Learning and memory after adrenalectomy-induced hippocampal dentate granule cell degeneration in the rat

Adrenalectomy (ADX) of normal adult rats causes selective hippocampal dentate granule cell degeneration that is prevented by corticosterone. The ability to destroy this one hippocampal cell type noninvasively made it possible to address the role of the dentate granule cells in learning and memory. Four months after ADX, 31 of 45 rats failed to show obvious granule cell loss and displayed behavior in the Morris water maze that was similar to 16 sham-operated control rats and 16 ADX rats maintained on corticosterone throughout the study. Conversely, 14 of the 45 ADX rats experienced a loss of granule cells that varied from minimal to extensive. Although there were no obvious differences between groups in motoric and motivational characteristics or search strategies, ADX rats with moderate to extensive granule cell loss acquired place learning slightly slower than controls or ADX rats with minimal or no obvious cell loss. Furthermore, the ADX rats with moderate to extensive cell loss were temporarily impaired following alteration of either intramaze or extramaze cues compared to controls. In contrast, the rats with granule cell loss remembered an old place and learned a new place as quickly as controls. These results suggest that a normal complement of dentate granule cells may not be necessary for the acquisition or retention of spatial information in the Morris water maze.     

The alteration of glucocorticoid receptor-immunoreactivity in the rat forebrain following short-term and long-term adrenalectomy

To examine the effect of short-term and long-term adrenalectomy (ADX) on the glucocorticoid receptor (GR) expression, we performed an immunohistochemical study on the rat forebrain. One day after ADX, the GR-immunoreactivity significantly decreased or disappeared in most forebrain structures, while relatively strong GR-immunoreactivity was still found within the hypothalamus especially in the arcuate nucleus (ARC) and the parvocellular paraventricular nucleus (PVN). Two weeks following ADX, GR-immunoreactive cells disappeared in many structures of the forebrain including most parts of hypothalamus while moderate GR-immunoreactivity was still observable in the ARC and PVN. More than 3 months after ADX, the rats still survived when they received replacement of corticosterone during the first 2 weeks following the operation. Moderate GR-immunoreactivity in the ARC and PVN of the hypothalamus was exhibited whereas no immunoreactive cells remained in the cerebral cortex, thalamus and other forebrain structures when these animals showed obvious cell death in the granule cells of the dentate gyrus, identified with the silver impregnation method for degenerating cells. Massive cell loss in this hippocampal region is an indicator of a complete ADX, in addition to the blood corticosterone level. These results demonstrate topographic differences of GR expression in the rat forebrain after ADX with only continuous immunoreactivity in the ARC and PVN of the hypothalamus, suggesting that some neurons in the ARC and PVN could keep active GR probably in order to maintain their survival after removing the adrenal gland.     

Adrenalectomy-induced granule cell degeneration in the hippocampus causes spatial memory deficits that are not reversed by chronic treatment with corticosterone or fluoxetine

Long-term adrenalectomy (ADX) causes a nearly complete and selective loss of granule cells in the dentate gyrus (DG) of the hippocampus. Previously, learning and memory deficits have been observed following ADX-induced granule cell degeneration for tasks that require the hippocampus. Our objective here was to determine whether corticosterone (CORT) replacement and treatment with the neurogenic compound fluoxetine could reverse behavioral deficits after ADX. We trained ADX and control rats in a moving, hidden platform version of the Morris water task before chronic administration (6 weeks) of CORT and either fluoxetine or vehicle. After treatment, all rats were retested in the Morris water task. Brains were labeled for the endogenous neurogenic markers Ki67 and doublecortin. Here we provide evidence that neurogenesis persists at a normal rate in the hippocampus after long-term ADX. After 8 weeks of CORT and fluoxetine administration, ADX-fluoxetine rats did not differ significantly compared to ADX-vehicle rats receiving CORT or compared to control rats in the number of Ki67 or doublecortin labeled cells. ADX-fluoxetine rats also did not significantly differ from ADX-vehicle rats in regards to granule cell layer thickness. Our results indicate that long-term ADX is associated with impaired spatial ability in the Morris water task and that neither chronic treatment with CORT, nor with CORT and fluoxetine are capable of altering the Morris water task deficit.     

