Replication Of Scrapie Agent Research Articles

Reduction of Prion Infectivity and Levels of Scrapie Prion Protein By Lithium Aluminum Hydride: Implications for RNA in Prion Diseases

Previous studies indicate that RNA may be required for proteinase-resistant prion protein (PrP) amplification and for infectious prion formation in vitro, suggesting that RNA molecules may function as cellular cofactors for abnormal PrP (PrPSc) formation and become part of the structure of the infectious agent. To address this question, we used chemicals that can cleave phosphodiester bonds of RNA and assessed their effects on the infectious agent. Lithium aluminum hydride, a reducing agent that can induce reductive cleavage of oxidized molecules such as carbonyls, carboxyl acids, esters, and phosphodiester bonds, did not affect cellular PrP degradation; however, it destroyed PrPSc, extended the scrapie incubation period, and markedly reduced total RNA concentrations. These results prompted us to investigate whether RNA molecules are cofactors for PrPSc propagation. RNase A treatment of partially purified PrP and of 263K scrapie brain homogenates was sufficient to increase the sensitivity of PrPSc to proteinase K degradation. This is the first evidence that suggests that RNA molecules are a component of PrPSc. Treatment with RNase A alone and PrP degradation by RNase A plus proteinase K in vitro, however, did not result in loss of scrapie infectivity compared with the effects of lithium aluminum hydride. Together, these data suggest that RNA molecules may be important for maintaining the structure of PrPSc and that oxidized molecules can be important in scrapie agent replication and prion infectivity.

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.

A Tailoring Activity Is Responsible for Generating Polyene Amide Derivatives in Streptomyces diastaticus var. 108

We recently characterized rimocidin B (3b) and CE-108B (4b) as two polyene amides with improved pharmacological properties, produced by genetically modified Streptomyces diastaticus var. 108. In this work, genetic and biochemical analysis of the producer strain show that the two amides are derived from the parental polyenes rimocidin (3a) and CE-108 (4a) by a post-PKS modification of the free side chain carboxylic acid. This modification is mediated by an amidotransferase activity operating after the biosynthesis of rimocidin (3a) and CE-108 (4a) are completed. Two polyenes, intermediates of the biosynthetic pathway of rimocidin (3a) and CE-108 (4a), were also isolated and shown to have some improved pharmacological properties compared with the final products.

In-situ analysis of cellular poly(ADP-ribose) production in scrapie-infected mouse neuroblastoma cells.

Transmissible spongiform encephalopathies (TSEs), also called prion diseases, are characterized by formation of the disease-associated isoform of prion protein (PrP(Sc)), which arises from a normal isoform termed PrP(c) by a post-translational conversion process occurring in an autocatalytic fashion. Oxidative stress has been proposed as a pathogenetic mechanism in TSEs and increased lipid peroxidation has recently been described in prion-infected cell cultures, suggesting an intrinsic link between the presence of prions and oxidative stress. We investigated if poly(ADP-ribose) formation can be detected in cultured cells upon prion infection, as this NAD+-consuming and DNA strand break-activated nuclear enzymatic reaction has the potential to cause rapid and lethal NAD+ depletion in cells under severe oxidative stress. Poly(ADP-ribose) production was analysed by immunofluorescence in freshly scrapie-infected Neuro2a-D11 mouse neuroblastoma cells, which had been confirmed by immunocytochemistry to produce PrP(Sc), and in uninfected controls. No spontaneous poly(ADP-ribose) specific signals were observed in infected or in uninfected cells, while both cell types readily reacted to H2O2 treatment with poly(ADP-ribose) synthesis in a dose-dependent manner, with no obvious difference in staining intensity at any dose tested. In summary, our data reveal that replication of scrapie agent in neuroblastoma cells can proceed without detectable stimulation of the cellular poly(ADP-ribosyl)ation system.

Role of spleen macrophages in the clearance of scrapie agent early in pathogenesis.

