NECAB1-3, parvalbumin, calbindin, and calretinin in the hippocampus of the European mole.

Many calcium-binding proteins are expressed in a region-and cell-type specific manner in the mammalian hippocampus. Neuronal calcium-binding proteins (NECABs) are also expressed in hippocampal neurons, but few species have been investigated, with partly controversial findings. We here describe NECAB1, NECAB2 and NECAB3 as well as parvalbumin, calbindin, and calretinin in the European mole, and compare staining patterns of these proteins with those in mouse and other species. While subtle differences are present, NECAB staining in the European mole was generally similar to those in mouse. Common to European moles, mice, and other species we investigated, large hilar polymorphic cells, likely to represent mossy cells, were positive for all three NECABs. NECAB1 and 2 are suitable as markers for these cells along the entire septotemporal axis of the hippocampus. In the European mole, parvalbumin, calbindin and calretinin showed traits that have been described in other species before, albeit in a unique combination. In summary, we provide the first description of distribution of these proteins in the hippocampus of the European mole. This subterranean, insectivorous, and solitary living species belongs to the Order of Eulipotyphla. Despite many similarities with other subterranean species from the rodent order in terms of lifestyle, its hippocampus is cytoarchitecturally much more elaborated than in, e.g., mole-rats. It remains an open question if the hippocampal structure of the European mole reflects evolutionary constraints or ecology. Our descriptive study highlights the diversity in hippocampal cytoarchitecture even in small mammalian species.

Context and space coding in mossy cell population activity.

The dentate gyrus plays a key role in the discrimination of memories by segregating and storing similar episodes. Whether hilar mossy cells, which constitute a major excitatory principal cell type in the mammalian hippocampus, contribute to this decorrelation function has remained largely unclear. Using two-photon calcium imaging of head-fixed mice performing a spatial virtual reality task, we show that mossy cell populations robustly discriminate between familiar and novel environments. The degree of discrimination depends on the extent of visual cue differences between contexts. A context decoder revealed that successful environmental classification is explained mainly by activity difference scores of mossy cells. By decoding mouse position, we reveal that in addition to place cells, the coordinated activity among active mossy cells markedly contributes to the encoding of space. Thus, by decorrelating context information according to the degree of environmental differences, mossy cell populations support pattern separation processes within the dentate gyrus.

Comparative Anatomy of the Dentate Mossy Cells in Nonhuman Primates: Their Spatial Distributions and Axonal Projections Compared With Mouse Mossy Cells.

Glutamatergic mossy cells (MCs) mediate associational and commissural connectivity, exhibiting significant heterogeneity along the septotemporal axis of the mouse dentate gyrus (DG). However, it remains unclear whether the neuronal features of MCs are conserved across mammals. This study compares the neuroanatomy of MCs in the DG of mice and monkeys. The MC marker, calretinin, distinguishes two subpopulations: septal and temporal. Dual-colored fluorescence labeling is utilized to compare the axonal projection patterns of these subpopulations. In both mice and monkeys, septal and temporal MCs project axons across the longitudinal axis of the ipsilateral DG, indicating conserved associational projections. However, unlike in mice, no MC subpopulations in monkeys make commissural projections to the contralateral DG. In monkeys, temporal MCs send associational fibers exclusively to the inner molecular layer, while septal MCs give rise to wide axonal projections spanning multiple molecular layers, akin to equivalent MC subpopulations in mice. Despite conserved septotemporal heterogeneity, interspecies differences are observed in the topological organization of septal MCs, particularly in the relative axonal density in each molecular layer along the septotemporal axis of the DG. In summary, this comparative analysis sheds light on both conserved and divergent features of MCs in the DG of mice and monkeys. These findings have implications for understanding functional differentiation along the septotemporal axis of the DG and contribute to our knowledge of the anatomical evolution of the DG circuit in mammals.

Maladaptation of dentate gyrus mossy cells mediates contextual discrimination deficit after traumatic stress.

