Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.     

Corticosterone replacement restores normal morphological features to the hippocampal dendrites, axons and synapses of adrenalectomized rats

A thorough evaluation of hippocampal dendrites, axons and synaptic contacts has not been undertaken following prolonged periods of absence of corticosteroids despite the marked granule cell loss which occurs in the dentate gyrus of adrenalectomized rats. Thus, we have applied morphometric techniques to analyse the dendrites of granule and pyramidal cells, the mossy fiber system, and the number and morphology of synapses between the mossy fibers and the excrescences of CA3 pyramidal cells in rats submitted to different periods of adrenalectomy. In addition, to search for the presence of neuritic reorganisation in the hippocampal formation once normal corticosteroid levels were re-established, we incorporated in this study a group of rats replaced with corticosterone one month after adrenalectomy. The results obtained in adrenalectomized rats showed a striking impoverishment of the dendrites of surviving granule cells, subtle alterations in the apical dendritic arborization of CA3 pyramidal cells and no changes in the apical dendrites of CA1 pyramidal cells. In addition, in adrenalectomized rats there was a progressive reduction in the total number of synapses established between mossy fibers and CA3 pyramids, as a consequence of a reduction in the volume of the suprapyramidal part of the mossy fiber system, and profound changes in the morphology of mossy fiber terminals and CA3 dendritic excrescences. A remarkable reorganisation of neurites was found to occur following the administration of low doses of corticosterone, completely reversing the adrenalectomy-induced synaptic loss and partially restoring the morphology of hippocampal axons and dendrites. These plastic mechanisms provide a sound structural basis for the reversibility of cognitive deficits observed after corticosterone administration to adrenalectomized rats.     

The Recurrent Mossy Fiber Pathway of the Epileptic Brain

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K⁺]_o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1–2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies <1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.     

Borna disease virus replication in organotypic hippocampal slice cultures from rats results in selective damage of dentate granule cells

In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a post-maturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.     

Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.     

Rapamycin suppresses the recurrent excitatory circuits of dentate gyrus in a mouse model of temporal lobe epilepsy

Recurrent excitatory circuits and abnormal recurrent excitatory inputs are essential in epileptogenesis. Studies in temporal lobe epilepsy have shown that mossy fiber sprouting, which represents synaptic reorganization, renders the formation of abnormal recurrent excitatory circuits and inputs. The mammalian target of rapamycin (mTOR) pathway has recently been proved important in mossy fiber sprouting. In the present study, rapamycin, a mTOR inhibiter, was injected into the mouse of temporal lobe epilepsy. Electrophysiological and histological properties of the hippocampus were investigated by whole cell patch clamp, extracellular recording and Timm staining. Following the development of epilepsy, frequency of spontaneous excitatory postsynaptic currents (EPSCs) and amplitude of antidromically evoked EPSCs in granule cells were remarkably increased, as well as the epileptiform activity and mossy fiber sprouting were detected, which indicated the formation of abnormal recurrent excitatory circuits. By the use of rapamycin, frequency of spontaneous EPSCs, amplitude of antidromically evoked EPSCs, the epileptiform activity and mossy fiber sprouting were all remarkably suppressed. Our findings suggested an anti-epileptogenic role of rapamycin by suppressing the recurrent excitatory circuits of dentate gyrus.     

Separable actions of acetylcholine and noradrenaline on neuronal ensemble formation in hippocampal CA3 circuits

In the hippocampus, episodic memories are thought to be encoded by the formation of ensembles of synaptically coupled CA3 pyramidal cells driven by sparse but powerful mossy fiber inputs from dentate gyrus granule cells. The neuromodulators acetylcholine and noradrenaline are separately proposed as saliency signals that dictate memory encoding but it is not known if they represent distinct signals with separate mechanisms. Here, we show experimentally that acetylcholine, and to a lesser extent noradrenaline, suppress feed-forward inhibition and enhance Excitatory–Inhibitory ratio in the mossy fiber pathway but CA3 recurrent network properties are only altered by acetylcholine. We explore the implications of these findings on CA3 ensemble formation using a hierarchy of models. In reconstructions of CA3 pyramidal cells, mossy fiber pathway disinhibition facilitates postsynaptic dendritic depolarization known to be required for synaptic plasticity at CA3-CA3 recurrent synapses. We further show in a spiking neural network model of CA3 how acetylcholine-specific network alterations can drive rapid overlapping ensemble formation. Thus, through these distinct sets of mechanisms, acetylcholine and noradrenaline facilitate the formation of neuronal ensembles in CA3 that encode salient episodic memories in the hippocampus but acetylcholine selectively enhances the density of memory storage.     