The involvement of spleen macrophages in the early stages of scrapie pathogenesis was studied by applying the 'macrophage-suicide technique' to scrapie-infected mice. This method comprises critically the intravenous administration to mice of dichloromethylene disphosphonate encapsulated into liposomes. Depletion of spleen macrophages before scrapie infection induced an increased amount of scrapie inoculum in the spleen, consequently leading to accelerated scrapie agent replication in the early phase of pathogenesis, as followed by PrPres accumulation, a specific hallmark of scrapie. The same effect was observed when spleen macrophages were depleted just before the beginning of scrapie agent replication. These findings suggest that macrophages may partly control scrapie infection in peripheral tissues by sequestration of the scrapie inoculum and may thus impair early scrapie agent replication in the spleen. In addition to macrophages, most follicular dendritic cells and B lymphocytes, which are thought to support scrapie agent replication, were also transiently depleted by dichloromethylene disphosphonate administration. This suggests that a compensatory mechanism is sufficient to ensure the persistence of infection in these early stages of pathogenesis.

Inhibition of prion-protein conversion: a therapeutic tool?

These results open the door to more physiological models of a prophylactic or therapeutic use of blocking peptides. A diverse range of compounds has been investigated for experimental TSE prophylaxis or therapy13xCongo red prolongs the incubation period in scrapie-infected hamsters. Ingrosso, L. et al. J. Virol. 1995; 69: 506–508PubMedSee all References, 14xProphylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. Farquhar, C. et al. Lancet. 1999; 353: 117Abstract | Full Text | Full Text PDF | PubMed | Scopus (90)See all References, 15xProlongation of scrapie incubation period by an injection of dextran sulphate 500 within the first month before or after injection. Farquhar, C. et al. J. Gen. Virol. 1986; 67: 463–473Crossref | PubMedSee all References, 16xAmphotericin B delays both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. McKenzie, D. et al. J. Virol. 1994; 68: 7534–7536PubMedSee all References, 17xMS-8209, a water soluble amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. Adjou, K.T. et al. J. Gen. Virol. 1999; 80: 1079–1085PubMedSee all References, 18xEffectiveness of anthracycline against experimental prion disease in Syrian hamsters. Tagliavini, F. et al. Science. 1997; 276: 1119–1122Crossref | PubMed | Scopus (149)See all References, 19xScrapie-induced neuronal loss is reduced by treatment with basic fibroblast growth factor. Fraser, J.R. et al. Neuroreport. 1997; 8: 2405–2409Crossref | PubMedSee all References (Table 1Table 1). Although most appear to influence the incubation period in experimental models, some (including pentosan polysulphate and MS-8209) have been shown to inhibit TSE infection when administered prophylactically under certain experimental conditions. A single study in which basic fibroblast growth factor was administered towards the end of the incubation period following intraocular injection of scrapie found enhanced survival of retinal photoreceptors, suggesting a potential late-stage therapeutic effect. These compounds are under further evaluation, but the conceptually elegant approach of Chesebro and colleagues10xSpecific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. Chabry, J. et al. J. Biol. Chem. 1998; 273: 13203–13207Crossref | PubMed | Scopus (133)See all References, 11xSpecies-independent inhibition of prion protein (PrP) formation by a peptide containing conserved PrP sequence. Chabry, J. et al. J. Virol. 1999; 73: 6245–6250PubMedSee all References also merits further study in vitro, and perhaps eventually in vivo. Given the probable widespread exposure of the UK human population to the BSE agent (which, in some cases, has resulted in variant CJD after an unknown but presumably lengthy incubation period), any compound which offers prophylactic potential should be rigorously explored. However, the logistics and ethics of implementing such a regime, even if of proven efficacy, will be formidable.Table 1Compounds which have a demonstrable prophylactic or potential therapeutic effect in experimental TSEaaCompoundExampleRef.Anionic dyes (amyloid stain)Congo red13xCongo red prolongs the incubation period in scrapie-infected hamsters. Ingrosso, L. et al. J. Virol. 1995; 69: 506–508PubMedSee all References13Sulphated glycansPentosan polysulphate14xProphylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. Farquhar, C. et al. Lancet. 1999; 353: 117Abstract | Full Text | Full Text PDF | PubMed | Scopus (90)See all References14Dextran sulphate 50015xProlongation of scrapie incubation period by an injection of dextran sulphate 500 within the first month before or after injection. Farquhar, C. et al. J. Gen. Virol. 1986; 67: 463–473Crossref | PubMedSee all References15Polyene antibioticsAmphotericin B16xAmphotericin B delays both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. McKenzie, D. et al. J. Virol. 1994; 68: 7534–7536PubMedSee all References16MS-820917xMS-8209, a water soluble amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. Adjou, K.T. et al. J. Gen. Virol. 1999; 80: 1079–1085PubMedSee all References17Anthracycline4-iodo-4-deoxy-doxorubicin18xEffectiveness of anthracycline against experimental prion disease in Syrian hamsters. Tagliavini, F. et al. Science. 1997; 276: 1119–1122Crossref | PubMed | Scopus (149)See all References18Growth factorsBasic fibroblast growth factor19xScrapie-induced neuronal loss is reduced by treatment with basic fibroblast growth factor. Fraser, J.R. et al. Neuroreport. 1997; 8: 2405–2409Crossref | PubMedSee all References19aAbbreviation: TSE, transmissible spongiform encephalopathy.

