Ectopic granule cells of the rat dentate gyrus

Granule cells of the mammalian dentate gyrus normally form a discrete layer, and virtually all granule cells migrate to this location. Exceptional granule cells that are positioned incorrectly, in 'ectopic' locations, are rare. Although the characteristics of such ectopic granule cells appear similar in many respects to granule cells located in the granule cell layer, their rare occurrence has limited a full evaluation of their structure and function. More information about ectopic granule cells has been obtained by studying those that develop after experimental manipulations that increase their number. For example, after severe seizures, the number of ectopic granule cells located in the hilus increases dramatically. These experimentally-induced ectopic granule cells may not be equivalent to normal ectopic granule cells necessarily, but the vastly increased numbers have allowed much more information to be obtained. Remarkably, the granule cells that are positioned ectopically develop intrinsic properties and an axonal projection that are similar to granule cells that are located normally, i.e., in the granule cell layer. However, dendritic structure and synaptic structure/function appear to differ. These studies have provided new insight into a rare type of granule cell in the dentate gyrus, and the plastic characteristics of dentate granule cells that appear to depend on the location of the cell body.     

Maturation of glutamatergic neurotransmission in dentate gyrus granule cells

We studied the development of glutamatergic neurotransmission in dentate gyrus granule cells (GCs) in hippocampal slices from 5 to 12-day-old rats. The active postnatal neuronogenesis in dentate permits GCs with staggered birthdates to be studied in situ in a single preparation. We recorded evoked responses to medial perforant path stimulation using visually-guided whole-cell patch clamping to select immature GCs, and biocytin filling to correlate electrophysiologic responses with maturational stage. Even within this immature cell population we found four distinct electrophysiologic patterns. Type 1 cells had no glutamatergic current; Type 2 cells had only N-methyl-D-aspartate receptor (NMDA) current; Type 3 cells had both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current although the NMDA component could be isolated at low stimulus intensity (NMDA threshold/=AMPA threshold. Type 1 cells were least mature, and Type 4 cells most mature as assessed by cell properties, dendritic arborization, and penetration of dendrites into the molecular layer. Thus NMDA-mediated currents predominate early in GC development as is consistent with their role in processes that determine dentate architecture - neuronal migration, dendritic outgrowth and regression, and synapse stabilization. By analogy with 'silent synapses' (i.e. synapses that contain only NMDA receptors), Type 2 cells are candidate 'silent cells' that may undergo activity-dependent acquisition of functional fast-conducting AMPA receptors with maturation.     

Learning deficits after lesions of dentate gyrus granule cells

The effect of destroying granule cells in the dentate gyrus on learning was examined with a behavioral testing protocol. These neurons were destroyed by microinjections of the selective neurotoxin colchicine in the hippocampal formation of rats. After a 30-day recovery period, the animals were trained in an operant chamber with an appetitive conditioning paradigm. The learning abilities of the animals with lesions were compared with two control groups—naive, unoperated rats and those with control injections of saline. The basic task required the animal to discriminate between two spatially separate visual stimuli which represented positive and negative cues. Testing and training was separated into four progressively more difficult phases with various stimulus schedules, contingencies of reinforcement, and stimulus positions. Colchicine-treated animals demonstrated significantly poorer performance than naive animals and those receiving saline control injections. None of the colchicine-treated animals achieved criterion performance in the stimulus position reversal paradigm, and half had difficulty with variable ratio schedules of reinforcement. Our experiments suggested that granule cells in the dentate gyrus played a pivotal role in certain learning tasks.     

Morphologic and electrophysiologic maturation in developing dentate gyrus granule cells

Dentate gyrus granule cells from immature (7-28 days) Sprague-Dawley rats were examined with whole cell patch clamp recordings and biocytin filling in in vitro hippocampal slice preparations. Although recordings were confined to the middle third of the suprapyramidal limb of the dentate, the granule cells exhibited marked variability in their physiologic properties: input resistance (IR) ranged from 250 MOmega to 3 GOmega, and resting membrane potential (RMP) from -82 to -41 mV. Both IR and RMP were inversely correlated with dendritic length, a morphometric indicator of cell maturity. Thus the highest IR cells were the youngest, and maturation was characterized by a progressive decrease in IR, hyperpolarization of RMP, and elongation of the dendritic arbor. When cells were grouped by IR, significant intergroup differences were found in RMP, dendritic length, and number of dendritic terminal branches. Although cells of all IR categories were examined throughout the age spectrum under study, none of the inter-IR group differences was age-dependent. These data suggest that IR provides a reasonable estimate of granule cell maturity and that maturation entails predictable changes in cell properties and morphology. These aspects of maturation correlate with each other, are independent of animal age, and most likely proceed according to a program related to cell birth.     

