Seizure induced dentate neurogenesis does not diminish with age in rats.

Neurogenesis in the mammalian dentate gyrus occurs throughout life, is believed to be important for the laying down of episodic memory and diminishes significantly with increasing age. Pathological insults such as seizures, hypoxia and traumatic brain injury increase dentate neurogenesis compared to age matched controls. Using unilateral intracerebroventricular kainate we show that although baseline neurogenesis is significantly lower in 3 month old rats compared to 1 month old rats, kainate increases neurogenesis to reach similar levels in both age groups. Additionally, this effect is bilateral after a unilateral intracerebroventricular kainate injection. We conclude that the potential for dentate neurogenesis is maintained despite diminishing baseline levels with increasing age and that injury signals override the age related suppression of neurogenesis.

Somatostatin- and neuropeptide Y-synthesizing neurones in the fascia dentata of humans with temporal lobe epilepsy.

We used in situ hybridization techniques to study the distribution of neurones synthesizing somatostatin mRNA and neuropeptide Y mRNA in the hilar region of the hippocampal formation of patients with temporal lobe epilepsy. In the dentate gyrus, somatostatin mRNA- and neuropeptide Y mRNA-synthesizing neurones were found to be exclusively located within the hilar region. Unlike animal models, no ectopic expression of either peptide was found in principal cells. The numbers of hilar interneurones expressing somatostatin mRNA and neuropeptide Y mRNA were compared with the degree of hilar cell loss determined by immunohistochemistry against neuronal nuclear antigen. The numbers of somatostatin and neuropeptide Y mRNA-synthesizing neurones varied considerably between patients, but both were found to be highly correlated to the total number of neuronal nuclear antigen-immunoreactive hilar neurones. These results suggest that loss of somatostatin and neuropeptide Y interneurones occurs in proportion to overall hilar cell loss, and therefore the hypothesis of a selective loss of these interneurones in temporal lobe epilepsy seems unlikely.

Neuroprotective role of monocarboxylate transport during glucose deprivation in slice cultures of rat hippocampus.

The effects of energy substrate removal and metabolic pathway block have been examined on neuronal and glial survival in organotypic slice cultures of rat hippocampus. Slice cultures resisted 24 h of exogenous energy substrate deprivation. Application of 0.5 mM alpha-cyano-4-hydroxycinnamate (4-CIN) for 24 h resulted in specific damage to neuronal cell layers, which could be reversed by co-application of 5 mM lactate. Addition of 10 mM 2-deoxyglucose in the absence of exogenous energy supply produced widespread cell death throughout the slice. This was partly reversed by co-application of 5 mM lactate. These effects of metabolic blockade on cell survival were qualitatively similar to the effects on population spikes recorded in the CA1 cell layer following 60 min application of these agents. The data suggest that monocarboxylate trafficking from glia to neurons is an essential route for supply of energy substrates to neurons particularly when exogenous energy supply is restricted.

Attenuation and augmentation of ischaemia-related neuronal death by tumour necrosis factor-alpha in vitro.

Upregulation of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF) occurs rapidly in the brain following ischaemia, although it is unclear whether this represents a neurotoxic or neuroprotective response. We have investigated whether TNF has different actions in the pre- and postischaemic periods in a tissue culture model of cerebral ischaemia. Organotypic hippocampal slice cultures were prepared from 8-10-day-old rats and maintained in vitro for 14 days. Neuronal damage was induced by either 1 h oxygen-glucose deprivation or 3 h exposure to NMDA or the superoxide generator duroquinone, and assessed after 24 h by propidium iodide fluorescence. TNF pretreatment was neuroprotective against both oxygen-glucose deprivation and duroquinone. This effect was associated with an activation of the transcription factor NFkappaB and upregulation of manganese superoxide dismutase, and was prevented by a free radical scavenger. When addition of TNF was delayed until the postinsult period, an exacerbation of neurotoxicity occurred, which was also prevented by a free radical scavenger. The actions of TNF are determined by whether TNF is present before or after an ischaemia-related insult. Both actions are mediated through the production of free radicals, and the response to TNF is determined by whether a cell is metabolically competent to respond by synthesis of antioxidant defences.

A macrophage hippocampal slice co-culture system: application to the study of HIV-induced brain damage.