Effect of chronic adrenalectomy on neuron loss and distribution of sulfated glycoprotein-2 in the dentate gyrus of prepubertal rats

This study extends the unexpected finding of Sloviter et al. (Science, 1989, 243: 535-538) that adrenalectomy (ADX) of young rats casues a loss of granule neurons in the dentate gyrus. In particular, we determined how the vulnerability of dentate granule neurons to the cytocidal effect of ADX is related to the completeness of the ADX and whether sulfated glycoprotein-2, a putative component of programmed cell death, is associated with the death of granule neurons after ADX. We report that 4 months after bilateral ADX of young (150-175 g) rats only ADX rats that had attenuated weight gain and less than 2 ng/ml of serum corticosterone lost granule neurons; whereas as little as 15 ng/ml of serum corticosterone was sufficient to protect granule neurons from cell death. In addition, by immunocytochemistry, SGP-2 was distributed as punctate deposits throughout the molecular layer of the dentate gyrus and in glial cells juxtaposed to surviving neurons in the dentate of ADX rats with a granule cell loss. However, immunoreactivity for SGP-2 was not found in granule neurons that exhibited morphological signs of cellular generation after ADX.     

Time course and distribution of neuronal degeneration in the dentate gyrus of rat after adrenalectomy: a silver impregnation study.

Recently, Sloviter et al. reported that adrenalectomy (ADX) of young adult rats after 3 months led to a selective loss of granule neurons in the dentate gyrus (DG) and that this loss could be prevented by low doses of corticosterone. In the present study, the ADX-induced neuronal degeneration was investigated in Wistar rats, using a silver impregnation method for degenerating neurons. To examine the time course and distribution of the ADX-induced degeneration, young adult male rats were allowed to survive 2, 3, and 5 days and 1, 2, and 3 weeks after ADX. Argyrophilic neurons were present in the dentate granule cell layer on the second day following ADX. Three days after ADX, the number of argyrophilic granule neurons was much more abundant, and it increased gradually with longer post-ADX survival times. Argyrophilia was specifically confined to dentate granule cells and was accompanied by the occurrence of pyknotic nuclei as observed in adjacent cresyl violet-stained sections. There were significant differences between individual rats in quantity of argyrophilia. About one fifth of the ADX rats showed sporadic or no argyrophilia, in spite of plasma corticosterone levels below the detection limit (10 ng/mL). Sham-operated rats and ADX rats receiving corticosterone (10 mg/L) or dexamethasone (15 mg/L) in their drinking water did not display any argyrophilic neurons in the dentate gyrus. The distribution of the argyrophilia within the DG was highly characteristic with the highest number of degenerating cells in the hidden blade of the middle and the temporal thirds of the DG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and corticosteroid regulation of glucocorticoid receptor in the brain of Xenopus laevis

Glucocorticoids (GCs) play essential roles in physiology, development, and behavior that are mediated largely by the glucocorticoid receptor (GR). Although the GR has been intensively studied in mammals, very little is known about the GR in nonmammalian tetrapods. We analyzed the distribution and GC regulation of GR in the brain of the frog Xenopus laevis by immunohistochemistry. GR-immunoreactive (GR-ir) cells were widely distributed, with the highest densities in the medial pallium (mp; homolog of the mammalian hippocampus), accumbens, anterior preoptic area (POA; homolog of the mammalian paraventricular nucleus), Purkinje cell layer of the cerebellum, and rostral anterior pituitary gland (location of corticotropes). Lower but distinct GR-ir was observed in the internal granule cell layer of the olfactory bulbs, dorsal and lateral pallium, striatum, various subfields of the amygdala, bed nucleus of the stria terminalis (BNST), optic tectum, various tegmental nuclei, locus coeruleus, raphe nuclei, reticular nuclei, and the nuclei of the trigeminal motor nerves. Treatment with corticosterone (CORT) for 4 days significantly decreased GR-ir in the POA, mp, medial amygdala (MeA), BNST, and rostral pars distalis. Treatment with the corticosteroid synthesis inhibitor metyrapone (MTP) also significantly reduced GR-ir in the POA, mp, MeA and BNST, but not in the rostral pars distalis. Replacement with a low dose of CORT in MTP-treated animals reversed these effects in brain. Thus, chronic increase or decrease in circulating corticosteroids reduces GR-ir in regions of the frog brain. Our results show that the central distribution of GR-ir and regulation by corticosteroids are highly conserved among vertebrates.     