Fear overgeneralization is a maladaptive response to traumatic stress that is associated with the inability to discriminate between threat and safety contexts, a hallmark feature of post-traumatic stress disorder (PTSD). However, the neural mechanisms underlying this deficit remain unclear. Here, we show that traumatic stress exposure impairs contextual discrimination between threat and safety contexts in the learned helplessness (LH) model. Mossy cells (MCs) in the dorsal hippocampus are suppressed in response to traumatic stress. Bidirectional manipulation of MC activity in the LH model reveals that MC inhibition is causally linked to impaired contextual discrimination. Mechanistically, MC inhibition increases the number of active granule cells in a given context, significantly overlapping context-specific ensembles. Our study demonstrates that maladaptive inhibition of MCs after traumatic stress is a substantial mechanism underlying fear overgeneralization with contextual discrimination deficit, suggesting a potential therapeutic target for cognitive symptoms of PTSD.

Intrinsic and Synaptic Contributions to Repetitive Spiking in Dentate Granule Cells.

Repetitive firing of granule cells (GCs) in the dentate gyrus (DG) facilitates synaptic transmission to the CA3 region. This facilitation can gate and amplify the flow of information through the hippocampus. High-frequency bursts in the DG are linked to behavior and plasticity, but GCs do not readily burst. Under normal conditions, a single shock to the perforant path in a hippocampal slice typically drives a GC to fire a single spike, and only occasionally more than one spike is seen. Repetitive spiking in GCs is not robust, and the mechanisms are poorly understood. Here, we used a hybrid genetically encoded voltage sensor to image voltage changes evoked by cortical inputs in many mature GCs simultaneously in hippocampal slices from male and female mice. This enabled us to study relatively infrequent double and triple spikes. We found GCs are relatively homogeneous and their double spiking behavior is cell autonomous. Blockade of GABA type A receptors increased multiple spikes and prolonged the interspike interval, indicating inhibitory interneurons limit repetitive spiking and set the time window for successive spikes. Inhibiting synaptic glutamate release showed that recurrent excitation mediated by hilar mossy cells contributes to, but is not necessary for, multiple spiking. Blockade of T-type Ca2+ channels did not reduce multiple spiking but prolonged interspike intervals. Imaging voltage changes in different GC compartments revealed that second spikes can be initiated in either dendrites or somata. Thus, pharmacological and biophysical experiments reveal roles for both synaptic circuitry and intrinsic excitability in GC repetitive spiking.

Neuronal activity dynamics in the dentate gyrus during early epileptogenesis.

Epileptogenesis is a complex process involving a multitude of changes at the molecular, cellular and network level. Previous studies have identified several key alterations contributing to epileptogenesis and the development of hyper-excitability in different animal models, but only a few have focused on the early stages of this process. For post status epilepticus (SE) temporal lobe epilepsy in particular, understanding network dynamics during the early phases might be crucial for developing accurate preventive treatments to block the development of chronic spontaneous seizures. In this study, we used a viral vector mediated approach to examine activity of neurons in the dentate gyrus of the hippocampus during early epileptogenesis. We find that while granule cells are active 8 h after SE and then gradually decrease their activity, Calretinin-positive mossy cells and Neuropeptide Y-positive GABAergic interneurons in the hilus show a delayed activation pattern starting at 24 and peaking at 48 h after SE. These data suggest that indirect inhibition of granule cells by mossy cells through recruitment of local GABAergic interneurons could be an important mechanisms of excitability control during early epileptogenesis, and contribute to our understanding of the complex role of these cells in normal and pathological conditions.

Global remapping in granule cells and mossy cells of the mouse dentate gyrus.