MS-8209, a water-soluble amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie.

Amphotericin B (AmB) has been shown to delay hamster scrapie. Infectivity studies have been performed previously using AmB in order to understand the relationship between the accumulation of an abnormal isoform (PrPres) of the prion protein and 263K scrapie agent replication in the brain. The first study reported that AmB had no effect upon agent replication, although it delayed the development of both clinical signs and PrPres accumulation. However, subsequent experiments using the same model showed a significant effect both on agent replication and PrPres accumulation early in infection. This fundamental discrepancy was assumed to be linked to differences in experimental protocols. In order to unravel the issue, a new experiment has been performed encompassing different protocols and using an AmB derivative, MS-8209, that can be used at higher doses because of its lower toxicity. The findings of this study exclude the suspected differences in the protocols as the reason for previous conflicting results, and suggest strongly that these discrepancies were due to a low dose of AmB causing a 'threshold effect'. Overall, this study indicates that, in this model, PrPres cannot be dissociated from infectivity by polyene antibiotics.

Amphotericin B delays both scrapie agent replication and PrP-res accumulation early in infection

Amphotericin B delays the onset of clinical symptoms in hamsters infected with scrapie agent strain 263K. Here we show that accumulation of a scrapie-specific isoform of the prion protein (PrP-res) and agent replication were delayed early in amphotericin B-treated animals. By 8 weeks postinfection, only untreated animals exhibited clinical symptoms of scrapie infection whereas PrP-res levels and titers were similar in treated and untreated animals. This suggests that although PrP-res accumulation and agent replication are linked, they are not the sole factors required for the onset of clinical disease.

SCID mouse spleen does not support scrapie agent replication

BALB/c and severe combined immunodeficiency (SCID) mice were inoculated intracerebrally or intraperitoneally with scrapie agent strain ME7 to examine the role of functional lymphocytes and follicular dendritic cells in splenic infectivity and PrPSc accumulation. Intracerebrally inoculated BALB/c and SCID mice developed the clinical signs and microscopic lesions characteristic of scrapie. Spleens from terminally affected BALB/c mice contained PrPSc which was detectable by immunoblot analysis; SCID mouse spleens did not contain detectable PrPSc. SCID mouse spleens collected during the first 90 days after intraperitoneal infection contained neither infectivity nor PrPSc.

Scrapie strain infection in vitro induces changes in neuronal cells.

