Transcript Performance of 3xTG AD mice on the T

Studying cognitive processes in
freely behaving rodents: neurons,
oscillations, and behaviour
(focusing on hippocampal formation)
Colin Lever
Institute of Psychological Sciences
University of Leeds
ART PhD student Day, 15th March 2011
Plan of the talk
Why focus on the hippocampus? Which regions
degenerate first in classic AD?
Outline characteristics of neurons supporting
spatial cognition and memory in Hippocampal
formation
Outline Theta oscillation-related changes in
environmental novelty (encoding-related
changes?)
THEN: 2 rodent AD models:
one with theta-related impairments,
one with CA1 place cell impairments
Why focus on the hippocampal formation?
Hippocampus has been linked to memory since
H.M.’s devestating memory loss following removal
of hippocampus & surrounding tissue
In animal literature, two key discoveries in the early
1970s:
1) LTP (Bliss and Lomo, 1973)
2) Place cells (O’Keefe and Dostrovsky, 1971)
The Hippocampus is the first region to degenerate
in ‘classic’ Alzheimer’s dementia
Stages in Alzheimer’s disease:
The spread from entorhinal cortex & CA1
Groups 1, 2, 3, 4, 5, 6, 7
Densities of Neurofibrillary tangles in mm2 in various brain regions amongst 7 groups defined by
patterns of damage. These groups are then used ‘post hoc’ to predict clinical features.
Groups 1, 2, 3, 4, 5, 6, 7
Corder et al, 2000, Exp Gerontol
Stages in Alzheimer’s disease:
The spread from entorhinal cortex & CA1
Groups 1, 2, 3, 4, 5, 6, 7
Group 1 = ‘normal aged’, Groups 2 & 3 = ‘possible AD’,
Group 4, 5, & 6 = ‘probable AD’
Group 7 = ‘definite AD’
Corder et al, 2000, Exp Gerontol
Layer II entorhinal cells are critical
Profound Loss of Layer II Entorhinal Cortex Neurons Occurs in Very Mild
Alzheimer's Disease
Teresa Gómez-Isla, Joseph L. Price, Daniel W. McKeel Jr., John C. Morris, John H.
Growdon, and Bradley T. Hyman
Journal of Neuroscience, 1996, 16: 4491-4500
‘A marked decrement of layer II neurons distinguishes even very mild AD from
nondemented aging’.
Basic findings replicated by:
Kordower et al, 2001, Annals of Neurology 49: 202-213
MCI and mild AD = fewer/atrophied Entorhinal layer II neurons
Layer II entorhinal cells are critical
No cog impairment
Layer 2 ‘islands’
Layer 2 ‘islands’
Mild cog impairment
Alzheimer’s disease
Kordower et al, 2001,
Annals of Neurology
49: 202-213
Layer II entorhinal cells are critical
No cog impairment
Layer 2 ‘islands’
Layer 2 ‘islands’
Mild cog impairment
Very few layer 2 neurons
Alzheimer’s disease
Kordower et al, 2001,
Annals of Neurology
49: 202-213
Layer II entorhinal cells are critical
No cog impairment
Layer 2 ‘islands’
Layer 2 ‘islands’
Mild cog impairment
Very few layer 2 neurons
Alzheimer’s disease
Very few layer 2 neurons
Kordower et al, 2001,
Annals of Neurology
49: 202-213
Stages in Alzheimer’s disease:
The spread from entorhinal cortex
No cognitive impairment -> Mild cognitive impairment ->
Early stage AD -> Developed AD
Entorhinal cortex (esp. layer 2) ->
CA1 ->
Subiculum
CA3 ->
MTL and temporal cortex ->
Other neocortex and subcortical regions
Where to focus in the hippocampal formation?
The Hippocampal formation (HF) is the first region
to degenerate in ‘classic’ Alzheimer’s dementia
Regions affected early on:
Entorhinal cortex, CA1, Subiculum
The HF is part of ‘septo-hippocampal’ theta
system. Medial Septum/DBB has an important role
in controlling hippocampal theta.