Hippocampal place cells exhibit spatially modulated firing, or place fields, which can remap to encode changes in the environment or other variables. Unique among hippocampal subregions, the dentate gyrus (DG) has two excitatory populations of place cells, granule cells and mossy cells, which are among the least and most active spatially modulated cells in the hippocampus, respectively. Previous studies of remapping in the DG have drawn different conclusions about whether granule cells exhibit global remapping and contribute to the encoding of context specificity. By recording granule cells and mossy cells as mice foraged in different environments, we found that by most measures, both granule cells and mossy cells remapped robustly but through different mechanisms that are consistent with firing properties of each cell type. Our results resolve the ambiguity surrounding remapping in the DG and suggest that most spatially modulated granule cells contribute to orthogonal representations of distinct spatial contexts.

Cannabinoid regulation of neurons in the dentate gyrus during epileptogenesis: Role of CB1R-associated proteins and downstream pathways.

The hippocampal formation plays a central role in the development of temporal lobe epilepsy (TLE), a disease characterized by recurrent, unprovoked epileptic discharges. TLE is a neurologic disorder characterized by acute long-lasting seizures (i.e., abnormal electrical activity in the brain) or seizures that occur in close proximity without recovery, typically after a brain injury or status epilepticus. After status epilepticus, epileptogenic hyperexcitability develops gradually over the following months to years, resulting in the emergence of chronic, recurrent seizures. Acting as a filter or gate, the hippocampal dentate gyrus (DG) normally prevents excessive excitation from propagating through the hippocampus, and is considered a critical region in the progression of epileptogenesis in pathological conditions. Importantly, lipid-derived endogenous cannabinoids (endocannabinoids), which are produced on demand as retrograde messengers, are central regulators of neuronal activity in the DG circuit. In this review, we summarize recent findings concerning the role of the DG in controlling hyperexcitability and propose how DG regulation by cannabinoids (CBs) could provide avenues for therapeutic interventions. We also highlight possible pathways and manipulations that could be relevant for the control of hyperexcitation. The use of CB compounds to treat epilepsies is controversial, as anecdotal evidence is not always validated by clinical trials. Recent publications shed light on the importance of the DG as a region regulating incoming hippocampal excitability during epileptogenesis. We review recent findings concerning the modulation of the hippocampal DG circuitry by CBs and discuss putative underlying pathways. A better understanding of the mechanisms by which CBs exert their action during seizures may be useful to improve therapies.

Disynaptic effect of hilar cells on pattern separation in a spiking neural network of hippocampal dentate gyrus.

We study the disynaptic effect of the hilar cells on pattern separation in a spiking neural network of the hippocampal dentate gyrus (DG). The principal granule cells (GCs) in the DG perform pattern separation, transforming similar input patterns into less-similar output patterns. In our DG network, the hilus consists of excitatory mossy cells (MCs) and inhibitory HIPP (hilar perforant path-associated) cells. Here, we consider the disynaptic effects of the MCs and the HIPP cells on the GCs, mediated by the inhibitory basket cells (BCs) in the granular layer; MC → BC → GC and HIPP → BC → GC. The MCs provide disynaptic inhibitory input (mediated by the intermediate BCs) to the GCs, which decreases the firing activity of the GCs. On the other hand, the HIPP cells disinhibit the intermediate BCs, which leads to increasing the firing activity of the GCs. In this way, the disynaptic effects of the MCs and the HIPP cells are opposite. We investigate change in the pattern separation efficacy by varying the synaptic strength K ( BC , X ) [from the pre-synaptic X (= MC or HIPP) to the post-synaptic BC]. Thus, sparsity for the firing activity of the GCs is found to improve the efficacy of pattern separation, and hence the disynaptic effects of the MCs and the HIPP cells on the pattern separation become opposite ones. In the combined case when simultaneously changing both K ( BC , MC ) and K ( BC , HIPP ) , as a result of balance between the two competing disynaptic effects of the MCs and the HIPP cells, the efficacy of pattern separation is found to become the highest at their original default values where the activation degree of the GCs is the lowest. We also note that, while the GCs perform pattern separation, sparsely synchronized rhythm is found to appear in the population of the GCs. Hence, we examine quantitative association between population and individual firing behaviors in the sparsely synchronized rhythm and pattern separation. They are found to be strongly correlated. Consequently, the better the population and individual firing behaviors in the sparsely synchronized rhythm are, the more pattern separation efficacy becomes enhanced.

