The University of Southampton
University of Southampton Institutional Repository

Using Hi-Spots to investigate in vitro network dysfunction in Cysteine String Protein ? knockout mice

Using Hi-Spots to investigate in vitro network dysfunction in Cysteine String Protein ? knockout mice
Using Hi-Spots to investigate in vitro network dysfunction in Cysteine String Protein ? knockout mice
Hi-Spot are highly re-aggregated neural cultures grown on PTFE membrane at the liquidair interface. The Hi-Spot protocol was developed by scientists at Capsant Neurotechnologies using embryonic neural tissue. We characterised Hi-Spots made using existing protocols to confirm they represented a maturing neural network with molecular, cellular and functional signatures. We have additionally modified existing protocols to allow use of postnatal tissue as the source for dissociation and re-aggregation. Hi-Spots made from postnatal day zero (P0) rats self organised into a complex 3D tissue-like structure containing anatomically synaptically-interconnected neurons, astroglia and microglia. This CNS analogue of brain tissue provides for the emergence of a co-ordinated excitatory and inhibitory network, demonstrating a maturing pattern of activity involving single spikes developing into bursting behaviour driven by intrinsic synaptic activity. This activity can be represented as frequency or averaged amplitude (RMS), increases with time in culture and is blocked by glutamate receptor antagonists and stimulated by the inhibitory receptor antagonist bicuculline. The validity of its more in vivo-like organization, not observed in more conventional dissociated cultures, is evidenced by a glutamate toxicity resistance in the Hi-Spot cultures. Further, the thesis describes the optimisations to the Hi-Spot protocol to allow viable high density re-aggregated cultures to be made from individual rather than pooled brain dissociations. Modifications to the protocol included dissociation of the mouse tissue using a protease papain kit and cell plating at an increased cell density. Using this as a platform we have gone on to investigate the neuronal dysfunction occurring in Cysteine String Protein (CSP)?-/- mice. CSP? is a presynaptic protein thought to have co-chaperone like functions, mice lacking CSP? are born alive but show progressive weakness and neuronal degeneration soon after birth (Fernandez-Chacon, Wolfel et al. 2004). Hi-Spots formed from CSP? -/- tissue did not show overt neurodegenerative characteristics compared to +/+ controls and functional analysis demonstrated that at~DIV14 CSP? +/+ and -/- mice displayed equal levels of basal spontaneous network activity. Addition of bicuculline (50?M) to +/+ cultures lead to a significantly increasedfrequency and RMS value. However, in -/- cultures there was no increase induced by bicuculline. This may be due to an inability of CSP? -/- cultures to sustain high frequency synaptic transmission that is associated with bursting activity, or a selective degeneration of a sub-population of inhibitory neurons and a homeostatic network plasticity. The data suggest CSP? may act to protect the ability of neurons for high frequency synaptic transmission and/ or protect inhibitory neurons from degeneration.
Bailey, Joanne Louise
636f885f-76d9-4405-a1ea-b5a038d13bec
Bailey, Joanne Louise
636f885f-76d9-4405-a1ea-b5a038d13bec
O'Connor, Vincent
8021b06c-01a0-4925-9dde-a61c8fe278ca
Chad, John
d220e55e-3c13-4d1d-ae9a-1cfae8ccfbe1
Perry, V. Hugh
8f29d36a-8e1f-4082-8700-09483bbaeae4
Biggs, T.E.
5ef9af49-c9ba-44ed-b9b1-2aacdc5bee2d

Bailey, Joanne Louise (2010) Using Hi-Spots to investigate in vitro network dysfunction in Cysteine String Protein ? knockout mice. University of Southampton, School of Biological Sciences, Doctoral Thesis, 281pp.

Record type: Thesis (Doctoral)

Abstract

Hi-Spot are highly re-aggregated neural cultures grown on PTFE membrane at the liquidair interface. The Hi-Spot protocol was developed by scientists at Capsant Neurotechnologies using embryonic neural tissue. We characterised Hi-Spots made using existing protocols to confirm they represented a maturing neural network with molecular, cellular and functional signatures. We have additionally modified existing protocols to allow use of postnatal tissue as the source for dissociation and re-aggregation. Hi-Spots made from postnatal day zero (P0) rats self organised into a complex 3D tissue-like structure containing anatomically synaptically-interconnected neurons, astroglia and microglia. This CNS analogue of brain tissue provides for the emergence of a co-ordinated excitatory and inhibitory network, demonstrating a maturing pattern of activity involving single spikes developing into bursting behaviour driven by intrinsic synaptic activity. This activity can be represented as frequency or averaged amplitude (RMS), increases with time in culture and is blocked by glutamate receptor antagonists and stimulated by the inhibitory receptor antagonist bicuculline. The validity of its more in vivo-like organization, not observed in more conventional dissociated cultures, is evidenced by a glutamate toxicity resistance in the Hi-Spot cultures. Further, the thesis describes the optimisations to the Hi-Spot protocol to allow viable high density re-aggregated cultures to be made from individual rather than pooled brain dissociations. Modifications to the protocol included dissociation of the mouse tissue using a protease papain kit and cell plating at an increased cell density. Using this as a platform we have gone on to investigate the neuronal dysfunction occurring in Cysteine String Protein (CSP)?-/- mice. CSP? is a presynaptic protein thought to have co-chaperone like functions, mice lacking CSP? are born alive but show progressive weakness and neuronal degeneration soon after birth (Fernandez-Chacon, Wolfel et al. 2004). Hi-Spots formed from CSP? -/- tissue did not show overt neurodegenerative characteristics compared to +/+ controls and functional analysis demonstrated that at~DIV14 CSP? +/+ and -/- mice displayed equal levels of basal spontaneous network activity. Addition of bicuculline (50?M) to +/+ cultures lead to a significantly increasedfrequency and RMS value. However, in -/- cultures there was no increase induced by bicuculline. This may be due to an inability of CSP? -/- cultures to sustain high frequency synaptic transmission that is associated with bursting activity, or a selective degeneration of a sub-population of inhibitory neurons and a homeostatic network plasticity. The data suggest CSP? may act to protect the ability of neurons for high frequency synaptic transmission and/ or protect inhibitory neurons from degeneration.

Text
Joanne_Louise_Bailey_Thesis.pdf - Other
Download (90MB)

More information

Published date: 30 September 2010
Organisations: University of Southampton

Identifiers

Local EPrints ID: 181815
URI: http://eprints.soton.ac.uk/id/eprint/181815
PURE UUID: ed05e050-51d9-493d-9e4d-345b3c038f1a
ORCID for Vincent O'Connor: ORCID iD orcid.org/0000-0003-3185-5709
ORCID for John Chad: ORCID iD orcid.org/0000-0001-6442-4281

Catalogue record

Date deposited: 23 May 2011 11:52
Last modified: 15 Mar 2024 03:04

Export record

Contributors

Author: Joanne Louise Bailey
Thesis advisor: Vincent O'Connor ORCID iD
Thesis advisor: John Chad ORCID iD
Thesis advisor: V. Hugh Perry
Thesis advisor: T.E. Biggs

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×