So to develop useful rodent AD models, we need
to establish normal physiology and function of
neurons and oscillations in the rodent HF.
How can we go about doing that?
Extracellular recording in freely moving rodent
Multi-site dual-drive extracellular recording (64ch)
Example configuration of 1 drive
e.g. one site is
CA1 pyramidal
layer
Electrodes
gradually
lowered to
target site over
days/weeks
e.g. other site
is Hpc fissure
Histology confirms
the recording sites
of the electrodes
Extracellular recording in freely moving rodent
Multi-site dual-drive extracellular recording (64ch)
Example configuration of 1 drive
e.g. one site is
CA1 pyramidal
layer
Electrodes
gradually
lowered to
target site over
days/weeks
Histology confirms
the recording sites
of the electrodes
camera
Spikes
& LFP
e.g. other site
is Hpc fissure
Track Head position & orientation:
LEDs on front & back of head
Extracellular recording in freely moving rodent
Multi-site dual-drive extracellular recording (64ch)
Example configuration of 1 drive
e.g. one site is
CA1 pyramidal
layer
Electrodes
gradually
lowered to
target site over
days/weeks
Histology confirms
the recording sites
of the electrodes
camera
Spikes
& LFP
e.g. other site
is Hpc fissure
Track Head position & orientation:
LEDs on front & back of head
Place cell Spike location plot
Recording
Environment
(bird’s eye view)
Place cell Firing rate map
10.1
peak rate (Hz)
HP
Extracellular recording in freely moving rodent
Multi-site dual-drive extracellular recording (64ch)
Example configuration of 1 drive
e.g. one site is
CA1 pyramidal
layer
Electrodes
gradually
lowered to
target site over
days/weeks
Histology confirms
the recording sites
of the electrodes
camera
Spikes
& LFP
e.g. other site
is Hpc fissure
Track Head position & orientation:
LEDs on front & back of head
Amplitude (mV)
LFP showing theta oscillation
Dashed Lines indicate theta peak
‘Raw’ theta (broad low-pass filter)
Analytic theta (apply offline 6-12 Hz
filter, then Hilbert transform)
Time (seconds)
Extracellular recording in freely moving rodent:
Recording many neurons simultaneously
Extracellular spike waveform
on each of 4 tetrode tips
Bird’s eye view of
recording environment
‘Place cells’ in CA1
Coloured square
indicates
where rat was
when cell fired
Firing rate maps
HP
All spikes
Averaged spike
(taking dwell time
into account)
What do neurons do in different hippocampal
regions?
CA1 pyramidal cells are ‘place cells’.
Entorhinal cortex contains different types of spatial
cells. Layer 2 cells are often ‘grid cells’.
Subiculum contains different types of spatial cells.
Some act like place cells. Some are boundary
vector cells. Some are grid cells.
We need to develop some idea of how neurons
function normally, before we know how to look for
impairment.
What do neurons do in region CA1?
CA1 pyramidal cells are ‘place cells’.
CA1 place cells show context-specific firing (later
slides).
Simultaneously recorded CA1 place cells
A few cells cover the whole
environment
The active cells in that
environment embody the
‘Cognitive Map’ of that
environment
They code for location AND
spatial context
Lever et al, Nature, 2002
What do neurons do in entorhinal cortex?
Entorhinal cortex cells are heterogenous
population:
Grid cells most striking discovery (Hafting et al,
Nature, 2005). Many Layer II stellate cells are grid
cells.
So this may be the first thing that goes wrong in
human AD. And if a rat AD model could
recapitulate human disease progression, you must
understand grid cells.