Activin A regulates the excitability of hippocampal mossy cells.

Mossy cells (MCs) in the hilus of the dentate gyrus (DG) receive increasing attention as a major player controlling information processing in the DG network. Furthermore, disturbed MC activity has been implicated in widespread neuropsychiatric disorders such as epilepsy and major depression. Using whole-cell patch-clamp recordings from MCs in acute hippocampal slices from wild type and transgenic mice, we demonstrate that activin, a member of the transforming growth factor-ß (TGF-ß) family, has a strong neuromodulatory effect on MC activity. Disruption of activin receptor signaling reduced MC firing, dampened their excitatory input and augmented their inhibitory input. By contrast, acute application of recombinant activin A strongly increased MC activity and promoted excitatory synaptic drive. Notably, similar changes of MC activity have been observed in a rodent model of depression and after antidepressant drug therapy, respectively. Given that a rise in activin signaling particularly in the DG has been proposed as a mechanism of antidepressant action, our data suggest that the effect of activin on MC excitability might make a considerable contribution in this regard.

Adaptive Mossy Cell Circuit Plasticity after Status Epilepticus.

Hilar mossy cells regulate network function in the hippocampus through both direct excitation and di-synaptic inhibition of dentate granule cells (DGCs). Substantial mossy cell loss accompanies hippocampal circuit changes in epilepsy. We examined the contribution of surviving mossy cells to network activity in the reorganized dentate gyrus after pilocarpine-induced status epilepticus (SE). To examine functional circuit changes, we optogenetically stimulated mossy cells in acute hippocampal slices from male mice. In control mice, activation of mossy cells produced monosynaptic excitatory and di-synaptic GABAergic currents in DGCs. In pilocarpine-treated mice, mossy cell density and excitation of DGCs were reduced in parallel, with only a minimal reduction in feedforward inhibition, enhancing the inhibition/excitation ratio. Surprisingly, mossy cell-driven excitation of parvalbumin-positive (PV+) basket cells, primary mediators of feed-forward inhibition, was maintained. Our results suggest that mossy cell outputs reorganize following seizures, increasing their net inhibitory effect in the hippocampus.SIGNIFICANCE STATEMENT Hilar mossy cell loss in epilepsy is associated with hippocampal hyperexcitability, potentially as a result of disrupted dentate microcircuit function. We used transgenic mice, translational mouse modeling, viral vectors, and optogenetics to selectively examine functional changes to mossy cell outputs following status epilepticus (SE). Interestingly, the outputs of surviving mossy cells exhibited adaptive plasticity onto target parvalbumin-positive (PV+) interneurons, resulting in a relative increase in their inhibitory control of dentate granule cells (DGCs). Our findings suggest that residual mossy cell outputs can reorganize in a homeostatic manner, which may provide clues for therapeutic targeting of this microcircuit.

Recurrent Excitatory Feedback From Mossy Cells Enhances Sparsity and Pattern Separation in the Dentate Gyrus via Indirect Feedback Inhibition.

It is generally appreciated that storing memories of specific events in the mammalian brain, and associating features of the environment with behavioral outcomes requires fine-tuning of the strengths of connections between neurons through synaptic plasticity. It is less understood whether the organization of neuronal circuits comprised of multiple distinct neuronal cell types provides an architectural prior that facilitates learning and memory by generating unique patterns of neuronal activity in response to different stimuli in the environment, even before plasticity and learning occur. Here we simulated a neuronal network responding to sensory stimuli, and systematically determined the effects of specific neuronal cell types and connections on three key metrics of neuronal sensory representations: sparsity, selectivity, and discriminability. We found that when the total amount of input varied considerably across stimuli, standard feedforward and feedback inhibitory circuit motifs failed to discriminate all stimuli without sacrificing sparsity or selectivity. Interestingly, networks that included dedicated excitatory feedback interneurons based on the mossy cells of the hippocampal dentate gyrus exhibited improved pattern separation, a result that depended on the indirect recruitment of feedback inhibition. These results elucidate the roles of cellular diversity and neural circuit architecture on generating neuronal representations with properties advantageous for memory storage and recall.

