Janet R. Fraser

Synaptic Plasticity in the CA1 Area of the Hippocampus of Scrapie-Infected Mice

Using conventional in vitro extracellular field potential recordings we have investigated both short- and long-term synaptic plasticity in the hippocampal CA1 area of mice infected with ME7 scrapie. In agreement with earlier studies, no changes were seen in the properties of the Schäffer collateralevoked field excitatory postsynaptic potential during the early stages of the disease (up to 160 days, post inoculation, d.p.i) after which time the recorded potentials were seen to attenuate. Also, up to this time no changes were seen in either paired-pulse facilitation or post-tetanic potentiation, which are short-term phenomena associated with brief elevations in presynaptic calcium levels. However, there was a significant shift from the ability of slices to maintain long-term potentiation (LTP) from 100 d.p.i. onwards. In all of these experiments short-term potentiation (STP) was preserved, suggesting that from the time that abnormal PrP becomes detectable, or perhaps even earlier, the mechanisms responsible for stabilizing the maintenance phase of LTP are impaired. This result is discussed in terms of the relationship between STP and LTP and how this might be compromised by the conversion of cellular prion protein (PrPC) to the scrapie, protease resistant form of PrP (PrPSc).

Scrapie infection alters the membrane and synaptic properties of mouse hippocampal CA1 pyramidal neurones.

1. Electrophysiological recordings using conventional intracellular and extracellular techniques were made from the CA1 region of the hippocampus of ME7 scrapie‐infected mice in a brain slice preparation at specific stages during the incubation period of the disease and compared with data obtained from age‐matched control animals. 2. Extracellular field EPSP recordings in the stratum radiatum showed a gradual increase in the effective stimulus threshold and a reduction in amplitude of the response 5 months after inoculation with scrapie. Terminal animals showed a complete loss of the field EPSP. 3. Intracellular recordings from CA1 pyramidal cells of scrapie‐infected animals after 5 months showed that the Schaffer collateral‐evoked EPSP was attenuated, the effective stimulus threshold was increased and the rise time was slower in slices from scrapie‐infected mice than in age‐matched control mice. Inhibitory potentials evoked by the same stimulus also appeared weaker in scrapie‐infected mice at this time. 4. To determine if the mechanisms of transmitter release during low‐frequency stimulation of the Schaffer collaterals were altered in scrapie‐infected mice, paired‐pulse experiments were performed, but failed to show any differences between cells from scrapie‐infected and control animals. 5. Pyramidal cells from scrapie‐infected mice showed depolarized resting potentials and an increased membrane resistance compared with age‐matched control cells. 6. The majority of scrapie‐infected cells were spontaneously active, showing both single spike and bursting activity. The observed bursting activity was abolished and the spontaneous discharge rate of infected cells was markedly reduced by removing the CA3 area from the slice. 7. The action potential of cells from scrapie‐infected mice showed a faster falling phase and larger amplitude fast and medium after‐hyperpolarizations (AHPs) than age‐matched control cells. In response to depolarizing current pulses cells from infected tissue showed a loss of early spike frequency adaptation. 8. Morphological observations of biocytin‐labelled neurones confirmed our recordings were from pyramidal cells and showed that CA1 cells from scrapie‐infected mice after 5 months showed a marked loss of dendritic spines and an abnormal dendritic morphology that included the appearance of vacuolar swellings. 9. The data show that membrane and synaptic abnormalities of the CA1 pyramidal neurones develop around 5 months after intracerebral infection of the mouse hippocampus with ME7 scrapie.

Apoptosis and dendritic dysfunction precede prion protein accumulation in 87V scrapie

The sequence of events involved in the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs) is not yet known. Using a murine scrapie model in which neurodegeneration in the hippocampus is restricted to CA2, we show that pyramidal neuron damage and death by an apoptotic mechanism occur early in the incubation period, prior to the appearance of CA2 disease-specific accumulation of PrP and the onset of clinical disease. We suggest that the initial hippocampal pathological event in this model is dendritic dysfunction and activation of an apoptotic pathway rather than PrP accumulation.

