Prenatal development of the human entorhinal cortex.

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP-expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders.

Cellular resolution anatomical and molecular atlases for prenatal human brains.

Increasing interest in studies of prenatal human brain development, particularly using new single-cell genomics and anatomical technologies to create cell atlases, creates a strong need for accurate and detailed anatomical reference atlases. In this study, we present two cellular-resolution digital anatomical atlases for prenatal human brain at postconceptional weeks (PCW) 15 and 21. Both atlases were annotated on sequential Nissl-stained sections covering brain-wide structures on the basis of combined analysis of cytoarchitecture, acetylcholinesterase staining, and an extensive marker gene expression dataset. This high information content dataset allowed reliable and accurate demarcation of developing cortical and subcortical structures and their subdivisions. Furthermore, using the anatomical atlases as a guide, spatial expression of 37 and 5 genes from the brains, respectively, at PCW 15 and 21 was annotated, illustrating reliable marker genes for many developing brain structures. Finally, the present study uncovered several novel developmental features, such as the lack of an outer subventricular zone in the hippocampal formation and entorhinal cortex, and the apparent extension of both cortical (excitatory) and subcortical (inhibitory) progenitors into the prenatal olfactory bulb. These comprehensive atlases provide useful tools for visualization, segmentation, targeting, imaging, and interpretation of brain structures of prenatal human brain, and for guiding and interpreting the next generation of cell census and connectome studies.

Heterogeneity of Age-Related Neural Hyperactivity along the CA3 Transverse Axis.

Age-related memory deficits are correlated with neural hyperactivity in the CA3 region of the hippocampus. Abnormal CA3 hyperactivity in aged rats has been proposed to contribute to an imbalance between pattern separation and pattern completion, resulting in overly rigid representations. Recent evidence of functional heterogeneity along the CA3 transverse axis suggests that proximal CA3 supports pattern separation while distal CA3 supports pattern completion. It is not known whether age-related CA3 hyperactivity is uniformly represented along the CA3 transverse axis. We examined the firing rates of CA3 neurons from young and aged, male, Long-Evans rats along the CA3 transverse axis. Consistent with prior studies, young CA3 cells showed an increasing gradient in mean firing rate from proximal to distal CA3. However, aged CA3 cells showed an opposite, decreasing trend, in that CA3 cells in aged rats were hyperactive in proximal CA3, but possibly hypoactive in distal CA3, compared with young (Y) rats. We suggest that, in combination with altered inputs from the entorhinal cortex and dentate gyrus (DG), the proximal CA3 region of aged rats may switch from its normal function that reflects the pattern separation output of the DG and instead performs a computation that reflects an abnormal bias toward pattern completion. In parallel, distal CA3 of aged rats may create weaker attractor basins that promote abnormal, bistable representations under certain conditions.SIGNIFICANCE STATEMENT Prior work suggested that age-related CA3 hyperactivity enhances pattern completion, resulting in rigid representations. Implicit in prior studies is the notion that hyperactivity is present throughout a functionally homogeneous CA3 network. However, more recent work has demonstrated functional heterogeneity along the CA3 transverse axis, in that proximal CA3 is involved in pattern separation and distal CA3 is involved in pattern completion. Here, we show that age-related hyperactivity is present only in proximal CA3, with potential hypoactivity in distal CA3. This result provides new insight in the role of CA3 in age-related memory impairments, suggesting that the rigid representations in aging result primarily from dysfunction of computational circuits involving the dentate gyrus (DG) and proximal CA3.

Regional Tau Effects on Prospective Cognitive Change in Cognitively Normal Older Adults.