Inhibition of neuronal nitric oxide synthase increases adrenalectomy-induced granule cell death in the rat dentate gyrus

Recent studies have shown that the expression of neuronal nitric oxide synthase (NOS) mRNA is increased after adrenalectomy (ADX). However, the role of increased NO production after ADX in the dentate gyrus is unknown. In this study, the relationship between NO inhibition and apoptosis in the dentate gyrus after ADX was examined. 7-Nitroindazole (7-NI; 30 mg/kg, i.p.), a selective inhibitor of neuronal NOS, was injected 1 day before ADX and subsequently once every 24 h. Then 4 days after ADX, dentate granule cell death was evaluated using silver impregnation and Nissl staining methods. Inhibition of neuronal NOS by 7-NI increased the number of dying granule cells approximately 4-fold in the dentate gyrus of the ADX rats, compared to vehicle-injected ADX controls. These results suggest that increased NO production after ADX may play an endogenous neuroprotective role in the dentate gyrus.     

Analysis of the mechanisms underlying increased histogenetic cell death in developing cerebellum of the hypothyroid rat: determination of the time required for granule cell death

The connection between the date of formation of granule cells and their final position in the internal granular layer of the cerebellum has been described previously. In rats made hypothyroid since the end of gestation, the distribution of the pyknotic cells in the internal granular layer of the cerebullar cortex was also previously found to be age-related. In 14-day-old hypothyroid rats, it was compared with that of the granule cells which were labeled after a pulse of [³H]thymidine at various stages of development. It appeared that the localization of the dying cells corresponded roughly to that of granule cells labeled on day 9. Therefore the maximum time required for granule cell death was about 5 days. Since the migratory phase through the molecular layer lasted about two days, the granule cells died after a maximum time of 3 days following their deposition in the internal granular layer. Information concerning the time of survival of the dying granule cells was important for subsequent investigation of the mechanisms underlying increased granule cell death in the hypothyroid cerebellum and the corrective effects of thyroid hormone.     

Long-term adrenalectomy reduces hippocampal granule cell excitability in vivo

Ten days after bilateral adrenalectomy (ADX), neural transmission between the perforant path and hippocampal dentate granule cells was severely impaired in the anaesthetized rat, in that the slope of the stimulus-response curve was reduced to less than half the value in sham controls, the stimulation current necessary to elicit a standard population spike (PS) field potential was increased approximately threefold, the amplitude of PS and its ratio to the slope of the field excitatory postsynaptic potential (EPSP) were reduced, and high-frequency tetanization (TET) of the perforant path resulted in potentiated PS with smaller amplitude and higher onset latency in ADX rats than in sham controls. However, the fractional increase of PS amplitude and its decay following TET were similar in 10 day-ADX and sham groups, from which it is inferred that long-term ADX entails a general decrease of dentate granule cell excitability, particularly at somatic membrane level, without specifically affecting the mechanism of long-term potentiation. None of the above changes occurred 24 h after ADX.     

Neurotrophin‐3 promotes cerebellar granule cell exit from the EGL

In the cerebellum, the mRNAs for neurotrophin-3 (NT-3) and its high-affinity tyrosine kinase receptor trkC are expressed by both the differentiated granule cells of the internal granule cell layer (IGL) and their precursors in the external germinal layer (EGL). We have investigated the effects of chronic application of exogenous NT-3 in vivo on cerebellar granule cell genesis and differentiation. NT-3 was applied to the posterior surface of the rat cerebellum from P6 onwards using Elvax implants. At P10 the EGL of cerebellar lobules VII and VIII was significantly reduced in thickness in NT-3 implanted rats when compared with controls. Immunocytochemical analysis of the EGL using antibodies to proliferating cell nuclear antigen (PCNA) revealed that the number of postmitotic, premigratory (PCNA-immunonegative) granule cell precursors was preferentially reduced in the NT-3 implanted rats. In situ DNA fragmentation labelling confirmed that this was not accompanied by increased cell death in the EGL. These results suggest that NT-3 promotes the differentiation of postmitotic, premigratory granule cell precursors, accelerating cell exit from the EGL.     

Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures

Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 microM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation.     