Alterations in Potassium Currents May Trigger Neurodegeneration in Murine Scrapie

Conventional electrophysiological intracellular recording techniques were used to test the hypothesis that enhanced calcium entry via voltage-gated calcium channels or the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor-channel complex may be a primary pathological mechanism triggering neurodegeneration in scrapie and related diseases. This study was carried out at a time when cell loss is known to occur and when hippocampal pyramidal cells in area CA1 are rendered hyperexcitable following scrapie infection. There was no change to the NMDA receptor-mediated component of the Schäffer collateral evoked excitatory postsynaptic potential (EPSP) or the level of spontaneous firing activity of CA1 cells following addition of the specific NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV, 20 microM), to the perfusate in scrapie-infected mice, indicating that the NMDA receptor-channel complex is not compromised by scrapie. There was also no change seen in the non-NMDA mediated component of the EPSP. The calcium spike of CA1 pyramidal cells was not significantly altered by scrapie infection, indicating that high threshold voltage-gated Ca2+ channel function is not compromised by scrapie. By contrast, cells from scrapie-infected mice fired calcium spikes repetitively and the long, slow AHP, which in control cells inhibited repetitive firing, was absent. Cells from scrapie-infected mice showed more depolarized membrane potentials than controls but this difference in potential was no longer observed after exposure to TEA. These data indicate a loss of TEA-insensitive and TEA-sensitive potassium conductances. We suggest that altered potassium currents rather than increased calcium entry via voltage-sensitive calcium channels or the NMDA receptor complex may be the primary pathological mechanism triggering neurodegeneration in scrapie and related diseases.

Activation of Fas and caspase 3 precedes PrP accumulation in 87V scrapie.

The sequence of events involved in the neurodegeneration caused by transmissible spongiform encephalopathies is not yet known. Using a murine scrapie model in which neurodegeneration in the hippocampus is restricted to the CA2, we show an up-regulation of the proapoptotic markers Fas and caspase 3 early in the incubation period prior to disease-specific prion protein (PrP) deposition and clinical signs. These results suggest that activation of Fas and caspase 3 are involved in the early pathological sequence of events during murine scrapie, and that these proapoptotic markers may be a specific method for early detection of neurodegeneration.

Immunolocalization of the prion protein in scrapie affected rodent retinas

Some mouse and hamster scrapie models are known to replicate infectivity in the retina, and this can be associated with photoreceptor atrophy. We used immuno-labelling to identify the cellular localization of the prion protein (PrP) in the retina and correlated this with infectivity titre. It is only with the 263K scrapie strain in hamsters that disease-associated PrP (PrP(Sc)) staining was always easily detectable. Very little PrP(Sc) immunolabelling was observed in any of the mouse models, even in those demonstrating scrapie-induced retinopathy in which high titres of infective agent are known to occur in the retina.

Scrapie-induced neuron loss is reduced by treatment with basic fibroblast growth factor.

Neuron loss can be a prominent feature of the pathology of transmissible spongiform encephalopathies (TSEs); recent evidence indicates that this loss occurs through apoptosis. Growth factor treatment of other neurodegenerative diseases has been shown to protect neurons destined for apoptosis, and several types of experimental retinopathy have been successfully treated with basic fibroblast growth factor (bFGF). In a murine scrapie model which develops a severe loss of photoreceptors, we administered a single intravitreal injection of bFGF four-fifths of the way through the disease process; this doubled the number of photoreceptors surviving for up to 5 weeks, i.e. to the terminal stages of the disease. This is the first time that a potential late-stage therapy for the TSEs has been demonstrated.