Modeling intrahippocampal effects of anterior hippocampal hyperactivity relevant to schizophrenia using chemogenetic excitation of long axis-projecting mossy cells in the mouse dentate gyrus.

BACKGROUND: The anterior hippocampus of individuals with early psychosis or schizophrenia is hyperactive, as is the ventral hippocampus in many rodent models for schizophrenia risk. Mossy cells (MCs) of the ventral dentate gyrus (DG) densely project in the hippocampal long axis, targeting both dorsal DG granule cells and inhibitory interneurons. Mossy cells are responsive to stimulation throughout hippocampal subfields, and thus may be suited to detect hyperactivity in areas where it originates such as CA1. Here we tested the hypothesis that hyperactivation of ventral MCs activates dorsal DG granule cells to influence dorsal hippocampal function. METHODS: In CD-1 mice, we targeted dorsal DG-projecting ventral MCs using an adeno-associated virus intersectional strategy. In vivo fiber photometry recording of ventral MCs was performed during exploratory behaviors. We used excitatory chemogenetic constructs to test the effects of ventral MC hyperactivation on long-term spatial memory during an object location memory task. RESULTS: Photometry revealed ventral MCs were activated during exploratory rearing. Ventral MCs made functional monosynaptic inputs to dorsal DG granule cells, and chemogenetic activation of ventral MCs modestly increased activity of dorsal DG granule cells measured by c-Fos. Finally, chemogenetic activation of ventral MCs during the training phase of an object location memory task impaired test performance 24 hours later, without effects on locomotion or object exploration. CONCLUSIONS: These data suggest that ventral MC activation can directly excite dorsal granule cells and interfere with dorsal DG function, supporting future study of their in vivo activity in animal models for schizophrenia featuring ventral hyperactivity.

The role of hippocampal mossy cells in novelty detection.

At the encounter with a novel environment, contextual memory formation is greatly enhanced, accompanied with increased arousal and active exploration. Although this phenomenon has been widely observed in animal and human daily life, how the novelty in the environment is detected and contributes to contextual memory formation has lately started to be unveiled. The hippocampus has been studied for many decades for its largely known roles in encoding spatial memory, and a growing body of evidence indicates a differential involvement of dorsal and ventral hippocampal divisions in novelty detection. In this brief review article, we discuss the recent findings of the role of mossy cells in the ventral hippocampal moiety in novelty detection and put them in perspective with other novelty-related pathways in the hippocampus. We propose a mechanism for novelty-driven memory acquisition in the dentate gyrus by the direct projection of ventral mossy cells to dorsal dentate granule cells. By this projection, the ventral hippocampus sends novelty signals to the dorsal hippocampus, opening a gate for memory encoding in dentate granule cells based on information coming from the entorhinal cortex. We conclude that, contrary to the presently accepted functional independence, the dorsal and ventral hippocampi cooperate to link the novelty and contextual information, and this dorso-ventral interaction is crucial for the novelty-dependent memory formation.

A neurogenic microenvironment defined by excitatory-inhibitory neuronal circuits in adult dentate gyrus.

Adult neurogenesis in the dentate gyrus plays a role in adaptive brain functions such as memory formation. Adding new neurons to a specific locus of a neural circuit with functional needs is an efficient way to achieve such an adaptive function. However, it is unknown whether neurogenesis is linked to local functional demands potentially specified by the activity of neuronal circuits. By examining the distribution of neurogenesis and different types of neuronal activity in the dentate gyrus of freely moving adult rats, we find that neurogenesis is positionally associated with active excitatory neurons, some of which show place-cell activity, but is positionally dissociated from a type of interneuron with high-burst tendency. Our finding suggests that the behaviorally relevant activity of excitatory-inhibitory neuronal circuits can define a microenvironment stimulating/inhibiting neurogenesis. Such local regulation of neurogenesis may contribute to strategic recruitment of new neurons to modify functionally relevant neural circuits.