Cysteamine potentiates entorhinal activation of dentate gyrus granule cells in rats

A dense plexus of somatostatin-positive fibers and varicosities is observed in the outer two-thirds of the dentate gyrus molecular layer where the glutamatergic perforant path afferents from the entorhinal cortex terminate. To test for a functional interaction between these two pathways, we examined the effects of Cysteamine, which enhances somatostatin release for a few hours after administration but produces subsequent depletion of somatostatin lasting several days, on perforant path evoked potentials recorded in the dentate gyrus. Cysteamine (50–400 mg/kg, IP) increased the population spike dose-dependently both in anesthetized and in awake rats, but the slope of the population excitatory postsynaptic potential (EPSP) was left unchanged or even decreased. The antidromic population spike evoked by mossy fiber stimulation was not changed by cysteamine. The change is thought to be due to the increase in slope of the EPSP-spike relationship. In the hippocampal slice preparation, a similar effect of the drug (1–5 mM) on dentate evoked potentials was observed, suggesting that cysteamine acts through its effects on somatostatin in the hippocampus itself. In chronically implanted awake animals, the perforant path population spike was increased 1 h after cysteamine but returned to the predrug level by 24 h when somatostatin seemed to be depleted. These results suggest that hippocampal somatostatin released by cysteamine potentiates the response of dentate granule cells to perforant path input, without directly affecting synaptic transmission or general cell excitability.     

Estimating the number of granule cells in the dentate gyrus with the disector

A practical example is given of how a newly developed stereological estimator of particle number, the disector, can be used to make estimates of neuron number in the dentate gyrus of rats. The estimates are free of biases related to lost caps, overprojection and assumptions about size, shape and orientation of the objects that are counted. The disector principle and the practical considerations relating to histological preparations and sampling are presented.     

AMPA and NMDA receptor-mediated currents in developing dentate gyrus granule cells

Granule cells (GCs) of the hippocampal dentate gyrus (DG) undergo postnatal neurogenesis such that cells at different maturational stages can be studied within an anatomically restricted region and a narrow animal age epoch. Using whole cell patch clamp recordings in hippocampal slices, we have previously found that input resistance (IR) correlates inversely with morphometric indicators of GC maturity. Using IR as an index of maturity we measured developmental changes in synaptic currents mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in GCs from 5- to 12-day rats. Peak NMDA and AMPA EPSC amplitudes increased, and the NMDA/AMPA ratio reversed with advancing cell age. NMDA EPSCs showed a maturational decrease in rise time but no change in decay time, whereas AMPA EPSCs showed neither rise nor decay time changes with development. Ifenprodil, a high affinity selective inhibitor of NR1/NR2B diheteromeric NMDA receptors, blocked approximately 50% of the peak amplitude of evoked NMDA EPSCs in all tested GCs regardless of their maturity and did not affect the measured kinetic properties. These data suggest that development of glutamatergic synapses follows distinct schedules. AMPA receptors possessed mature kinetics and became the dominant glutamatergic current within the age epoch studied, whereas NMDA receptors showed maturational changes in rise times but not decay kinetics. The reported modifications of EPSC properties are consistent with changes in receptor and synapse number, and relative quantities of AMPA and NMDA receptors. Changes in the subunit composition that determines NMDA decay kinetics may occur beyond the early neonatal period.     

Balloon cells associated with granule cell dispersion in the dentate gyrus in hippocampal sclerosis

Granule cell dispersion (GCD) is a common finding in hippocampal sclerosis in patients with intractable focal epilepsy. It is considered to be an acquired, post-developmental rather than a pre-existing abnormality, involving dispersion of either mature or newborn neurones, but the precise factors regulating it and its relationship to seizures are unknown. We present two cases of GCD with associated CD34-immunopositive balloon cells, a cell phenotype associated with focal cortical dysplasia type IIB, considered to be a developmental cortical lesion promoting epilepsy. This observation opens up the debate regarding the origin of balloon cells and CD34 expression and their temporal relationship to seizures.     