Neuropeptide Y in the recurrent mossy fiber pathway

In the epileptic brain, hippocampal dentate granule cells become synaptically interconnected through the sprouting of mossy fibers. This new circuitry is expected to facilitate epileptiform discharge. Prolonged seizures induce the long-lasting neoexpression of neuropeptide Y (NPY) in mossy fibers. NPY is released spontaneously from recurrent mossy fiber terminals, reduces glutamate release from those terminals by activating presynaptic Y2 receptors, and depresses granule cell epileptiform activity dependent on the recurrent pathway. These effects are much greater in rats than in C57BL/6 mice, despite apparently equivalent mossy fiber sprouting and neoexpression of NPY. This species difference can be explained by contrasting changes in the expression of mossy fiber Y2 receptors; seizures upregulate Y2 receptors in rats but downregulate them in mice. The recurrent mossy fiber pathway may synchronize granule cell discharge more effectively in humans and mice than in rats, due to its lower expression of either NPY (humans) or Y2 receptors (mice).     

Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP⁺ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP⁺ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.     

Electroconvulsive seizure promotes spine maturation in newborn dentate granule cells in adult rat

Neurogenesis continues in the dentate gyrus of the hippocampus throughout life in mammals. This process is influenced by daily activities such as exercise, learning, and stress and may contribute to certain forms of hippocampus-dependent learning and memory. Adult hippocampal neurogenesis is also subject to regulation by antidepressant treatment, including chronic treatment with antidepressant drugs or electroconvulsive seizure (ECS) therapy. Here we investigated how the connectivity of newborn and mature granule cells is influenced by ECS administration in rats. Specifically, we examined the dendritic spine morphology of newborn and mature granule cells in rats and found that ECS administration promoted the maturation of dendritic spines in newborn cells and increased spine density in mature cells. These changes could potentially lead to alteration in dentate circuitry and may partially contribute to the functional effects of ECS.     

Short-term environmental enrichment enhances adult neurogenesis, vascular network and dendritic complexity in the hippocampus of type 1 diabetic mice

Background

Several brain disturbances have been described in association to type 1 diabetes in humans. In animal models, hippocampal pathological changes were reported together with cognitive deficits. The exposure to a variety of environmental stimuli during a certain period of time is able to prevent brain alterations and to improve learning and memory in conditions like stress, aging and neurodegenerative processes.

Methodology/Principal Findings

We explored the modulation of hippocampal alterations in streptozotocin-induced type 1 diabetic mice by environmental enrichment. In diabetic mice housed in standard conditions we found a reduction of adult neurogenesis in the dentate gyrus, decreased dendritic complexity in CA1 neurons and a smaller vascular fractional area in the dentate gyrus, compared with control animals in the same housing condition. A short exposure -10 days- to an enriched environment was able to enhance proliferation, survival and dendritic arborization of newborn neurons, to recover dendritic tree length and spine density of pyramidal CA1 neurons and to increase the vascular network of the dentate gyrus in diabetic animals.

Conclusions/Significance

The environmental complexity seems to constitute a strong stimulator competent to rescue the diabetic brain from neurodegenerative progression.     

Structural Remodeling of the Neuronal Network in the Dentate Gyrus of the Epileptically Transformed Murine Hippocampus

Immunohistochemical analysis of the hippocampus of a transgene mutant line of the mice (genetic model of temporal epilepsy) demonstrated a progressive increase in the level of brain-derived neurotrophic factor (BDNF) in granular cells of the dentate gyrus and their axons; this increase correlated with an enhancement of the level of epileptic activity. Epileptogenic reprojection of mossy fibers toward an internal zone of the molecular layer of the hippocampus was also observed. In addition, we observed excessive spreading of the zone of axon branching of GABA-ergic basket cells, as compared with the norm; their maximum collateralization was found within an internal zone of the molecular layer of the dentate gyrus. This allows us to hypothesize that the processes of sprouting of the mossy fibers and recovery of recurrent inhibition of the granular cells are interrelated.     

Monosynaptic GABAergic signaling from dentate to CA3 with a pharmacological and physiological profile typical of mossy fiber synapses

Mossy fibers are the sole excitatory projection from dentate gyrus granule cells to the hippocampus, where they release glutamate, dynorphin, and zinc. In addition, mossy fiber terminals show intense immunoreactivity for the inhibitory neurotransmitter GABA. Fast inhibitory transmission at mossy fiber synapses, however, has not previously been reported. Here, we show that electrical or chemical stimuli that recruit dentate granule cells elicit monosynaptic GABA(A) receptor-mediated synaptic signals in CA3 pyramidal neurons. These inhibitory signals satisfy the criteria that distinguish mossy fiber-CA3 synapses: high sensitivity to metabotropic glutamate receptor agonists, facilitation during repetitive stimulation, and NMDA receptor-independent long-term potentiation. GABAergic transmission from the dentate gyrus to CA3 has major implications not only for information flow into the hippocampus but also for developmental and pathological processes involving the hippocampus.     

