title: Investigation into the effect of the general anaesthetics etomidate and ketamine on long-range coupling of population activity in the mouse neocortical slice

authors: Voss, Logan J.

subjects: ketamine

abstract: General anaesthetics have been hypothesised to ablate consciousness by decoupling intracortical neural connectivity. We explored this by investigating the effect of etomidate and ketamine on coupling of neural population activity using the low magnesium neocortical slice model. Four extracellular electrodes (50 μm) were positioned in mouse neocortical slices (400 μm thick) with varying separation. The effect of etomidate (24 μM) and ketamine (16 μM) on the timing of population activity recorded between channels was analysed. No decoupling was observed at the closest electrode separation of 0.2 mm. At 4 mm separation, decoupling was observed in 50% and 42% of slices during etomidate and ketamine delivery, respectively (P<0.0001 and P=0.002, compared to 0.2 mm separation). A lower rate of decoupling was observed with 1 mm separation (21% and 8%, respectively, P<0.03 for etomidate compared to 0.2 mm separation). The data support the hypothesis that mechanistically diverse general anaesthetics disrupt neuronal connectivity across widely distributed intracortical networks.

text: Investigation into the effect of the general anaesthetics etomidate and ketamine on long-range coupling of population activity in the mouse neocortical slice General anaesthetics have been hypothesised to ablate consciousness by decoupling intracortical neural connectivity. We explored this by investigating the effect of etomidate and ketamine on coupling of neural population activity using the low magnesium neocortical slice model. Four extracellular electrodes (50 μm) were positioned in mouse neocortical slices (400 μm thick) with varying separation. The effect of etomidate (24 μM) and ketamine (16 μM) on the timing of population activity recorded between channels was analysed. No decoupling was observed at the closest electrode separation of 0.2 mm. At 4 mm separation, decoupling was observed in 50% and 42% of slices during etomidate and ketamine delivery, respectively (P<0.0001 and P=0.002, compared to 0.2 mm separation). A lower rate of decoupling was observed with 1 mm separation (21% and 8%, respectively, P<0.03 for etomidate compared to 0.2 mm separation). The data support the hypothesis that mechanistically diverse general anaesthetics disrupt neuronal connectivity across widely distributed intracortical networks.

year: 2012

title: Protein Dynamics and its Correlation to Protein Activity and Stability

authors: Clement, David

subjects: Incoherent neutron scattering

abstract: The aim of this thesis is to investigate the role of fast protein dynamics (picosecond
timescale) in enzyme activity and stability, and specifically to test the
hypothesis that enzyme activity and stability are inversely related by their internal
dynamics.
Activity   Dynamics (flexibility)   1/Stability
In order to test this hypothesis, the well known anti-cancer drug: methotrexate
was used as an informative ligand in the network established between these
properties. A multidisciplinary approach combining neutron scattering, circular
dichroism, UV absorption, isothermal titration calorimetry and X-ray crystallography
was undertaken to examine the current paradigm using the enzyme: dihydrofolate
reductase as a model.
As inferred by neutron spectroscopy, the binding of MTX influences the
dynamical behavior of DHFR. Macromolecular dynamics such as the resilience: lt;kgt;
(i.e. structural rigidity) was found to be increased and, inversely, the flexibility
decreased upon MTX binding. In addition, as revealed by circular dichroism, this
dynamical dependency upon MTX binding was correlated with an enhanced thermal
stability. Compared to the free enzyme, the melting temperature was found to be
increased by 13.8  C in the presence of MTX. The inhibitory power of MTX was also
examined by steady state kinetics and isothermal titration calorimetry. The Ki for
MTX was found to be in the nanomolar range Ki= 10.9 nM. Using isothermal
titration calorimetry, the binding thermodynamic signature between MTX and DHFR
was characterized. The binding event was found to be largely favourable (DGb=-12.1
Kcal mol-1), enthalpy driven (DHb= -16.8 Kcal mol-1) with an unfavourable entropy
DSb=-15.6 cal K-1mol-1.
In conclusion, the modulation of the macromolecular dynamics may reflect
how specific conformations are favoured for subsequent protein function in response
of the binding of specific ligand and how conformational substates approach to
protein function. In this context the unprecedented power of transition state analogs
such as MTX on protein function might therefore be dependent on fast protein
dynamics.

text: Protein Dynamics and its Correlation to Protein Activity and Stability The aim of this thesis is to investigate the role of fast protein dynamics (picosecond
timescale) in enzyme activity and stability, and specifically to test the
hypothesis that enzyme activity and stability are inversely related by their internal
dynamics.
Activity   Dynamics (flexibility)   1/Stability
In order to test this hypothesis, the well known anti-cancer drug: methotrexate
was used as an informative ligand in the network established between these
properties. A multidisciplinary approach combining neutron scattering, circular
dichroism, UV absorption, isothermal titration calorimetry and X-ray crystallography
was undertaken to examine the current paradigm using the enzyme: dihydrofolate
reductase as a model.
As inferred by neutron spectroscopy, the binding of MTX influences the
dynamical behavior of DHFR. Macromolecular dynamics such as the resilience: lt;kgt;
(i.e. structural rigidity) was found to be increased and, inversely, the flexibility
decreased upon MTX binding. In addition, as revealed by circular dichroism, this
dynamical dependency upon MTX binding was correlated with an enhanced thermal
stability. Compared to the free enzyme, the melting temperature was found to be
increased by 13.8  C in the presence of MTX. The inhibitory power of MTX was also
examined by steady state kinetics and isothermal titration calorimetry. The Ki for
MTX was found to be in the nanomolar range Ki= 10.9 nM. Using isothermal
titration calorimetry, the binding thermodynamic signature between MTX and DHFR
was characterized. The binding event was found to be largely favourable (DGb=-12.1
Kcal mol-1), enthalpy driven (DHb= -16.8 Kcal mol-1) with an unfavourable entropy
DSb=-15.6 cal K-1mol-1.
In conclusion, the modulation of the macromolecular dynamics may reflect
how specific conformations are favoured for subsequent protein function in response
of the binding of specific ligand and how conformational substates approach to
protein function. In this context the unprecedented power of transition state analogs
such as MTX on protein function might therefore be dependent on fast protein
dynamics.

year: 2009

title: In vitro cell culture to study microglial inflammation

authors: Newland, Julia

subjects: CNs

abstract: Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

text: In vitro cell culture to study microglial inflammation Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

year: 2016