Studies suggest that tau deposition starts in the anterolateral entorhinal cortex (EC) with normal aging, and that the presence of ß-amyloid (Aß) facilitates its spread to neocortex, which may reflect the beginning of Alzheimer's disease (AD). Functional connectivity between the anterolateral EC and the anterior-temporal (AT) memory network appears to drive higher tau deposition in AT than in the posterior-medial (PM) memory network. Here, we investigated whether this differential vulnerability to tau deposition may predict different cognitive consequences of EC, AT, and PM tau. Using 18F-flortaucipir (FTP) and 11C-Pittsburgh compound-B (PiB) positron emission tomography (PET) imaging, we measured tau and Aß in 124 cognitively normal human older adults (74 females, 50 males) followed for an average of 2.8 years for prospective cognition. We found that higher FTP in all three regions was individually related to faster memory decline, and that the effects of AT and PM FTP, but not EC, were driven by Aß+ individuals. Moreover, when we included all three FTP measures competitively in the same model, only AT FTP significantly predicted memory decline. Our data support a model whereby tau, facilitated by Aß, transits from EC to cortical regions that are most closely associated with the anterolateral EC, which specifically affects memory in the initial stage of AD. Memory also appears to be affected by EC tau in the absence of Aß, which may be less clinically consequential. These findings may provide clarification of differences between normal aging and AD, and elucidate the transition between the two stages.SIGNIFICANCE STATEMENT Tau and ß-amyloid (Aß) are hallmarks of Alzheimer's disease (AD) but are also found in cognitively normal people. It is unclear whether, and how, this early deposition of tau and Aß may affect cognition in normal aging and the asymptomatic stage of AD. We show that tau deposition in the entorhinal cortex (EC), which is common in advanced age, predicts memory decline in older adults independent of Aß, likely reflecting normal, age-related memory loss. In contrast, tau in anterior-temporal (AT) regions is most predictive of memory decline in Aß+ individuals. These data support the idea that tau preferentially spreads to specific cortical regions, likely through functional connections, which plays a primary role in memory decline in the early stage of AD.

Postnatal development of the entorhinal cortex: A stereological study in macaque monkeys.

The entorhinal cortex is the main gateway for interactions between the neocortex and the hippocampus. Distinct regions, layers, and cells of the hippocampal formation exhibit different profiles of structural and molecular maturation during postnatal development. Here, we provide estimates of neuron number, neuronal soma size, and volume of the different layers and subdivisions of the monkey entorhinal cortex (Eo, Er, Elr, Ei, Elc, Ec, Ecl) during postnatal development. We found different developmental changes in neuronal soma size and volume of distinct layers in different subdivisions, but no changes in neuron number. Layers I and II developed early in most subdivisions. Layer III exhibited early maturation in Ec and Ecl, a two-step/early maturation in Ei and a late maturation in Er. Layers V and VI exhibited an early maturation in Ec and Ecl, a two-step and early maturation in Ei, and a late maturation in Er. Neuronal soma size increased transiently at 6 months of age and decreased thereafter to reach adult size, except in Layer II of Ei, and Layers II and III of Ec and Ecl. These findings support the theory that different hippocampal circuits exhibit distinct developmental profiles, which may subserve the emergence of different hippocampus-dependent memory processes. We discuss how the early maturation of the caudal entorhinal cortex may contribute to path integration and basic allocentric spatial processing, whereas the late maturation of the rostral entorhinal cortex may contribute to the increased precision of allocentric spatial representations and the temporal integration of individual items into episodic memories.

Postnatal Development of Functional Projections from Parasubiculum and Presubiculum to Medial Entorhinal Cortex in the Rat.

Neurons in parasubiculum (PaS), presubiculum (PrS), and medial entorhinal cortex (MEC) code for place (grid cells) and head direction. Directional input has been shown to be important for stable grid cell properties in MEC, and PaS and PrS have been postulated to provide this information to MEC. In line with this, head direction cells in those brain areas are present at postnatal day 11 (P11), having directional tuning that stabilizes shortly after eye opening, which is before premature grid cells emerge in MEC at P16. Whether functional connectivity between these structures exists at those early postnatal stages is unclear. Using anatomical tracing, voltage-sensitive dye imaging and single-cell patch recordings in female and male rat brain slices between P2 and P61, we determined when the pathways from PaS and PrS to MEC emerge, become functional, and how they develop. Anatomical connections from PaS and PrS to superficial MEC emerge between P4 and P6. Monosynaptic connectivity from PaS and PrS to superficial MEC was measurable from P9 to P10 onward, whereas connectivity with deep MEC was measurable from P11 to P12. From P14/P15 on, reactivity of MEC neurons to parasubicular and presubicular inputs becomes adult-like and continues to develop until P28-P30. The maturation of the efficacy of both inputs between P9 and P21 is paralleled by maturation of morphological properties, changes in intrinsic properties of MEC principal neurons, and changes in the GABAergic network of MEC. In conclusion, synaptic projections from PaS and PrS to MEC become functional and adult-like before the emergence of grid cells in MEC.SIGNIFICANCE STATEMENT Head direction information, crucial for grid cells in medial entorhinal cortex (MEC), is thought to enter MEC via parasubiculum (PaS) and presubiculum (PrS). Unraveling the development of functional connections between PaS, PrS, and MEC is key to understanding how spatial navigation, an important cognitive function, may evolve. To gain insight into the development, we used anatomical tracing techniques, voltage-sensitive dye imaging, and single-cell recordings. The combined data led us to conclude that synaptic projections from PaS and PrS to MEC become functional and adult-like before eye opening, allowing crucial head direction information to influence place encoding before the emergence of grid cells in rat MEC.

