1. Lolitrem B Intoxication Activates Neuronal Stress Pathways
M.D Combs, G.E Rogers, A.S Hamlin, & J.C Quinn
Results: Anatomy
Administration of Lolitrem B (2mg/kg, IP) up-regulated c-Fos in neuronal stress pathways, including the paraventricular hypothalamus, central amygdala, nucleus tractus solitarius and ventrolateral medulla. No c-Fos was detected within the cerebellum or the olivary nucleus.
Conclusion
Lolitrem B induced a pattern of c-Fos immunoreactivity that is consistent with previous clinical and experimental observations of anxiety, allodynia and hyperaesthesia seen with intoxication of grazing animals . This is this first time activation of these pathways has been directly associated with a BK channel blocker.
Mass drowning is a feature of the clinical syndrome perennial ryegrass toxicosis (PRGT) in Australia. These findings suggest lolitrem B activation of neuronal stress pathways may alter normal herding behaviours and play a significant aetiological role in mass drowning. Anxiety and decreased voluntary movement are likely to alter normal feeding/drinking behaviour thereby increasing physiological stressors. Millions of animals suffer PRGT each year making these findings of critical animal welfare significance.
Introduction Lolitrem B is an indole–diterpenoid alkaloid toxin found primarily in perennial ryegrass and is a potent inhibitor of BK channels. Cerebellar ataxia, tremor and Purkinje cell lesions have been extensively reported in grazing livestock however it has been suggested that a more widespread syndrome exists including possible anxiety, hyperaesthesia, allodynia and cognitive dysfunction. This study used c-Fos up-regulation to determine the level of activation in neuronal stress pathways following acute lolitrem B exposure in the mouse. It was hypothesised that lolitrem B intoxication would activate neural pathways associated with stress, pain and hyperaesthesia in addition to prolonged induction of a tremor and cerebellar ataxia.
Methods
8 adult mice were injected with lolitrem B (2mg/kg, i.p.) (n=4) or vehicle (90 % DMSO) (n=4). Tremor analysis was performed 1 hour post injection using a pressure sensor and PowerLab™( ADI Instruments Pty.) analyser. At 3 hours post injection mice were anaesthetised and transcardially perfused with 4% paraformaldehyde. Brains were sectioned at 40μm and c-Fos immunoreactivity was revealed using the avidin-biotin-horseradish peroxidase technique (1:5000, Santa Cruz Biotechnologies, TX, USA). An additional cohort of mice was tested to characterise the period and severity of induced tremor. Tremor was analysed by selecting one minute epochs for Fast Fourier Transform (FFT) analysis. The ratio of power output at likely tremor frequencies (9-20Hz) to total power output (0-50Hz) was used to assess severity of tremor.
Results
Analysis confirmed a tremor in the range of 10-19Hz. Lolitrem B induced a tremor of prolonged duration. Induction of tremor is also accompanied by a marked decrease in voluntary movement.
Blind unilateral counts of c-Fos-positive neurons revealed a significant up-regulation of c-Fos in the paraventricular hypothalamus, central amygdala, nucleus tractus solitarius and ventrolateral medulla following i.p. injection of 2mg/ml lolitrem B. Bars show the total Mean (±SEM) of c-Fos-positive neurons counted across the rostro-caudal extent of each nucleus (*p<0.05).
Photomicrographs showing c-Fos-positive labelling in the paraventricular hypothalamus (PVN), central amygdala (CeA), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM), parabrachial nucleus (PB) and locus coeruleus (LC). 2mg/kg i.p lolitrem B dramatically increased the number of c-fos-positive nuclei in the PVN, CeA, NTS and VLM. Scale bar = 200mm. * *
Tremor analysis: FFT frequency analysis from a mouse prior to (A) and 6 hours post lolitrem B injection 2mg/kg i.p.(B). Low frequency (0-7Hz) power output represents primarily movement. This is markedly decreased in the intoxicated animal whereas there is a distinct peak in a tremor around 13Hz. (C) Tremor-Motion power ratio reveals tremor accounting for an increasing proportion of movement even at 6 hours. (D) Tremor-Motion power ratio at one hour post in c-Fos tested mice (*p<0.05, lolitrem B (2mg/kg i.p. vs vehicle). A B C D