Increased inhibition in dentate gyrus granule cells following exposure to GABA-uptake blockers

Rats anesthetized with urethane had stimulating and recording electrodes placed in the perforant pathway and in the dentate gyrus. They were then exposed to increasing doses of either the vehicle control dimethylsulfoxide (DMSO) or one of two gamma-aminobutyric acid (GABA)-uptake blockers (SKF-100330A or SKF-89976A). Analysis of evoked field potentials from dentate granule cells indicated that the only effect of the GABA uptake blockers was to increase the threshold for evoking the field population spikes (PS). No other measure of excitatory postsynaptic potentials (EPSPs) or PS's were significantly affected. The lack of effect on evoked EPSP by these drugs suggests no direct effect on transmitter release at this synapse, while the increase in PS threshold suggests a slight decrease in granule cell excitability. The effects of the two GABA-uptake blockers on synaptically mediated facilitation and inhibition was tested by using paired-pulse paradigms. Both GABA-uptake blockers increased early GABA-mediated inhibition to a greater extent than they reduced synaptically mediated facilitation. Neither GABA uptake blocker appeared to effect the late inhibition seen at paired-pulse intervals of 400-1000 ms which is presumably associated with calcium-activated increases in potassium conductance. These effects on granule cell responses occurred at doses found previously not to be associated with side effects and yet to be anticonvulsant in unanesthetized rats. These data confirm in vivo that SKF-100330A and SKF-89976A increase GABA-mediated inhibition. The effect on granule cell excitability and late inhibition are minimal. Although facilitation was reduced by exposure to these drugs, the mechanism of this reduction (direct or prolongation of early inhibition) cannot be determined.     

Fate of afferents to the dentate gyrus following destruction of granule cells with colchicine

Granule cells of the dentate gyrus can be selectively destroyed by intrahippocampal injections of colchicine. The present study evaluates the consequences of this selective neuronal destruction on the afferent axon terminals which have been deprived of their normal targets. The area of the neuropil in the dentate gyrus (the molecular layer) was evaluated in sections stained using the Timm's method for heavy metals, which selectively marks the terminal fields of the different afferent systems. The molecular layer was examined electron microscopically to determine the fate of afferent terminals. Anterograde transport of HRP or [3H]proline was used to define the location and extent of afferent terminal fields of the entorhinal and commissural projections to the dentate gyrus in which the granule cells had been destroyed. There was a substantial reduction in the size of the dentate gyrus molecular layer after destruction of granule cells with colchicine. Electron microscopic analyses revealed that there were very few axon terminals or synapses remaining in the shrunken molecular layer. Tract tracing methods revealed that both the entorhinal and commissural pathways were still present in their normal terminal zones in the dentate gyrus, however, the density of the projections was greatly reduced. There was no evidence to suggest the formation of ectopic projections to unusual locations, such as the contralateral dentate gyrus. Pathways passing through the hippocampus appeared to survive the colchicine injections. These results suggest that target destruction in adult animals leads to the disappearance of the afferent axon terminals which normally innervate the cells which die.     

Early-life status epilepticus induces ectopic granule cells in adult mice dentate gyrus

A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these “ectopic” GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.     

Selective lesions of granule cells by fluid injections into the dentate gyrus

Granule cells were selectively lesioned by injections of fluid into the infragranular cleavage plane in the dentate gyrus. The granule cells were axotomized by the cavity created by the fluid and 6 days after the injection there were no granule cells at the injection site. The size of the granule cell loss could be altered by varying the volume and rate of the injection. The loss of granule cells led to a shrinkage of the molecular layer and to a reactive gliosis. The lesion also caused an increase in the density of AChE and Timm staining in the molecular layer above the lesion. Although the increased density of AChE and Timm staining may have been due in part to the shrinkage of the molecular layer, part was due to the growth of inputs in response to the loss of granule cells and/or to the axotomy of the input terminals. The changes seen in the molecular layer above the lesion site ended abruptly at the margins of the lesion and the adjacent molecular and granule cell layers appeared normal.     

Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus

Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. To assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous γ-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units.     

Corticosteroid effects on serotonin responses in granule cells of the rat dentate gyrus