We have developed an in vitro system that allows the study of the effects of factors released from macrophages on neuronal and glial survival in cultured hippocampal slices. Organotypic hippocampal slice cultures are grown on semi-permeable membranes in stationary co-culture with a murine macrophage cell line (RAW 264.7). The two culture systems are separated by a semi-permeable membrane specifically allowing the study of diffusable factors between the two culture systems. The use of the fluorescent exclusion dye propidium iodide as an in vitro marker of cell viability allows the study of progressive toxicity as it evolves in the slice cultures. We demonstrate that the HIV-1 derived nuclear regulatory protein Tat induces toxicity in slice cultures via the production of soluble mediators. The advantages of organotypic cultures over other in vitro systems is discussed as well as the general applicability of this method to the study of other brain pathologies, where macrophage derived factors are thought to play a role in neuronal survival.

A soluble factor produced by macrophages mediates the neurotoxic effects of HIV-1 Tat in vitro.

OBJECTIVES: There is now a strong consensus that the neurotoxic properties of HIV-1 are likely to be mediated by an indirect mechanism in which neurones are damaged by infected mononuclear cells. The aim of this study was to determine the ability of HIV-1 Tat to induce neurotoxic properties in a murine macrophage cell line RAW264.7. DESIGN: Simple culture systems using dissociated neurones may not provide the appropriate microenvironment in which to observe the complex cell-cell interactions that occur in the brain. We have therefore developed a more physiological model in which rat organotypic hippocampal slices are co-cultured with the murine macrophage cell line RAW264.7. Effects of Tat were studied by using a stable Tat expressing RAW264.7 cell line or by addition of recombinant Tat protein to co-cultures. METHODS: Organotypic hippocampal slices prepared from 8-10 day rat pups were grown on membrane inserts that were placed into six-well plates on which RAW264.7 cells were growing as an adherent monolayer. Cell death in the slices was assessed using propidium iodide. Specific astrocytic (glial fibrillary acidophilic protein; GFAP) and neuronal (microtubule-associated protein; MAP2) markers were visualized by immunocytochemistry. RESULTS: RAW264.7 cells that either expressed or were exposed to HIV-1 Tat protein, produced a soluble factor that caused profound degeneration in brain slice cultures involving loss of both glial cells and neurones. By contrast treatment of slice cultures with Tat in the absence of RAW264.7 cells was not neurotoxic. CONCLUSIONS: The neurotoxic properties previously attributed to HIV-1 Tat are likely to be mediated via induction of macrophage derived soluble factor(s).

Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose.

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.

Mossy fibre innervation is not required for the development of kainic acid toxicity in organotypic hippocampal slice cultures.

The glutamate analogue kainic acid (KA) generates convulsions when applied systemically or directly into the brain and produces lesions comparable to those seen in Ammon's horn sclerosis, observed in many patients with temporal lobe epilepsy. The neurotoxic actions of KA in-vivo appear to be mediated by a combination of direct effects on neurons and indirect effects mediated by seizures. Understanding the contribution of both direct and indirect effects of KA towards neuronal cell death is important for elucidating excitotoxic mechanisms, which may represent a common final pathway in a variety of neurodegenerative disorders including stroke, traumatic brain injury and epilepsy. We have investigated the effects of mossy fibre innervation on the development of KA toxicity in organotypic hippocampal slice cultures in order to assess the role of this input pathway on the specific toxicity of KA toward CA3 pyramidal neurones in vitro.

Kainic acid increases the proliferation of granule cell progenitors in the dentate gyrus of the adult rat.

Granule cell progenitors in the dentate gyrus of the hippocampal formation have the unusual capacity to be able to divide in the brains of adult rats and primates. The basal proliferation rate of granule cell progenitors in the adult rat is low compared with development, however, it is possible that this rate may become significantly altered under pathological conditions such as epilepsy. We have investigated whether the proliferation of granule cell progenitors is increased in adult rats in a model of temporal lobe epilepsy, by using systemic bromodeoxyuridine injections to label dividing cells. We report here for the first time that granule cell neurogenesis is increased bilaterally 1 week after a single unilateral intracerebroventricular injection of kainic acid. Bromodeoxyuridine labeled neurons increased at least 6-fold on the side ipsilateral to the kainic acid injection compared to controls, but significantly, were also increased, by at least 3-fold on the side contralateral to the injection. The dividing cells in the subgranular zone were identified as neurons since they expressed Class III beta tubulin but not glial fibrillary acidic protein.

A new protocol for the transmission of physiological signals by digital telemetry.

A method has been devised to transmit physiological signals from the brains of rats using a new digital telemetry transmission protocol. The chief advantage of the present system over existing systems is that the circuit only consumes power during the transmission of a brief pulse of information. This results in a very much extended battery life allowing the device to be implanted and recordings to be carried out over a longer period of time.

