional roles of eSPWs compared to adult SPWs.

Synaptic Origin of Early Sensory-evoked Oscillations in the Immature Thalamus.

During the critical period of postnatal development, brain maturation is extremely sensitive to external stimuli. Newborn rodents already have functional somatosensory pathways and the thalamus, but the cortex is still forming. Immature thalamic synapses may produce large postsynaptic potentials in immature neurons, while non-synaptic membrane currents remain relatively weak and slow. The thalamocortical system generates spontaneous and evoked early gamma and spindle-burst oscillations in newborn rodents. How relatively strong synapses and weak intrinsic currents interact with each other and how they contribute to early thalamic activities remains largely unknown. Here, we performed local field potential (LFP), juxtacellular, and patch-clamp recordings in the somatosensory thalamus of urethane-anesthetized rat pups at postnatal days 6-7 with one whisker stimulation. We removed the overlying cortex and hippocampus to reach the thalamus with electrodes. Deflection of only one (the principal) whisker induced spikes in a particular thalamic cell. Whisker deflection evoked a group of large-amplitude excitatory events, likely originating from lemniscal synapses and multiple inhibitory postsynaptic events in thalamocortical cells. Large-amplitude excitatory events produced a group of spike bursts and could evoke a depolarization block. Juxtacellular recordings confirmed the partial inactivation of spikes. Inhibitory events prevented inactivation of action potentials and gamma-modulated neuronal firing. We conclude that the interplay of strong excitatory and inhibitory synapses and relatively weak intrinsic currents produces sensory-evoked early gamma oscillations in thalamocortical cells. We also propose that sensory-evoked large-amplitude excitatory events contribute to evoked spindle-bursts.

Anoxic spreading depolarization in the neonatal rat cortex in vitro.

Anoxic spreading depolarization (aSD) is a hallmark of ischemic injury in the cerebral cortex. In adults, aSD is associated with rapid and nearly complete neuronal depolarization and loss of neuronal functions. While ischemia also evokes aSD in the immature cortex, developmental aspects of neuronal behavior during aSD remain largely unknown. Here, using oxygen-glucose deprivation (OGD) ischemia model in slices of the postnatal rat somatosensory cortex, we found that immature neurons displayed much more complex behaviors: they initially moderately depolarized during aSD, then transiently repolarised (for up to tens of minutes), and only then passed to terminal depolarization. The ability to fire action potentials was maintained in neurons mildly depolarized during aSD without reaching the level of depolarization block, and these functions were regained in the majority of immature neurons during post-aSD transient repolarization. The amplitude of depolarization and the probability of depolarization block during aSD increased, whereas transient post-SD repolarization levels and duration, and associated recovery in neuronal firing decreased with age. By the end of the first postnatal month, aSD acquired an adult-like phenotype, where depolarization during aSD merged with terminal depolarization and the phase of transient recovery was lost. Thus, changes in neuronal function during aSD undergo remarkable developmental changes that may contribute to lower susceptibility of the immature neurons to ischemia.

Diversity of cortical activity changes beyond depression during Spreading Depolarizations.

Spreading depolarizations (SDs) are classically thought to be associated with spreading depression of cortical activity. Here, we found that SDs in patients with subarachnoid hemorrhage produce variable, ranging from depression to booming, changes in electrocorticographic activity, especially in the delta frequency band. In rats, depression of activity was characteristic of high-potassium-induced full SDs, whereas partial superficial SDs caused either little change or a boom of activity at the cortical vertex, supported by volume conduction of signals from spared delta generators in the deep cortical layers. Partial SDs also caused moderate neuronal depolarization and sustained excitation, organized in gamma oscillations in a narrow sub-SD zone. Thus, our study challenges the concept of homology between spreading depolarization and spreading depression by showing that SDs produce variable, from depression to booming, changes in activity at the cortical surface and in different cortical layers depending on the depth of SD penetration.

On the accuracy of cell-attached current-clamp recordings from cortical neurons.