Construction and disruption of spatial memory networks during development.

Spatial memory, the aspect of memory involving encoding and retrieval of information regarding one's environment and spatial orientation, is a complex biological function incorporating multiple neuronal networks. Hippocampus-dependent spatial memory is not innate and emerges during development in both humans and rodents. In children, nonhippocampal dependent egocentric (self-to-object) memory develops before hippocampal-dependent allocentric (object-to-object) memory. The onset of allocentric spatial memory abilities in children around 22 mo of age occurs at an age-equivalent time in rodents when spatially tuned grid and place cells arise from patterned activity propagating through the entorhinal-hippocampal circuit. Neuronal activity, often driven by specific sensory signals, is critical for the normal maturation of brain circuits This patterned activity fine-tunes synaptic connectivity of the network and drives the emergence of specific firing necessary for spatial memory. Whereas normal activity patterns are required for circuit maturation, aberrant neuronal activity during development can have major adverse consequences, disrupting the development of spatial memory. Seizures during infancy, involving massive bursts of synchronized network activity, result in impaired spatial memory when animals are tested as adolescents or adults. This impaired spatial memory is accompanied by alterations in spatial and temporal coding of place cells. The molecular mechanisms by which early-life seizures lead to disruptions at the cellular and network levels are now becoming better understood, and provide a target for intervention, potentially leading to improved cognitive outcome in individuals experiencing early-life seizures.

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.

Development of Parvalbumin-Expressing Basket Terminals in Layer II of the Rat Medial Entorhinal Cortex.

Grid cells in layer II of the medial entorhinal cortex (MEC LII) generate multiple regular firing fields in response to the position and speed of an individual within the environment. They exhibit a protracted postnatal development and, in the adult, show activity differences along the dorsoventral axis (DVA). Evidence suggests parvalbumin-positive (PV+) interneurons, most of which are perisomatic-targeting cells, play a crucial role in generation of the hexagonal grid cell activity pattern. We therefore hypothesized that the development and organization of PV+ perisomatic terminals in MEC LII reflect the postnatal emergence of the hexagonal firing pattern and dorsoventral differences seen in grid cell activity. We used immuno-electron microscopy to examine the development of PV+ perisomatic terminals and their target somata within dorsal and ventral MEC LII in rats of postnatal day (P)10, P15, and P30. We demonstrate that in dorsal and ventral MEC LII, the cross-sectional area of somata and number and density of perisomatic PV+ terminals increase between P10 and P15. A simultaneous decrease was observed in cross-sectional area of PV+ terminals. Between P15 and P30, both MEC regions showed an increase in PV+ terminal size and percentage of PV+ terminals containing mitochondria, which may enable grid cell activity to emerge and stabilize. We also report that dorsal somata are larger and apposed by more PV+ terminals than ventral somata at all stages, suggesting a protracted maturation in the ventral portion and a possible gradient in soma size and PV+ basket innervation along the DVA in the adult.

Path integration in place cells of developing rats.

Place cells in the hippocampus and grid cells in the medial entorhinal cortex rely on self-motion information and path integration for spatially confined firing. Place cells can be observed in young rats as soon as they leave their nest at around 2.5 wk of postnatal life. In contrast, the regularly spaced firing of grid cells develops only after weaning, during the fourth week. In the present study, we sought to determine whether place cells are able to integrate self-motion information before maturation of the grid-cell system. Place cells were recorded on a 200-cm linear track while preweaning, postweaning, and adult rats ran on successive trials from a start wall to a box at the end of a linear track. The position of the start wall was altered in the middle of the trial sequence. When recordings were made in complete darkness, place cells maintained fields at a fixed distance from the start wall regardless of the age of the animal. When lights were on, place fields were determined primarily by external landmarks, except at the very beginning of the track. This shift was observed in both young and adult animals. The results suggest that preweaning rats are able to calculate distances based on information from self-motion before the grid-cell system has matured to its full extent.