Induction of 72 kDa heat-shock protein following sub-lethal oxygen deprivation in organotypic hippocampal slice cultures.

The phenomenon of induced tolerance to a normally lethal episode of ischaemia by preconditioning with sub-lethal ischaemia has been linked to induction of the 72 kDa heat-shock protein (HSP72). However, a direct correlation between HSP72 expression and ischaemic preconditioning in vivo has not been proven. Using an in vitro model of ischaemia-related neuronal damage we have investigated whether HSP72 protein expression is temporally correlated with subsequent tolerance to a normally lethal ischaemic episode. Organotypic hippocampal slice cultures were maintained in vitro for 14 days before being exposed to hypoxia for 15-180 min. Periods of hypoxia shorter than 60 min did not produce neuronal damage. No HSP72 immunoreactivity was observed in either untreated cultures or in those exposed to hypoxia for 15 min. Following 30 and 45 min hypoxia a significant induction of HSP72 occurred in neurons of both the CA1 and CA3/4 regions of the pyramidal cell layer. A significant number of microglia were positively stained with HSP72. The peak of HSP72 expression occurred 18 h after the induction of hypoxia but remained significantly elevated for 48 h post-hypoxia. Prolonged hypoxia (60 or 180 min) produced a selective lesion of the CA1 pyramidal cell layer which was not associated with an induction of HSP72. Pre-conditioning with 45 min hypoxia 18 h prior to 180 min hypoxia did not reduce the neuronal damage associated with 180 min hypoxia alone. These data strongly suggest that HSP72 does not directly confer tolerance in this in vitro model of ischaemia-related neuronal death.

Induction of tolerance and mossy fibre neuropeptide-Y expression in the contralateral hippocampus following a unilateral intrahippocampal kainic acid injection in the rat.

We have previously reported an ectopic expression of neuropeptide-Y (NPY) immunoreactivity in mossy fibres (MFs) in the contralateral hippocampus following a unilateral intrahippocampal (IH) injection of kainic acid (KA). In the present study we report that, in addition to MF NPY expression, unilateral IH KA injections also induce tolerance towards a subsequent intracerebroventricular (ICV) contralateral KA injection, resulting in a reduction in the number of overt seizures and degree of cell loss.

Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia.

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Selective N-type calcium channel antagonist omega conotoxin MVIIA is neuroprotective against hypoxic neurodegeneration in organotypic hippocampal-slice cultures.

BACKGROUND AND PURPOSE: Neuroprotection by antagonists of both L-type and N-type calcium channels occurs in in vivo models of ischemia. The site of action of calcium channel antagonists is unclear, however, and it is likely that a combination of vascular and direct neuronal actions occurs. We have investigated the effects of blocking neuronal calcium channels using an organotypic hippocampal-slice model of ischemia. METHODS: Organotypic hippocampal-slice cultures prepared from 10-day-old rats were maintained in vitro for 14 days. Cultures were exposed to either 3 hours of oxygen deprivation (hypoxia) or 1 hour of combined oxygen and glucose deprivation (ischemia). Neuronal damage was quantified after 24 hours by propidium iodide fluorescence. RESULTS: Three hours of anoxia produced damage exclusively in CAT pyramidal cells. This damage was prevented by preincubation with omega conotoxin MVIIA, a selective N-type calcium channel blocker, and omega conotoxin MVIIC, which blocks N-type and other presynaptic neuronal calcium channels. The dihydropyridine nifedipine and the mixed calcium channel blocker SB201823-A were not protective. Furthermore, if addition of conotoxin MVIIA was delayed until after the hypoxic episode, a dose-dependent neuroprotective effect was observed, with an IC50 of 50 nmol/L. In contrast to hypoxia, none of the compounds was neuroprotective in the model of oxygen-glucose deprivation, although it was determined that extracellular calcium was essential for the generation of ischemic damage. CONCLUSIONS: These studies present clear evidence that neuroprotection by selective N-type calcium channel antagonists is mediated directly through neuronal calcium channels. In contrast, the neuroprotective effects of dihydropyridines may be mediated through vascular calcium channels or indirectly through actions in other brain regions.

Widespread ectopic neuropeptide-Y immunoreactivity in contralateral mossy fibres after a unilateral intrahippocampal kainic acid injection in the rat.