Cell-attached current-clamp (CA/CC) recordings have been proposed to measure resting membrane potential and synaptic/agonist responses in neurons without disrupting the cell membrane, thus avoiding the intracellular dialysis that occurs in conventional whole-cell recordings (WC). However, the accuracy of CA/CC recordings in neurons has not been directly assessed. Here, we used concomitant CA and WC current clamp recordings from cortical neurons in brain slices. Resting membrane potential values and slow voltage shifts showed variability and were typically attenuated during CA/CC recordings by ~10-20% relative to WC values. Fast signals were slowed down and their amplitude was greatly reduced: synaptic potentials by nearly 2-fold, and action potentials by nearly 10-fold in CA/CC mode compared to WC. The polarity of GABAergic postsynaptic responses in CA/CC mode matched the responses in WC, and depolarising GABAergic potentials were predominantly observed during CA/CC recordings of intact neonatal CA3 hippocampal pyramidal neurons. Similarly, CA/CC recordings reliably detected neuronal depolarization and excitation during network-induced giant depolarizing potentials in the neonatal CA3 hippocampus, and revealed variable changes, from depolarization to hyperpolarization, in CA1 pyramidal cells during sharp wave ripples in the adult hippocampus. Thus, CA/CC recordings are suitable for assessing membrane potential but signal distortion, probably caused by leakage via the seal contact and RC filtering should be considered.

Depth-profile of impairments in endothelin-1 - induced focal cortical ischemia.

The development of ischemic lesions has primarily been studied in horizontal cortical space. However, how ischemic lesions develop through the cortical depth remains largely unknown. We explored this question using direct current coupled recordings at different cortical depths using linear arrays of iridium electrodes in the focal epipial endothelin-1 (ET1) ischemia model in the rat barrel cortex. ET1-induced impairments were characterized by a vertical gradient with (i) rapid suppression of the spontaneous activity in the superficial cortical layers at the onset of ischemia, (ii) compartmentalization of spreading depolarizations (SDs) to the deep layers during progression of ischemia, and (iii) deeper suppression of activity and larger histological lesion size in superficial cortical layers. The level of impairments correlated strongly with the rate of spontaneous activity suppression, the rate of SD onset after ET1 application, and the amplitude of giant negative ultraslow potentials (∼-70 mV), which developed during ET1 application and were similar to the tent-shaped ultraslow potentials observed during focal ischemia in the human cortex. Thus, in the epipial ET1 ischemia model, ischemic lesions develop progressively from the surface to the cortical depth, and early changes in electrical activity at the onset of ET1-induced ischemia reliably predict the severity of ischemic damage.

Bone conducted responses in the neonatal rat auditory cortex.

Rats are born deaf and start hearing at the end of the second postnatal week, when the ear canals open and low-intensity sounds start to evoke responses in the auditory cortex. Here, using µECoG electrode arrays and intracortical silicon probe recordings, we found that bone-conducted (BC) sounds evoked biphasic responses in the auditory cortex starting from postnatal day (P) 8. The initial phase of these responses, generated by thalamocortical input, was followed by intracortical propagation within supragranular layers. BC-evoked responses co-localized with the responses evoked by electrical stimulation of the cochlea and the deepest layers of the inferior colliculus prior to onset of low-threshold hearing (P13), as well as with the responses evoked by high-frequency (30 kHz) low-intensity (70 dB) air-conducted sounds after that. Thus, BC signals reach high-frequency processing regions of the auditory cortex well before the onset of low-threshold hearing, reflecting early integrity of the auditory system.

Full-Band EEG Recordings Using Hybrid AC/DC-Divider Filters.

Full-band DC recordings enable recording of slow electrical brain signals that are severely compromised during conventional AC recordings. However, full-band DC recordings may be limited by the amplifier's dynamic input range and the loss of small amplitude high-frequency signals. Recently, Neuralynx has proposed full-band recordings with inverse filtering for signal reconstruction based on hybrid AC/DC-divider RRC filters that enable only partial suppression of DC signals. However, the quality of signal reconstruction for biological signals has not yet been assessed. Here, we propose a novel digital inverse filter based on a mathematical model describing RRC filter properties, which provides high computational accuracy and versatility. Second, we propose procedures for the evaluation of the inverse filter coefficients, adapted for each recording channel to minimize the error caused by the deviation of the real values of the RRC filter elements from their nominal values. We demonstrate that this approach enables near 99% reconstruction quality of high-potassium-induced cortical spreading depolarizations (SDs), endothelin-induced ischemic negative ultraslow potentials (NUPs), and whole-cell recordings of membrane potential using RRC filters. The quality of the reconstruction was significantly higher than with the existing inverse filtering procedures. Thus, RRC filters with inverse filtering are optimal for full-band EEG recordings in various applications.