MicroRNAs contribute to postnatal development of laminar differences and neuronal subtypes in the rat medial entorhinal cortex.

The medial entorhinal cortex (MEC) is important in spatial navigation and memory formation and its layers have distinct neuronal subtypes, connectivity, spatial properties, and disease susceptibility. As little is known about the molecular basis for the development of these laminar differences, we analyzed microRNA (miRNA) and messenger RNA (mRNA) expression differences between rat MEC layer II and layers III-VI during postnatal development. We identified layer and age-specific regulation of gene expression by miRNAs, which included processes related to neuron specialization and locomotor behavior. Further analyses by retrograde labeling and expression profiling of layer II stellate neurons and in situ hybridization revealed that the miRNA most up-regulated in layer II, miR-143, was enriched in stellate neurons, whereas the miRNA most up-regulated in deep layers, miR-219-5p, was expressed in ependymal cells, oligodendrocytes and glia. Bioinformatics analyses of predicted mRNA targets with negatively correlated expression patterns to miR-143 found that miR-143 likely regulates the Lmo4 gene, which is known to influence hippocampal-based spatial learning.

Stellate cells drive maturation of the entorhinal-hippocampal circuit.

The neural representation of space relies on a network of entorhinal-hippocampal cell types with firing patterns tuned to different abstract features of the environment. To determine how this network is set up during early postnatal development, we monitored markers of structural maturation in developing mice, both in naïve animals and after temporally restricted pharmacogenetic silencing of specific cell populations. We found that entorhinal stellate cells provide an activity-dependent instructive signal that drives maturation sequentially and unidirectionally through the intrinsic circuits of the entorhinal-hippocampal network. The findings raise the possibility that a small number of autonomously developing neuronal populations operate as intrinsic drivers of maturation across widespread regions of the cortex.

Postnatal development of retrosplenial projections to the parahippocampal region of the rat.

The rat parahippocampal region (PHR) and retrosplenial cortex (RSC) are cortical areas important for spatial cognition. In PHR, head-direction cells are present before eye-opening, earliest detected in postnatal day (P)11 animals. Border cells have been recorded around eye-opening (P16), while grid cells do not obtain adult-like features until the fourth postnatal week. In view of these developmental time-lines, we aimed to explore when afferents originating in RSC arrive in PHR. To this end, we injected rats aged P0-P28 with anterograde tracers into RSC. First, we characterized the organization of RSC-PHR projections in postnatal rats and compared these results with data obtained in the adult. Second, we described the morphological development of axonal plexus in PHR. We conclude that the first arriving RSC-axons in PHR, present from P1 onwards, already show a topographical organization similar to that seen in adults, although the labeled plexus does not obtain adult-like densities until P12.

Cell-Type Specific Phase Precession in Layer II of the Medial Entorhinal Cortex.

The identity of phase-precessing cells in the entorhinal cortex is unknown. Here, we used a classifier derived from cell-attached recordings to separate putative pyramidal cells and putative stellate cells recorded extracellularly in layer II of the medial entorhinal cortex in rats. Using a novel method to identify single runs as temporal periods of elevated spiking activity, we find that both cell types show phase precession but putative stellate cells show steeper slopes of phase precession and larger phase ranges. As the two classes of cells have different projection patterns, phase precession is differentially passed on to different subregions of the hippocampal formation. SIGNIFICANCE STATEMENT: It is a great challenge for neuroscience to reveal the cellular basis of cognitive functions. One such function is the ability to learn and recollect temporal sequences of events. The representation of sequences in the brain is thought to require temporally structured activity of nerve cells. How different types of neurons generate temporally structured activity is currently unknown. In the present study, we use a computational classification procedure to separate different cell types and find that a subpopulation of cells, so-called stellate neurons, exhibits clear temporal coding. Contrary to the stellate cells, pyramidal cells show weaker temporal coding. This discovery sheds light on the cellular basis of temporal coding in the brain.

Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers.

