Granule Cell Dispersion in Human Temporal Lobe Epilepsy: Proteomics investigation of neurodevelopmental migratory pathways

Granule cell dispersion (GCD) is a common pathological feature observed in the hippocampus of patients with Mesial Temporal Lobe Epilepsy (MTLE). Pathomechanisms underlying GCD remain to be elucidated, but one hypothesis proposes aberrant reactivation of neurodevelopmental migratory pathways, possibly triggered by febrile seizures. This study aims to compare the proteomes of basal and dispersed granule cells in the hippocampus of eight MTLE patients with GCD to identify proteins that may mediate GCD in MTLE. Quantitative proteomics identified 1882 proteins, of which 29% were found in basal granule cells only, 17% in dispersed only and 54% in both samples. Bioinformatics analyses revealed upregulated proteins in dispersed samples were involved in developmental cellular migratory processes, including cytoskeletal remodelling, axon guidance and signalling by Ras homologous (Rho) family of GTPases (P<0.01). The expression of two Rho GTPases, RhoA and Rac1, was subsequently explored in immunohistochemical and in situ hybridisation studies involving eighteen MTLE cases with or without GCD, and three normal post mortem cases. In cases with GCD, most dispersed granule cells in the outer-granular and molecular layers have an elongated soma and bipolar processes, with intense RhoA immunolabelling at opposite poles of the cell soma, while most granule cells in the basal granule cell layer were devoid of RhoA. A higher density and percentage of cells expressing RhoA was observed in cases with GCD than without GCD (P<0.004). In GCD cases, the density and percentage of cells expressing RhoA was significantly higher in the inner molecular layer than granule cell layer (P<0.026), supporting proteomic findings. In situ hybridisation studies using probes against RHOA and RAC1 mRNAs revealed fine peri- and nuclear puncta in granule cells of all cases. The density of cells expressing RHOA mRNAs were significantly higher in the inner molecular layer of cases with GCD than without GCD(P=0.05). In summary, our study has found limited evidence for ongoing adult neurogenesis in the hippocampus of patients with MTLE, but evidence of differential dysmaturation between dispersed and basal granule cells has been demonstrated, and elevated expression of Rho GTPases in dispersed granule cells may contribute to the pathomechanisms underpinning GCD in MTLE

Temporal profile of hilar basal dendrite formation on dentate granule cells after status epilepticus.

Granule cells with hilar basal dendrites (HBDs) are found after status epilepticus (SE) in three rat models of temporal lobe epilepsy. These granule cells are commonly located at the hilar border and could be newly born granule cells based on their location. The aim of this study was to determine how long it takes for HBDs to form on granule cells after SE. Pilocarpine was injected to induce SE and rats were killed at different times: 3 days, 1, 2, and 3 weeks after SE. Biocytin was injected into CA3 stratum lucidum of hippocampal slices to label granule cells with HBDs. The number, morphology, and length of HBDs were analyzed at the different time points. Basal processes of granule cells from rats killed 3 days after pilocarpine injection were judged not to be HBDs because they were short in length and did not ramify in the hilus. "True" HBDs were detected as early as 7 and 8 days after pilocarpine-induced SE. Similar frequencies of granule cells with HBDs were observed at the later time points. This study shows that HBDs can form on granule cells as early as 1 week following SE. These results are consistent with the hypothesis that HBDs on granule cells may be generated from seizure-induced, de novo granule cells, however, alternative explanations that some or all HBDs arise from pre-SE generated granule cells cannot be ruled out at this time and will require further examination

Ultrastructural features of primate granule cell bodies show important differences from those of rats: axosomatic synapses, somatic spines and infolded nuclei.

Granule cells of the primate dentate gyrus were examined in the electron microscope where they displayed significantly less axosomatic synapses than granule cells in rodents. In addition, primate granule cells frequently had infolded cell nuclei and somal spines which are features that are both rare in rodents. Since the granule cell body is an important site for gamma-aminobutyric acid (GABA)ergic inhibitory control, the reduced number of axosomatic synapses in monkeys suggests that local inhibitory connections of primate granule cells are less than that of rodents. Together, these differences may indicate that the primate granule cells are physiologically more active than rat granule cells

Atypical features of rat dentate granule cells: recurrent basal dendrites and apical axons.