Granule cells of the dentate gyrus can express neuropeptide-Y (NPY) in several models of epilepsy involving limbic seizures, however, the nature of this ectopic expression is not well understood at present. We have studied the expression of NPY-immunoreactivity in mossy fibres contralateral to a unilateral intrahippocampal injection of kainic acid and report that ectopic mossy fibre NPY-immunoreactivity is observed throughout the contralateral hippocampus.

Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures.

We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

Pre-exposure to subtoxic levels prevents kainic acid lesions in organotypic hippocampal slice cultures: effects of kainic acid on parvalbumin-immunoreactive neurons and expression of heat shock protein 72 following the induction of tolerance.

The effects of kainic acid on the survival of principal neurons and parvalbumin-immunoreactive (PARV-IR) neurons, and on the expression of heat shock protein 72 immunoreactivity (HSP72-IR) were investigated in organotypic hippocampal slice cultures. Untreated cultures displayed an organotypic organization and the development and morphology of PARV-IR neurons in the hippocampus paralleled that reported to occur in vivo, with the exception of the hilar region of the dentate gyrus which exhibited a marked lack of PARV-IR neurons. No constitutive expression of HSP72 was found in untreated cultures. The lesion of CA3 neurons and the reduction in numbers of PARV-IR neurons in both CA3 and CA1 after chronic exposure to 5 microM kainic acid were similar to those reported to occur in vivo. Exposure to 1 microM doses of kainic acid resulted in a widespread appearance of HSP72-IR and the induction of tolerance to a previously toxic dose of kainic acid. These results suggest the presence of endogenous neuroprotective mechanisms, activated by a stress response which induces HSP72, and is reminiscent of the induced tolerance reported to occur after a mild ischaemic insult.

Aberrant Timm-stained fibres in the dentate gyrus following tetanus toxin-induced seizures in the rat.

The present study addresses whether seizures, which result from the chronic block of inhibition caused by an intrahippocampal injection of tetanus toxin, induce axonal sprouting of the hippocampal mossy fibres. Timm stain was used to identify the mossy fibre terminals. In nine of 15 animals killed at 1 month or later after an injection of tetanus toxin, Timm-stained terminals were observed bilaterally in the inner molecular layer and in seven animals a meshwork of Timm-stained fibres/ terminals was also observed bilaterally in the outer molecular layer of the fascia dentata. None of these changes were observed in any of the 12 saline-injected controls. There was no obvious correlation between the number of motor fits an animal exhibited and the amount of Timm-stained fibre sprouting present in either the inner or outer molecular layer. The Timm-stained axonal sprouting into the outer molecular layer of the fascia dentata may simply reflect the reinnervation of sites on the granule cell dendrites, previously occupied by the terminals of the hilar somatostatin-containing cells. These hilar somatostatin-containing cells which are believed to project to the outer molecular layer are known to succumb to the seizure activity in this animal model of epilepsy.

Loss of hilar somatostatin neurons following tetanus toxin-induced seizures.

A loss of inhibitory interneurons has been reported in the hippocampus following seizure activity in various animal models of epilepsy and in human epileptic tissue. The question of whether particular populations of inhibitory neurons are similarly affected by the chronic block of inhibition that results after tetanus toxin injections directly into the brain has not previously been addressed. In the present study a unilateral intrahippocampal injection of tetanus toxin into the ventral hippocampus was used to produce a chronic epileptic syndrome characterised by brief seizures that recurred intermittently for 6-8 weeks. The results reveal, for the first time, the morphological changes in somatostatin interneurons following tetanus toxin-induced seizures in the rat. A bilateral short-term increase in immunoreactivity of somatostatin neurons is present 1 week after injection. This is accompanied by an increased intensity of somatostatin-immunoreactive axon terminals in the outer molecular layer of the dentate gyrus, which is more marked on the contralateral side. A chronic and significant loss of somatostatin-immunoreactive neurons was noted in the hilus of the dentate gyrus 2 months later. The significance of the chronic loss of the hilar somatostatin neurons in the control of excitatory activity in the dentate gyrus and whether the acute morphological changes are due to a direct action of the toxin on release mechanisms or as a result of seizure activity are discussed.

Propidium iodide in vivo: an early marker of neuronal damage in rat hippocampus.

We have investigated the use of the fluorescent exclusion dye propidium iodide as a marker for acutely degenerating cells in vivo, and report here that combined injection of kainic acid and propidium iodide into the lateral cerebral ventricle results in labelling of CA3 pyramidal cells 1 and 6 h after injection. Alternate sections stained with thionin at these early times revealed little evidence of histologically detectable cell damage.