The transmissible spongiform encephalopathies: pathogenic mechanisms and strategies for therapeutic intervention

Primary neurodegenerative diseases tend to be intractable and largely affect the elderly. There is rarely the opportunity to identify individuals at risk and the appearance of clinical symptoms usually signifies the occurrence of irreversible neurological damage. This situation describes sporadic Creutzfeldt-Jakob disease which occurs world-wide, affecting one person per million per annum. The epidemic of bovine spongiform encephalopathy in the UK in the 1980s and the subsequent causal appearance of variant Creutzfeldt-Jakob disease in young UK residents in the 1990s has refocused attention on this whole group of diseases, known as the transmissible spongiform encephalopathies or prion diseases. The potentially lengthy incubation period of variant Creutzfeldt-Jakob disease, including perhaps an obligate peripheral phase, prior to neuroinvasion, marks variant Creutzfeldt-Jakob disease out as different from sporadic Creutzfeldt-Jakob disease. The formal possibility of detecting individuals infected with the bovine spongiform encephalopathy agent during this asymptomatic peripheral phase provides a strong incentive for the development of therapies for transmissible spongiform encephalopathies. This review focuses on recent advances in the understanding of the pathogenesis of these diseases, with particular reference to in vitro and animal model systems. Such systems have proved invaluable in the identification of potential therapeutic strategies that either specifically target the prion protein or more generally target peripheral pathogenesis. Furthermore, recent experiments in animal models suggest that even after neuroinvasion there may be pharmacological avenues to explore that might retard or even halt the degenerative process.

Electrophysiological properties of dorsal lateral geniculate neurons in brain slices from ME7 scrapie-infected mice

Electrophysiological recordings using conventional intracellular techniques were obtained from dorsal lateral geniculate nucleus (dLGN) neurons in brain slices from ME7 scrapie-infected mice at specific time points throughout the incubation period of the disease. Comparisons were made with age-matched control mice. A number of dLGN neurons from control and scrapie-infected mice were injected with biocytin in order to examine their cellular morphology. Mice were infected with ME7 scrapie by an intraocular route and the mean (+/- SEM) incubation period of the disease was 276 +/- 3.5 days. Our results indicate that there were no differences in the electrophysiological or morphological parameters of neurons recorded in ME7 scrapie-infected and age-matched control mice at any stage of the disease up to 240 days postinoculation. After this time, however, no detectable electrical activity was recorded in the dLGN. This study demonstrates that in the ME7 scrapie-infected dLGN, relay neurons with normal physiological and morphological properties are present even at an advanced stage of the disease at a time when the dLGN is known to be subject to marked pathological changes and a profound neuronal loss.

Microdissection: A method developed to investigate mechanisms involved in transmissible spongiform encephalopathy pathogenesis

BackgroundThe transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting both human and animals. The neuroanatomical changes which occur in the central nervous system (CNS) of TSE infected animals include vacuolation, gliosis, neuronal loss and the deposition of a disease specific protein, PrPSc. Experimental murine models of scrapie, a TSE of sheep, have revealed that pathology may be confined to specific brain areas with targeting of particular neuronal subsets depending on route of injection and scrapie isolate.To assess the biochemical changes which are taking place in these targeted areas it was necessary to develop a reliable sampling procedure (microdissection) which could be used for a variety of tests such as western blotting and magnetic resonance spectroscopy.MethodsThe method described is for the microdissection of murine brains. To assess the usefulness of this dissection technique for producing similar sample types for analysis by various down-stream biochemical techniques, the areas dissected were analysed for PrPSc by western blotting and compared to immunocytochemical (ICC) techniques.ResultsResults show that the method generates samples yielding a consistent protein content which can be analysed for PrPSc. The areas in which PrPSc is found by western blotting compares well with localisation visualised by immunocytochemistry.ConclusionThe microdisssection method described can be used to generate samples suitable for a range of biochemical techniques. Using these samples a range of assays can be carried out which will help to elucidate the molecular and cellular mechanisms underlying TSE pathogenesis. The method would also be useful for any study requiring the investigation of discrete areas within the murine brain.