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.     

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.     

GABAergic signaling in young granule cells in the adult rat and mouse dentate gyrus

Throughout most of the developing brain, including the hippocampus, GABAergic synapses are the first to become functional. Several features of GABAergic signaling change across development, suggesting that this signaling in the immature brain may play important roles in the growth of young neurons and the establishment of networks. To determine whether GABA(A) receptor (GABA(A)R)-containing synapses in new neurons born in the adult dentate gyrus have similar immature features, we examined spontaneous and evoked GABA(A)R-mediated synaptic currents in young (POMC-EGFP or doublecortin-immunostained) granule cells in acute slice preparations from adult mice and rats. Spontaneous inhibitory postsynaptic currents (IPSCs) were observed in nearly all immature granule cells, but their frequency was considerably lower and their decay time constant was nearly two times longer than in neighboring mature (doublecortin-non-immunoreactive or EGFP-non-expressing) granule cells within the sub-granular zone. Evoked IPSCs (eIPSCs) in mature granule cells, but not immature granule cells, were sensitive to zolpidem, suggesting a maturational increase in GABA(A)R alpha1-subunit expression. Perforated-patch recording revealed that eIPSCs depolarized young neurons, but hyperpolarized mature neurons. The early establishment of synaptic GABAergic inputs slow IPSC decay time, and depolarizing action of eIPSCs are remarkably similar to features previously seen in neurons during development, suggesting that they are intrinsic features of immature neurons and not functions of the surrounding circuitry. These developmental features in adult-born granule cells could play a role in maturational processes such as developmental cell death. However, treatment of adult mice with GABA(A)R agonists and an inverse agonist did not significantly alter the number of 4- to 14-day-old BrdU-labeled cells.     

Quantitative analysis of the dendritic branching pattern of granule cells from adult rat dentate gyrus

The three-dimensional structure of Golgi-impregnated cells of the hippocampal dentate gyrus was studied using modern data-collecting techniques. Branch length and branch angle distributions were examined and found to have a wide range of observed values. The distributions imply a stochastic design for the branching pattern of dendrites. Examination of the ordered branch lengths suggests that the local dendritic branching probability may be dependent on the density or specificity of afferent projections. Analysis of the total branch lengths and number of branch nodes strongly suggests that quantitative parameters related to the total dendritic arborization may be more important in the structural design than parameters related to the individual dendrites.     

Electrophysiological characterization of associational pathway terminating on dentate gyrus granule cells in the rat.

The functional topography and parameters of excitation and inhibition were determined in the in situ associational pathway of the rat dentate gyrus. The functional topography was found to be consistent with previous anatomical studies. The greatest amplitude population spikes and the strongest paired-pulse inhibition were generated with the stimulating electrode placed in the hilus at least 1.5 mm caudal to the ipsilateral dentate gyrus recording electrode. With this standard electrode configuration, neither long-term potentiation of the population spike nor of the population excitatory postsynaptic potential occurred. Hilar associational pathway activation of dentate gyrus granule cells elicited paired-pulse responses similar to those produced in granule cells by perforant path stimulation. Thus, the associational pathway provides another way to assess dentate granule cell function electrophysiologically.     

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.     

GABAergic synaptic boutons in the granule cell layer of rat dentate gyrus

GABAergic synapses in the granule cell layer of the rat dentate gyrus were examined light and electron microscopically with glutamate decarboxylase (GAD) immunocytochemistry. GAD-immunoreactive synaptic boutons formed synapses with axon initial segments and somatic spines as well as somata and dendritic shafts of the granule cell. Most of these synapses were symmetrical, while a few were asymmetrical.     

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.     

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.     

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.     

Quantitative, three-dimensional analysis of granule cell dendrites in the rat dentate gyrus

The three-dimensional organization of dentate granule cell dendritic trees has been quantitatively analyzed with the aid of a computerized microscope system. The dendrites were visualized by iontophoretic injection of horseradish peroxidase into individual granule cells in the in vitro hippocampal slice preparation. Selection criteria insured that the analyzed cells were completely stained and that only neurons with two or fewer cut dendrites in the distal portion of the molecular layer were analyzed. Twenty-nine of the 48 sampled granule cells had no cut dendrites. The granule cells had between one and four primary dendrites. Granule cell dendritic branches were covered with spines and most extended to the hippocampal fissure or pial surface. The mean total dendritic length was 3,221 microns with a range from 2,324 microns to 4,582 microns. The dendrites formed an elliptical plexus with the transverse spread averaging 325 microns and the spread in the septotemporal axis averaging 176 microns. On individual neurons, the maximum branch order ranged from four to eight and the number of dendritic segments ranged from 22 to 40. Approximately 63% of the dendritic branch points occurred in a zone that included the granule cell layer and the inner one-third of the molecular layer. The dendritic tree was organized so that, on average, 30% of the length was in the granule cell layer and proximal third of the molecular layer, 30% was in the middle third, and 40% was in the distal third. Comparisons were made between the dendrites of granule cells in the suprapyramidal and infrapyramidal blades of the dentate gyrus. Suprapyramidal cells had a significantly greater total dendritic length than infrapyramidal cells, their transverse spread was higher, and they had a greater number of dendritic segments. When neurons in the suprapyramidal blade were further subdivided on the basis of somal position within the depth of the cell body layer, superficial neurons were found to have a greater number of primary dendrites, more elliptical trees, and larger transverse spreads of their dendrites. There were no significant differences in dendritic segment number or total dendritic length between superficial and deep cells.     

Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer

Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20–40 μm from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 μm in diameter and they tapered down to 2 μm at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy fibers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This finding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL.     

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.     

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.     

Computational models of dentate gyrus with epilepsy-induced morphological alterations in granule cells

Temporal lobe epilepsy provokes a number of different morphological alterations in granule cells of the hippocampus dentate gyrus. These alterations may be associated with the hyperactivity and hypersynchrony found in the epileptic dentate gyrus, and their study requires the use of different kinds of approaches including computational modeling. Conductance-based models of both normal and epilepsy-induced morphologically altered granule cells have been used in the construction of network models of dentate gyrus to study the effects of these alterations on epilepsy. Here, we review these models and discuss their contributions to the understanding of the association between alterations in neuronal morphology and epilepsy in the dentate gyrus.This article is part of a Special Issue entitled “NEWroscience 2013”.     

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.