Early Hippocampal Synaptic Loss Precedes Neuronal Loss and Associates with Early Behavioural Deficits in Three Distinct Strains of Prion Disease.

Prion diseases are fatal neurodegenerative diseases of the CNS that are associated with the accumulation of misfolded cellular prion protein. There are several different strains of prion disease defined by different patterns of tissue vacuolation in the brain and disease time course, but features of neurodegeneration in these strains have not been extensively studied. Our previous studies using the prion strains ME7, 79A and 22L showed that infected mice developed behavioural deficits in the same sequence and temporal pattern despite divergent end-stage neuropathology. Here the objective was to address the hypothesis that synaptic loss would occur early in the disease in all three strains, would precede neuronal death and would be associated with the early behavioural deficits. C57BL/6 mice inoculated with ME7, 79A, or 22L-infected brain homogenates were behaviourally assessed on species typical behaviours previously shown to change during progression and euthanised when all three strains showed statistically significant impairment on these tasks. A decrease in labelling with the presynaptic marker synaptophysin was observed in the stratum radiatum of the hippocampus in all three strains, when compared to control animals. Negligible cell death was seen by TUNEL at this time point. Astrocyte and microglial activation and protease resistant prion protein (PrP(Sc)) deposition were assessed in multiple brain regions and showed some strain specificity but also strongly overlapping patterns. This study shows that despite distinct pathology, multiple strains lead to early synaptic degeneration in the hippocampus, associated with similar behavioural deficits and supports the idea that the initiation of synaptic loss is a primary target of the misfolded prion agent.

Neurotrophin-induced preprotachykinin-A gene promoter modulation in organotypic rat spinal cord culture.

To study regulation of the preprotachykinin-A gene promoter, we utilised a biolistic gene transfer protocol to deliver a DNA construct that incorporates a portion of the preprotachykinin-A gene promoter and an enhanced green fluorescent protein reporter gene into neonatal rat spinal cord organotypic slices. The ability of the neurokinin-1 receptor agonist [Sar9,Met(O2)11]-substance P, nerve growth factor and brain derived neurotrophic factor to modulate positively preprotachykinin-A gene promoter construct activity, as indicated by de novo enhanced green fluorescent protein expression, was determined. Treatment of organotypic slices with [Sar9, Met(O2)11]-substance P (10 microm, P < 0.05), nerve growth factor (200 ng/mL, P < 0.001) or brain derived neurotrophic factor (200 ng/mL, P < 0.02) significantly increased the proportion of cytomegaloviral promoter-DsRed transfected cells (used to visualise total transfected cells) that co-expressed enhanced green fluorescent protein. The distribution of enhanced green fluorescent protein/DsRed-positive neurones across spinal laminae was broadly in line with the known distribution of spinal Trk and neurokinin-1 receptors. These data suggest a modulated activity of the preprotachykinin-A gene promoter in spinal neurones in vitro by substance P and/or neurotrophins. The functional consequences of such transcriptional changes within central peptidergic circuitry and their relevance to chronic pain are considered.

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.

The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and beta-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K(+) (10 microM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar(9),Met (O(2))(11)]-substance P (10 microM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.