Suppressor of Cytokine Signalling 2 (SOCS2) Regulates Numbers of Mature Newborn Adult Hippocampal Neurons and Their Dendritic Spine Maturation

Overexpression of suppressor of cytokine signalling 2 (SOCS2) has been shown to promote hippocampal neurogenesis in vivo and promote neurite outgrowth of neurons in vitro. In the adult mouse brain, SOCS2 is most highly expressed in the hippocampal CA3 region and at lower levels in the dentate gyrus, an expression pattern that suggests a role in adult neurogenesis. Herein we examine generation of neuroblasts and their maturation into more mature neurons in SOCS2 null (SOCS2KO) mice. EdU was administered for 7 days to label proliferative neural precursor cells. The number of EdU-labelled doublecortin⁺ neuroblasts and NeuN⁺ mature neurons they generated was examined at day 8 and day 35, respectively. While no effect of SOCS2 deletion was observed in neuroblast generation, it reduced the numbers of EdU-labelled mature newborn neurons at 35 days. As SOCS2 regulates neurite outgrowth and dentate granule neurons project to the CA3 region, alterations in dendritic arborisation or spine formation may have correlated with the decreased numbers of EdU-labelled newborn neurons. SOCS2KO mice were crossed with Nes-CreER^T2/mTmG mice, in which membrane eGFP is inducibly expressed in neural precursor cells and their progeny, and the dendrite and dendritic spine morphology of newborn neurons were examined at 35 days. SOCS2 deletion had no effect on total dendrite length, number of dendritic segments, number of branch points or total dendritic spine density but increased the number of mature “mushroom” spines. Our results suggest that endogenous SOCS2 regulates numbers of EdU-labelled mature newborn adult hippocampal neurons, possibly by mediating their survival and that this may be via a mechanism regulating dendritic spine maturation.     

Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus

Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell–specific knockdown of cyclin-dependent kinase 5 (cdk5) activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.     

Quantitative studies on the neuron structure of the rat fascia dentata]

1. In 6 month old male rats the structure of dendrites and the distribution of spines on the apical dendrites of granular cells of the dentate gyrus were investigated by light microscopy and statistical methods. 2. The number of dendrites of the first, second and third order of granular cells increases in this sequence in a ratio of 1:2:3; the total length of the dendrites increases correspondently in a ratio of 1:4:5. 3. The mean number of origin points of dendritic branches is 10, the mean number of free dendritic endings is 12. 4. The number of spines per a 25 mum dendritic segment near to the pericaryon (dendritic segment A), in the middle of the dendrite (dendritic segment B) and in the peripheral dendritic part (dendritic segment C) as well as the distribution of spines in the whole apical dendrite was evaluated. The total mean of spines of granular cell apical dendrites of the dentate gyrus (superior respectively inferior) is 12 respectively 10 for the dendritic segment A, 18 respectively 17 for the dendritic segment B and 17 respectively 15 for the dendritic segment C calculated for a dendritic length of 25 mum. 5. The spine density in each case depends upon the distance pericaryondendritic segment and is in close relation to the adjacent layers with their specific afferents. 6. The averaged total number of spines per 1 mum of dendritic length is 0,62 spines/mum for the dentate gyrus (superior) and 0,57 spines/mum for the dentate gyrus (inferior). 7. The granular cells of the dentate gyrus (superior) have a mean dendritic length of a total of 357 mum with a total of 226 visible spines; the granular cells of the dentate gyrus (inferior) have a mean dendritic length of a total of 450 mum with a total of 258 visible spines.     

Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.     

Early natural stimulation through environmental enrichment accelerates neuronal development in the mouse dentate gyrus

The dentate gyrus is the primary afferent into the hippocampal formation, with important functions in learning and memory. Granule cells, the principle neuronal type in the dentate gyrus, are mostly formed postnatally, in a process that continues into adulthood. External stimuli, including environmental enrichment, voluntary exercise and learning, have been shown to significantly accelerate the generation and maturation of dentate granule cells in adult rodents. Whether, and to what extent, such environmental stimuli regulate the development and maturation of dentate granule cells during early postnatal development is largely unknown. Furthermore, whether natural stimuli affect the synaptic properties of granule cells had been investigated neither in newborn neurons of the adult nor during early development. To examine the effect of natural sensory stimulation on the dentate gyrus, we reared newborn mice in an enriched environment (EE). Using immunohistochemistry, we showed that dentate granule cells from EE-reared mice exhibited earlier morphological maturation, manifested as faster peaking of doublecortin expression and elevated expression of mature neuronal markers (including NeuN, calbindin and MAP2) at the end of the second postnatal week. Also at the end of the second postnatal week, we found increased density of dendritic spines across the entire dentate gyrus, together with elevated levels of postsynaptic scaffold (post-synaptic density 95) and receptor proteins (GluR2 and GABA(A)Rγ2) of excitatory and inhibitory synapses. Furthermore, dentate granule cells of P14 EE-reared mice had lower input resistances and increased glutamatergic and GABAergic synaptic inputs. Together, our results demonstrate that EE-rearing promotes morphological and electrophysiological maturation of dentate granule cells, underscoring the importance of natural environmental stimulation on development of the dentate gyrus.