The stereotyped morphology of dentate granule cells in rodents consists of apical dendrites arborizing in the molecular layer and an axon arising from the opposite pole of the soma. Recently, we showed that epilepsy induces the formation of basal dendrites on granule cells and that these dendrites extend into the hilus of the dentate gyrus. The present Golgi study of granule cells from adult rats shows two atypical features for granule cells in control rats. One is the occurrence of recurrent basal dendrites (RBDs) that are defined as basal dendrites arising at or near the hilar pole of the soma and then curving back to the molecular layer. The frequency of granule cells with RBDs was 3.8% in control rats. The second is apical axons of granule cells that were observed to originate from either the apical pole of the soma or an apical dendrite. The incidence of these "apical" axons was about 1%. These morphological findings in the present study suggest that rat granule cells are more heterogeneous than previously indicated. Furthermore, their frequency was not increased in epileptic rats

Biosensor measurement of purine release from cerebellar cultures and slices

We have previously described an action-potential and Ca2+-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7–8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K+ concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca2+dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7–25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca2+ dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage

Expression of the AMPA Receptor Subunits GluR1 and GluR2 is Associated with Granule Cell Maturation in the Dentate Gyrus

The dentate gyrus produces new granule neurons throughout adulthood in mammals from rodents to humans. During granule cell maturation, defined markers are expressed in a highly regulated sequential process, which is necessary for directed neuronal differentiation. In the present study, we show that α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptor subunits GluR1 and GluR2 are expressed in differentiated granule cells, but not in stem cells, in neonatal, and adult dentate gyrus. Using markers for neural progenitors, immature and mature granule cells, we found that GluR1 and GluR2 were expressed mainly in mature cells and in some immature cells. A time-course analysis of 5-bromo-2′-deoxyuridine staining revealed that granule cells express GluR1 around 3 weeks after being generated. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, a putative animal model of schizophrenia and bipolar disorder in which dentate gyrus granule cells fail to mature normally, GluR1 and GluR2 immunoreactivities were substantially downregulated in the dentate gyrus granule cells. In the granule cells of mutant mice, the expression of both presynaptic and postsynaptic markers was decreased, suggesting that GluR1 and GluR2 are also associated with synaptic maturation. Moreover, GluR1 and GluR2 were also expressed in mature granule cells of the neonatal dentate gyrus. Taken together, these findings indicate that GluR1 and GluR2 expression closely correlates with the neuronal maturation state, and that GluR1 and GluR2 are useful markers for mature granule cells in the dentate gyrus

A Transgenic Marker for Newly Born Granule Cells in Dentate Gyrus

Neurogenesis in the dentate gyrus continues into adulthood, yet little is known about the function of newly born neurons or how they integrate into an existing network of mature neurons. We made transgenic mice that selectively and transiently express enhanced green fluorescent protein (EGFP) in newly born granule cells of the dentate gyrus under the transcriptional control of proopiomelanocortin (POMC) genomic sequences. Analysis of transgenic pedigrees with truncation or deletion mutations indicated that EGFP expression in the dentate gyrus required cryptic POMC promoter regions dispensable for arcuate hypothalamic or pituitary expression. Unlike arcuate neurons, dentate granule cells did not express the endogenous POMC gene. EGFP-positive neurons had immature properties, including short spineless dendrites and small action potentials. Colocalization with bromodeoxyuridine indicated that EGFP-labeled granule cells were 2 weeks postmitotic. EGFP-labeled cells expressed markers for immature granule cells but not the glial marker GFAP. The number of EGFP-labeled neurons declined with age and increased with exercise, paralleling neurogenesis. Our results indicate that POMC-EGFP marks immature granule cells and that adult-generated granule cells integrate quite slowly into the hippocampal circuitry