Short-term and long-term survival of new neurons in the rat dentate gyrus

New neurons continue to be generated in the dentate gyrus throughout adulthood. Previous studies have shown that a significant proportion of new granule cells labeled with the thymidine analogue bromodeoxyuridine (BrdU) are lost from the adult dentate gyrus within 2 weeks. How long this loss continues and the extent to which it represents cell death, as opposed to dilution of label, is unclear. To address these questions, adult rats were injected with BrdU, and BrdU labeling in the dentate gyrus was compared at several survival time points. Double labeling with BrdU and the cell cycle marker Ki-67 showed that BrdU is detectable for up to 4 days in some cells that continue to divide, indicating that any decrease in the number of BrdU-labeled cells after 4 days is likely to reflect cell death rather than BrdU dilution. Death of new cells in the granule cell layer occurred at a steady rate between 6 and 28 days after labeling, resulting in loss of 50% of BrdU-labeled cells over this 22-day period. New granule cells that survived this first month lived for at least 5 additional months. In contrast, 26% of the granule cells labeled with BrdU at the peak of dentate gyrus development on postnatal day (P) 6 died between 1 and 6 months after labeling. These findings suggest that granule cells born during adulthood that become integrated into circuits and survive to maturity are very stable and may permanently replace granule cells born during development.     

The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat

Glucocorticoid receptor activation inhibits granule cell proliferation in the hippocampus, but little is known about the role of mineralocorticoid receptors in this process. Here we administered aldosterone to adrenalectomized (ADX) rats, and monitored neurogenesis by BrdU immunohistochemistry. ADX significantly increased the number of BrdU-positive cells and aldosterone replacement further augmented BrdU-positivity. Our results indicate that aldosterone, most probably acting through mineralocorticoid receptors, may positively influence the proliferation and survival of newly-generated granule cells.     

Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy

Granule neurons of the rat dentate gyrus are born in adulthood as well as during development. Apoptotic cell death also occurs normally in this population throughout the life of the rat. Removal of adrenal steroids results in both increased production and increased degeneration of dentate gyrus granule cells. In order to determine whether the age of a cell affects its response to adrenalectomy (ADX), the numbers of dentate gyrus cells of different ages were assessed following ADX or sham operation. Older cells, i.e., those labeled with the thymidine analog bromodeoxyuridine (BrdU) on postnatal day (P) 6, were reduced in number following ADX on P60, and some had the morphologic characteristics of degenerating cells, indicating that significant numbers of mature cells die in response to ADX. In contrast, the number of younger cells, labeled with ³H-thymidine or BrdU in adulthood, 24 hours or 2 weeks before ADX, did not decrease, suggesting that these less mature cells do not die following ADX. An increase in the number of cells that are immunoreactive for proliferating cell nuclear antigen, a marker of dividing or recently mitotic cells, indicates that immature dentate gyrus cells divide following ADX. These results suggest that following ADX, mature cells born during the 1st postnatal week die, whereas immature cells divide. © 1996 Wiley-Liss, Inc.     

Cystatin C expression is associated with granule cell dispersion in epilepsy

Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis, during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater numbers of polysialylated neural cell adhesion molecule-positive newborn cells in the molecular layer, although the overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule cells.     

Patterns of dentate granule cell responses to perforant path stimulation in epileptic mice with granule cell dispersion

In adult mice, intrahippocampal administration of kainic acid induces a structural modification of the granule cell layer reminiscent of granule cell dispersion (GCD) seen in humans with temporal lobe epilepsy. We tested that GCD might be involved in the patterns of granule cell responses to perforant path stimulation by recording field potentials in vivo after kainic acid-induced status epilepticus until the phase of chronic seizure activity in presence of GCD or after its alteration by K252a co-treatment, an inhibitor of tyrosine kinase activities. Stimulation triggered bursts of multiple population spikes, the number of which progressively increased with time whereas their amplitude decreased in parallel with the progressive decrease in granule cell density. The population spike threshold was reached for a lower excitatory synaptic drive than in controls, as assessed by the initial slope of the field excitatory post-synaptic potential. This indicates that, for identical synaptic responses, granule cells were closer to the firing threshold. Fast inhibition, assessed by paired pulse stimulation, was compromised immediately after the initial status epilepticus, consistent with the rapid loss of most hilar cells. Neither the epileptic course nor the epileptiform responses of the granule cells were modified and manipulation by alteration following GCD manipulation while granule cell neuropeptide-Y immunostaining was substantially decreased. In this mouse model of TLE, granule cells display a progressive increase in epileptiform responses to afferent input until the occurrence of spontaneous seizures. The population spike amplitude decreases in parallel with GCD while the granule cell excitability is enhanced. Consequently, data from field potentials in epilepsy experiments should be interpreted with care, taking into account the possible variations in the neuronal density in the recorded area.     

Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids.

The regulation of prodynorphin gene expression by glucocorticoids in the hippocampus was examined in rats that were adrenalectomized (ADX) either 7, 30, 60 and 90 days prior to sacrifice. Peptide levels in the hippocampus of ADX rats were determined by radioimmunoassay and immunocytochemistry. Prodynorphin (PDYN) mRNA was measured by Northern blot analysis and in situ hybridization. A time-dependent decrease in dynorphin A(1-8)(DYN) levels in the hippocampus (18% at 7 days; 44% at 30 days; 58% at 60 days) of ADX rats was found, which was accompanied by a comparable decrease in the abundance of PDYN mRNA. An in situ hybridization analysis revealed that both the number of positively hybridized cells and the number of silver grains per cell were decreased in the dentate gyrus after ADX. The administration of dexamethasone after surgery reversed the peptide and mRNA attenuation induced by ADX. ADX had no effect on the expression of proenkephalin mRNA or [Met5]-enkephalin immunoreactivity in the hippocampus. Examination of thionin-counterstained tissue showed that the dentate granule cell layer was intact. The decrement of DYN expression in this system is proposed to have resulted from the removal of glucocorticoid input and not dentate granule cell loss. This study provides the strong evidence for a differential susceptibility of these two opioid peptides in the hippocampus to the removal of glucocorticoids. In addition, these data provide support for a potentially selective, glucocorticoid-permissive component in PDYN gene expression.     

Population dynamics of adult-formed granule neurons of the rat olfactory bulb

The population dynamics of internal granule cells in the rat olfactory bulb during adult life were analyzed in histological sections and in autoradiograms with (1) counts of granule cells, (2) counts of labeled granule cells 1 month after injection of 3H-thymidine at various ages, and (3) counts of labeled granule cells at varying survival times (up to 18 months) after injection at 3 months and 24 months. The total number of granule cells increases linearly throughout life, approximately doubling between 3 and 31 months. Autoradiographic studies show that the rate of production of new granule cells decreases from 3 to 12 months and then is approximately constant during the rest of the life span. The number of labeled cells found 6 months after injection at 3 and 24 months is about one-fourth and one-half, respectively, that of the number at a 1-month survival, suggesting that many of the cells produced to do not survive. However, at least some granule cells labeled at 3 months survive for 18 months. A model is suggested in which granule cells are produced continuously throughout life and control of the total number of granule cells is effected chiefly through the rate of cell death.     

Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb

In the adult rat olfactory bulb, neurons are continually generated from progenitors that reside in the lateral ventricle wall. This study investigates long-term survival and cell death of newly generated cells within the adult olfactory bulb. After injecting rats at 2 months of age with 5-bromodeoxyuridine (BrdU), the newly generated cells were quantified over a period of 19 months. A peak of BrdU-positive cells was reached in the olfactory bulb 1 month after BrdU injection, when all new cells have finished migrating from the ventricle wall. Thereafter, a reduction of BrdU-positive cells to about 50% was observed and it was confirmed by dUTP-nick end-labelling (TUNEL) that progenitors and young neurons undergo programmed cell death. However, cells that survived the first 3 months after BrdU injection persisted for up to 19 months. The majority of the BrdU-positive cells that reach the olfactory bulb differentiate into granule cells, but a small fraction migrate further into the glomerular layer. These newborn cells differentiate more slowly into periglomerular interneurons, with a delay of more than 1 month when compared to the granule cells. The newly generated periglomerular neurons, among them a significant fraction of dopaminergic cells, showed a similar decline in number compared to the granule cell layer and long-term survival for the remaining new neurons of up to 19 months. Rather than replacing old neurons, this data suggests that adult olfactory bulb neurogenesis utilizes the overproduction and turnover of young neurons, which is reminiscent of the cellular dynamics observed during brain development.