Patterns of Spontaneous Local Network Activity in Developing Cerebral Cortex: Relationship to Adult Cognitive Function.

Detecting neurodevelopµental disorders of cognition at the earliest possible stages could assist in understanding them mechanistically and ultimately in treating them. Finding early physiological predictors that could be visualized with functional neuroimaging would represent an important advance in this regard. We hypothesized that one potential source of physiological predictors is the spontaneous local network activity prominent during specific periods in development. To test this we used calcium imaging in brain slices and analyzed variations in the frequency and intensity of this early activity in one area, the entorhinal cortex (EC), in order to correlate early activity with level of cognitive function later in life. We focused on EC because of its known role in different types of cognitive processes and because it is an area where spontaneous activity is prominent during early postnatal development in rodent models of cortical development. Using rat strains (Long-Evans, Wistar, Sprague-Dawley and Brattleboro) known to differ in cognitive performance in adulthood we asked whether neonatal animals exhibit corresponding strain-related differences in EC spontaneous activity. Our results show significant differences in this activity between strains: compared to a high cognitive-performing strain, we consistently found an increase in frequency and decrease in intensity in neonates from three lower performing strains. Activity was most different in one strain considered a model of schizophrenia-like psychopathology. While we cannot necessarily infer a causal relationship between early activity and adult cognition our findings suggest that the pattern of spontaneous activity in development could be an early predictor of a developmental trajectory advancing toward sub-optimal cognitive performance in adulthood. Our results further suggest that the strength of dopaminergic signaling, by setting the balance between excitation and inhibition, is a potential underlying mechanism that could explain the observed differences in early spontaneous activity patterns.

Laminar and dorsoventral molecular organization of the medial entorhinal cortex revealed by large-scale anatomical analysis of gene expression.

Neural circuits in the medial entorhinal cortex (MEC) encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations.

Glutamatergic system controls synchronization of spontaneous neuronal activity in the murine neonatal entorhinal cortex.

Synchronized spontaneous neuronal activity is a characteristic feature of the developing brain. Rhythmic network discharges in the neonatal medial entorhinal cortex (mEC) in vitro depend on activation of ionotropic glutamate receptors, but spontaneously active neurons are required for their initiation. Field potential recordings revealed synchronized neuronal activity in the mEC in vivo developmentally earlier than in vitro. We suggested that not only ionotropic receptors, but also other components of the glutamatergic system modulate neuronal activity in the mEC. Ca(2+) imaging was used to record neuronal activity in neonatal murine brain slices. Two types of spontaneous events were distinguished: global synchronous discharges (synchronous activity) and asynchronously (not synchronized with global discharges) active cells (asynchronous activity). AMPA receptor blockade strongly reduced the frequency of synchronous discharges, while NMDA receptor inhibition was less effective. AMPA and NMDA receptor blockade or activation of group 2/3 metabotropic glutamate receptors (mGluR2/3) completely suppressed synchronous discharges and increased the number of active cells. Blockade of glutamate transporters with DL-TBOA led to NMDA receptor-mediated hyper-synchronization of neuronal activity. Inhibition of NMDA receptors in the presence of DL-TBOA failed to restore synchronous discharges. The latter were partially reestablished only after blockade of mGluR2/3. We conclude that the glutamatergic system can influence neuronal activity via different receptors/mechanisms. As both NMDA and mGluR2/3 receptors have a high affinity for glutamate, changes in extracellular glutamate levels resulting for instance from glutamate transporter malfunction can balance neuronal activity in the mEC, affecting in turn synapse and network formation.

Representation of geometric borders in the developing rat.

Local space is represented by a number of functionally specific cell types, including place cells in the hippocampus and grid cells, head direction cells, and border cells in the medial entorhinal cortex (MEC). These cells form a functional map of external space already at the time when rat pups leave the nest for the first time in their life, at the age of 2.5 weeks. However, while place cells have adult-like firing fields from the outset, grid cells have irregular and variable fields until the fourth week, raising doubts about their contribution to place cell firing at young age. Recording in MEC of juvenile rats, we show that, unlike grid cells, border cells express adult-like firing fields from the first days of exposure to an open environment, at postnatal days 16-18. Thus, spatial signals from border cells may be sufficient to maintain spatially localized firing in juvenile hippocampal place cells.