PC12 cells, in the presence of nerve growth factor (NGF), support replication of the mouse-derived scrapie strains 139A and ME7, with the former yielding 100-1000-fold higher levels of infectivity. Infectivity remained cell-associated and cells did not show any gross morphological alterations, although changes were observed by electron microscopy in the form of an increased number of lipid droplets in 139A-infected cultures. Analysis of phospholipid metabolism in 139A infected cells indicated that scrapie replication did not change the inositol phosphate levels, but did stimulate phosphoinositide synthesis. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Since scrapie-infected cultures did not exhibit cell death or any gross changes, any scrapie-induced effects would probably be manifested in nonvital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, as well as the GABA synthetic pathway; however, the adrenergic pathway was unaffected by scrapie infection. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. No neurotransmitter-related enzymatic changes were detected in 263K- or 139R-infected PC12 cells. The enzymatic changes observed in ME7-infected PC12 cells and in Chandler agent-infected mouse neuroblastoma cells suggest that the significant changes in neurotransmitter levels in cultures exhibiting low infectivity titers must involve factors other than, but not excluding, replication of the agent. The role of additional factors is also suggested in studies of protein kinase C activity in 139A- and 139R-infected PC12 cells. These studies emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and, in addition, support the concept of variation among scrapie strains.

Scrapie-associated PrP accumulation and agent replication: effects of sulphated glycosaminoglycan analogues.

An abnormally protease-resistant and apparently neuropathogenic form of PrP accumulates in the brains of hosts with scrapie and related transmissible spongiform encephalopathies. Studies with scrapie-infected neuroblastoma cells have highlighted dramatic differences in the metabolism of the normal (protease-sensitive) and scrapie-associated (protease-resistant) isoforms of PrP. Furthermore, this model has been useful in identifying inhibitors of protease-resistant PrP accumulation and scrapie agent replication which are valuable as potential therapeutic agents and as probes of the mechanism of protease-resistant PrP formation. These inhibitors include the amyloid stain Congo red and certain sulphated glycans which are glycosaminoglycans themselves or glycosaminoglycan analogues. The relative potencies of various sulphated glycans correlate with their previously determined anti-scrapie activities in vivo, suggesting that the prophylactic effects of sulphated polyanions is due to inhibition of protease-resistant PrP accumulation. These and other observations suggest that an interaction of PrP with endogenous sulphated glycosaminoglycans or proteoglycans is important in protease-resistant PrP accumulation, and raise the possibility that therapies for transmissible spongiform encephalopathies and other amyloidoses could be based on blocking (pre)amyloid-glycosaminoglycan interactions.

Protease-resistant PrP accumulation and scrapie agent replication: a role for sulphated glycosaminoglycans?

Protease-resistant PrP accumulation and scrapie agent replication: a role for sulphated glycosaminoglycans?

Alterations in neurotransmitter-related enzyme activity in scrapie-infected PC12 cells

Enzyme activities associated with the neurotransmitter pathways in nerve growth factor-treated, 139A scrapie strain-infected PC12 cells were examined. Since these cells show no morphological alterations during the time of agent replication, any scrapie-induced effects would have to be associated with non-vital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, choline acetyltransferase and acetylcholinesterase. However, the adrenergic pathway was unaffected by scrapie infection as evidenced by unaltered tyrosine hydroxylase activity, the putative rate-limiting enzyme in the synthesis of catecholamines. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. Furthermore, the altered enzymic activities observed were not the result of contaminating material in the scrapie brain homogenate because similar results were obtained when partially purified scrapie preparations were used as the inoculum. These scrapie agent-induced alterations in specific neuronal properties suggest a mechanism for the clinical manifestations observed in scrapie and perhaps other related central nervous system disorders.