Calpain-2 activation in mouse hippocampus plays a critical role in seizure-induced neuropathology.

Calpain has been proposed to play a critical role in the development of epilepsy. Here we used conditional calpain-2 knock-out (C2CKO) mice in a C57/Bl6 background and a selective calpain-2 inhibitor to analyze the role of calpain-2 in epilepsy. Neurodegeneration was evident in various hippocampal subfields, in particular in mossy cells in the hilus of the dentate gyrus (DG) in C57/Bl6 mice 7 days after kainic acid (KA)-induced seizures. Calpain-2 activation was still observed in mossy cells 7 days after seizures. Calpain activation, astroglial and microglial activation, neurodegeneration, and cognitive impairment were absent in C2CKO mice and in C57/Bl6 mice treated with a selective calpain-2 inhibitor for 7 days after seizure initiation. Levels of the potassium chloride cotransporter 2 (KCC2) were decreased in mossy cells 7 days after seizures and this decrease was prevented by calpain-2 deletion or selective inhibition. Our results indicate that prolonged calpain-2 activation plays a critical role in neuropathology following seizures. A selective calpain-2 inhibitor could represent a therapeutic treatment for seizure-induced neuropathology.

Ventro-dorsal Hippocampal Pathway Gates Novelty-Induced Contextual Memory Formation.

Novelty facilitates memory formation and is detected by both the dorsal and ventral hippocampus. Although dentate granule cells (GCs) in the dorsal hippocampus are known to mediate the formation of novelty-induced contextual memories, the required pathways and mechanisms remain unclear. Here we demonstrate that a powerful excitatory pathway from mossy cells (MCs) in the ventral hippocampus to dorsal GCs is necessary and sufficient for driving dorsal GC activation in novel environment exploration. In vivo Ca2+ imaging in freely moving mice indicated that this pathway relays environmental novelty. Furthermore, manipulation of ventral MC activity bidirectionally regulates novelty-induced contextual memory acquisition. Our results show that ventral MC activity gates contextual memory formation through an intra-hippocampal interaction activated by environmental novelty.

Mossy Cells in the Dorsal and Ventral Dentate Gyrus Differ in Their Patterns of Axonal Projections.

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.

Neurophotonics Approaches for the Study of Pattern Separation.

Successful memory involves not only remembering over time but also keeping memories distinct. Computational models suggest that pattern separation appears as a highly efficient process to discriminate between overlapping memories. Furthermore, lesion studies have shown that the dentate gyrus (DG) participates in pattern separation. However, these manipulations did not allow identifying the neuronal mechanism underlying pattern separation. The development of different neurophotonics techniques, together with other genetic tools, has been useful for the study of the microcircuit involved in this process. It has been shown that less-overlapped information would generate distinct neuronal representations within the granule cells (GCs). However, because glutamatergic or GABAergic cells in the DG are not functionally or structurally homogeneous, identifying the specific role of the different subpopulations remains elusive. Then, understanding pattern separation requires the ability to manipulate a temporal and spatially specific subset of cells in the DG and ideally to analyze DG cells activity in individuals performing a pattern separation dependent behavioral task. Thus, neurophotonics and calcium imaging techniques in conjunction with activity-dependent promoters and high-resolution microscopy appear as important tools for this endeavor. In this work, we review how different neurophotonics techniques have been implemented in the elucidation of a neuronal network that supports pattern separation alone or in combination with traditional techniques. We discuss the limitation of these techniques and how other neurophotonic techniques could be used to complement the advances presented up to this date.