Granule cells in the rat dentate gyrus contain mineralocorticoid and glucocorticoid receptors to which the adrenal hormone corticosterone binds with differential affinity. These cells also express various receptor-subtypes for serotonin (5-HT), including the 5-HT1A receptor which mediates a membrane hyperpolarization accompanied by a decrease in membrane resistance. Earlier studies have shown that removal of corticosterone by adrenalectomy, particularly in the dentate gyrus, results in enhanced expression of the 5-HT1A receptor mRNA and increased 5-HT1A receptor binding capacity. This was normalized by activation of mineralocorticoid receptors or concurrent activation of both receptor types. In the present, intracellular recording study in vitro, we examined if the altered levels of 5-HT1A receptor mRNA and protein are associated with changes in the response to 5-HT. We found that the hyperpolarization and resistance decrease induced in granule cells by a submaximal (10 microM) dose of 5-HT were unaltered 2-4 days after adrenalectomy, indicating a dissociation between corticosteroid actions on 5-HT1A receptor mRNA/protein levels and functional responses to 5-HT. Subsequent occupation of mineralocorticoid receptors in vitro significantly suppressed the 5-HT induced change in resistance, 1-4 h after steroid application. Compared to this, concurrent activation of glucocorticoid receptors led to large responses to 5-HT. This modulation by steroids was not observed with a higher dose of 5-HT (30 microM). The data suggest that with moderate amounts of 5-HT, corticosteroids affect the information flow through the dentate gyrus such that excitatory transmission is promoted with predominant mineralocorticoid receptor activation and attenuated with additional glucocorticoid receptor occupation.     

Potential mechanisms underlying the destruction of dentate gyrus granule cells by colchicine

Microinjections of 1 to 6 nmol of colchicine into the hippocampal formation of rats selectively destroyed granule cells of the dentate gyrus and their axons over several days. Immediately after such microinjections, epileptiform discharges appeared in focal EEG recordings from the hippocampal formation. Vinblastine produced neuropathologic changes like colchicine, and lumicolchicine was devoid of neurotoxicity. These findings suggest that the selective cytotoxicity of colchicine for dentate gyrus granule cells may depend on its ability to bind to tubulin and is associated with an acute epileptogenic effect.     

Differential effect of norepinephrine upon granule cells and interneurons in the dentate gyrus

The effect of locally applied norepinephrine upon dentate granule cells and neighboring interneurons was examined in urethane-anesthetized rats. Norepinephrine inhibited the spontaneous firing of physiologically identified granule cells, but excited interneurons. These results demonstrate that two coexisting hippocampal cell types, which have many physiological properties and behavioral correlates in common, may be differentiated using a pharmacological criterion.     

Rapid integration of young newborn dentate gyrus granule cells in the adult hippocampal circuitry

Newborn dentate gyrus granule cells (DGCs) are integrated into the hippocampal circuitry and contribute to the cognitive functions of learning and memory. The dendritic maturation of newborn DGCs in adult mice occurs by the first 3–4 weeks, but DGCs seem to receive a variety of neural inputs at both their dendrites and soma even shortly after their birth. However, few studies on the axonal maturation of newborn DGCs have focused on synaptic structure. Here, we investigated the potentiality of output and input in newborn DGCs, especially in the early period after terminal mitosis. We labeled nestin-positive progenitor cells by injecting GFP Cre-reporter adenovirus into Nestin-Cre mice, enabling us to trace the development of progenitor cells by their GFP expression. In addition to GABAergic input from interneurons, we observed that the young DGCs received axosomatic input from the medial septum as early as postinfection day 7 (PID 7). To evaluate the axonal maturation of the newborn DGCs compared with mature DCGs, we performed confocal and electron microscopic analyses. We observed that newborn DGCs projected their mossy fibers to the CA3 region, forming small terminals on hilar or CA3 interneurons and large boutons on CA3 pyramidal cells. These terminals expressed vesicular glutamate transporter 1, indicating they were glutamatergic terminals. Intriguingly, the terminals at PID 7 had already formed asymmetric synapses, similar to those of mature DGCs. Together, our findings suggest that newborn DGCs may form excitatory synapses on both interneurons and CA3 pyramidal cells within 7 days of their terminal mitosis.     

Dendrites of dentate gyrus granule cells contribute to pattern separation by controlling sparsity

The hippocampus plays a key role in pattern separation, the process of transforming similar incoming information to highly dissimilar, nonverlapping representations. Sparse firing granule cells (GCs) in the dentate gyrus (DG) have been proposed to undertake this computation, but little is known about which of their properties influence pattern separation. Dendritic atrophy has been reported in diseases associated with pattern separation deficits, suggesting a possible role for dendrites in this phenomenon. To investigate whether and how the dendrites of GCs contribute to pattern separation, we build a simplified, biologically relevant, computational model of the DG. Our model suggests that the presence of GC dendrites is associated with high pattern separation efficiency while their atrophy leads to increased excitability and performance impairments. These impairments can be rescued by restoring GC sparsity to control levels through various manipulations. We predict that dendrites contribute to pattern separation as a mechanism for controlling sparsity. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.