Further characterization of scrapie replication in PC12 cells

The rat pheochromocytoma cell line, PC12, undergoes neuron-like morphological, biochemical and electrophysiological differentiation, in the presence of low concentrations of nerve growth factor (NGF). NGF-treated PC12 cells have been shown previously to support 139A scrapie agent replication. In the present report we extended these findings and analysed the cellular conditions necessary for agent replication. Following the infection of differentiated PC12 cells, scrapie replicated to relatively high titres as determined by an incubation period assay. The removal of NGF, which causes the gradual dedifferentiation of PC12 cells, resulted in the inability of scrapie to replicate. The scrapie infectivity detected in PC12 cultures is cell-associated and not released into the medium. Cells in infected cultures did not show any change in morphology when compared to cells in mock-infected cultures. Titration studies of scrapie infectivity in PC12 cells have indicated that up to 4 LD50 units per cell can be obtained although a yield of 1 LD50 per cell was more common. Using an approximate m.o.i. of 1, only differentiated PC12 cells supported 139A scrapie agent replication when compared to two other differentiated, neuronal cell types, indicating that PC12 cells are more susceptible to agent replication. These studies support further the suitability of using differentiated PC12 cells as an in vitro model to study scrapie agent replication.

Resistance of Scrapie Infectivity to Steam Autoclaving after Formaldehyde Fixation and Limited Survival after Ashing at 360 C: Practical and Theoretical Implications

Scrapie-infected hamster brains and their extracted amyloid fibrils were subjected to formaldehyde and steam autoclaving, alone or in combination. Treatment with formaldehyde before autoclaving stabilized infectivity, whereas treatment after autoclaving was either inactive or further reduced infectivity. In additional experiments on specimens (not treated with formaldehyde) that were subjected to dry heat, a small amount of infectivity still survived a 1-h exposure to temperatures as high as 360 degrees C. These results are consistent with the operation of a comparatively primitive molecular mechanism for the initiation of scrapie agent replication (perhaps an inorganic crystal nucleation step) and the subsequent participation of an organic macromolecule (scrapie amyloid protein) that is susceptible to intramolecular cross-stabilization by formaldehyde.

Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis.

Following intracerebral infection of hamsters with scrapie agent replication started with or without a very short lag phase. Infectivity titres increased exponentially within 35 to 40 days post-infection to a maximum level of 3 x 10(9) LD50 per brain and then remained constant until death. Minimal detectable amounts of scrapie-associated fibrils (SAF) appeared at 42 days and reached high levels 56 days after inoculation. The first clinical symptoms were diagnosed at about 65 days and animals died after 85 to 95 days. These data confirm earlier results in which peripheral infection first revealed agent replication, then SAF formation and finally clinical disease. Unconventional virus diseases, therefore, can best be described as virus-induced, organ-specific amyloidoses.

Prolongation of Scrapie Incubation Period by an Injection of Dextran Sulphate 500 within the Month Before or After Infection

A single intraperitoneal injection of 250 micrograms dextran sulphate 500 (DS500) reduced the susceptibility of mice to scrapie given by the same route. A lower dose (25 micrograms) was less effective but still produced significant incubation period lengthening, while a high dose (2.5 mg) further increased the degree of prolongation. This reduced susceptibility occurred with DS500 administered up to at least 4 weeks prior to intraperitoneal scrapie inoculation and up to at least 2 weeks after scrapie inoculation. A reduced average effect, but more variable between mice, was obtained with DS500 given 1 month or 2 months after scrapie. The effective scrapie titre was reduced by 90% when DS500 was injected either 72 h before or 7 h after ME7 scrapie. Using a relatively lower but normally still fatal dose of the 22A strain of scrapie approximately 50% of the treated mice survived. The effective 90% loss of titre was consistent with either of these strains of scrapie in 11 different inbred strains of mice (BALB/c, BSC, BRVR, C3H, C57BL, IM, LM, MM, RIII, VL and VM). No significant increase in the prolongation effect was obtained using multiple DS500 doses in two different time combinations. DS500 causes long-term interference in both the early processing and the replication of scrapie agent, unlike those immunomodulators which increase susceptibility.

Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly.

After intracerebral (i.c.) infection of hamsters, the 263K strain of scrapie replicated at a nearly constant exponential rate until clinical disease developed when titres in brain averaged 9.8 log10 LD50 i.c. units/g. After intraperitoneal infection, scrapie replication was first detected in spleen, then in thoracic spinal cord and finally in lumbar cord and brain. This pattern suggests that invasion of the central nervous system occurs by spread of infection along certain visceral autonomic nerves. Infectivity was detected in the thoracic cord only 3 to 4 weeks after infection (incubation period 16 weeks) indicating the exceptional neuroinvasiveness of this scrapie model. This observation and the failure of splenectomy to lengthen incubation period raises the possibility of direct infection of nerve tissue in the peritoneum and transport to the thoracic cord with minimal prior replication of scrapie agent extraneurally. After intraocular infection of the right eye, replication (or accumulation) of scrapie was detected in the right optic nerve and left superior colliculus, then in the right superior colliculus and finally in the left optic nerve and medulla. This pattern shows that scrapie infection can spread along nerves, possibly by intra-axonal transport. The duration of agent replication in brain (between detectable onset of replication and clinical disease) was shortest after intraperitoneal infection (51 to 58 days), longer after intracerebral infection (81 to 88 days) and longest after intraocular infection (greater than 121 days). These differences may reflect the relative efficiency of the neural pathways by which infectivity spreads from different sites of entry in the brain to the postulated 'clinical target areas'.

Agent replication dynamics in a long incubation period model of mouse scrapie.

The dynamics of scrapie agent replication in brain and spleen following intracerebral or intraperitoneal infection were investigated in a model with particularly long incubation periods [IM mice (Sincp7) infected with 87V scrapie]. In contrast to other mouse scrapie models previously investigated, there was no delay in onset of replication ('zero phase') in spleen using either route of infection, For both routes infectivity levels reached a plateau in spleen, which was maintained for at least 250 days in intraperitoneally infected mice. An infectivity plateau was also apparent in brain following intracerebral infection, suggesting that there may be constraints on agent replication in brain during the later part of the incubation period. In addition, the efficiency of the intraperitoneal route to produce disease was found to be particularly low for this model.

Changes in the nigrostriatal system following microinjection of an unconventional agent

The effect of apomorphine, a direct dopamine receptor agonist, was studied in golden hamsters after unilateral injection of scrapie agent (strain 263K) into the nigrostriatal system. Twenty-five recieved an injection into the left striatum and 24 into the left substantia nigra. Varying volumes, (5, 1 and 0.5 μl) and dilutions of the homogenate (10 −2, 10 −3, 10 −4) were used. The duration of the incubation period (delay of appearance of clinical signs of the disease) was dependent on the dose of the agent used: it varied within a range of72 ± 5days(5 μl10 −4and123 ± 8days(0.5 μl10 −4). Apomorphine-induced circling behavior appeared after an average of84 ± 15 days post-injection and this delay was closely related to the dose of agent. The direction of the rotation depended on the site of injection: most of the animals circled towards the inoculated side after striatal injection and away from it after inoculation in the substantia nigra. In the group of animals inoculated in the substantia nigra with 0.5 × 10 −4 μl, 6 presented the clinical phase of the disease and showed circling behavior but no clinical signs when they were killed 200 days post-injection. However, neuropathological studies revealed characteristic foci of vacuolation in the brain stem. Apomorphine-induced circling behavior can only be contained by replication of scrapie agent into the nigrostriatal neurons and the subsequent imbalance between the two sides which it reveals. Circling behavior and clinical signs of the disease can be temporally dissociated: low doses of agent slightly extended the circling behavior latency, whereas the incubation period was considerably increased. Circling behavior and neuropathological lesions in the nigrostriatal system were not closely related.