Grid cells (found in Entorhinal Ctx, presubiculum,
parasubiculum, and subiculum)
17.5
13.2 Hz
Large scale
Long distance between peaks
~ 100 cm
9.7
Intermediate scale
5.8
Small scale
Short distance between peaks
~30 cm
Grid cells (found in Entorhinal Ctx, presubiculum,
parasubiculum, and subiculum)
17.5
13.2 Hz
Large scale
Long distance between peaks
~ 100 cm
Mammalian brain divides the
environment into triangular grids
(broadly equilateral)
9.7
Intermediate scale
5.8
Small scale
Short distance between peaks
~30 cm
Each grid cell has a characteristic
spatial scale
Theta frequency & gain of movement-speed signal
Grid cells
17.5
13.2 Hz
Large scale
Spatial scale related to systematic
variation in the gain of a
movement-speed signal (theta
frequency changes)
Long distance between peaks
~ 100 cm
Lower theta frequency MPOs in
ventral Entorhinal grids, where
grids have large spatial scale
9.7
Intermediate scale
5.8
Small scale
Short distance between peaks
~30 cm
Higher theta frequency MPOs in
dorsal EC grids, where grids have
small spatial scale
Grids seem to provide a strong
spatial metric signal, encode
distance travelled?
Head direction cells (presubiculum, entorhinal ctx)
Code for Head Direction irrespective of location
e.g. the 4 quadrants of a cylinder
The brain’s
compass
Parallel vectors
The four vectors do not
converge on a point in
the distance
Burgess et al Hippocampus 2005
What do neurons do in Subiculum?
Subiculum contains different types of spatial cells.
Some act like place cells (shown).
Some are grid cells (shown)
Some are boundary vector cells (next slides).
Boundary Vector cells in the Subiculum
(Lever et al, 2009, Journal of Neuroscience)
What constitutes a boundary?
Wall-less Environments
13.2 Hz
50-cm high walls
No walls (drop)
No walls (drop)
10 cm gap between the 3
squares
What constitutes a boundary?
Wall-less Environments
13.2 Hz
50-cm high walls
No walls (drop)
No walls (drop)
10 cm gaps between the 3
squares
Rat walks across drop
What constitutes a boundary?
Wall-less Environments
13.2 Hz
50-cm high walls
No walls (drop)
No walls (drop)
10 cm gaps between the 3
squares
Rat walks across drop
What constitutes a boundary?
Wall-less Environments
13.2 Hz
50-cm high walls
No walls (drop)
No walls (drop)
10 cm gaps between the 3
squares
What constitutes a boundary?
Wall-less Environments
13.2 Hz
So Subicular boundary
vector cells appear to
function as high-level spatial
perceptual cells
Wall and drop don’t share the
same visual properties. And
BVCs fire in darkness.
Function?
Spatial Inputs to place cells
Anchor grids to external
boundaries?
Are these cell types found in humans?
Yes, and if not, seems very probable.
Place cells: monkeys, humans (Ekstrom et al,
Nature, 2003)
Head direction cells: in monkey presubiculum.
Grid cells: Indirect fMRI evidence (Doeller et al,
Nature, 2010)
Boundary vector cells: not yet looked for (recent
discovery)
Population signal of predicted grid
cell activity in right entorhinal
cortex
Strong links between spatial/context memory
system in rats and autobiographical memory
in humans
So if we understand the hippocampal system in
rodents at the level of neurons and oscillations
we will be able to create more precise rodent AD
models of episodic/autobiographical memory
deficits
and provide a more accurate platform for testing
therapeutic agents
Do hippocampal neurons show learning?
What does it look like at the neuron level?
Contextual discrimination learning
Square vs Circle
Do hippocampal neurons show learning?
What does it look like at the neuron level?
Slow Contextual discrimination learning:
Can we observe learning develop over time?
Can we see memory after a delay?
Incidental learning paradigm:
Experimenter does nothing to encourage the discrimination learning
Do hippocampal neurons show learning?
What does it look like at the neuron level?
Slow Contextual discrimination learning:
Quite a hard task for the rat?
Like too-similar floors in car park? –
Takes a while to discriminate.
Contextual discrimination in place cells
1
2
4.4
D1
3
3.6
2.8
2.6
2.1
Fields initially similar
5.1
1
4
2
5
6
7
8
3.1
2.6
2.9
8.1
5.4
1.5
2.1
0.5
0.0
0.2
0.2
0.6
1
2
3
5
D3
6.2
9
1.0
10
3.2
D5
2.3
5.3
2.0
0.6
3.9
3.1
0.2
0.7
4
5
1.1
3.1
0.2
0.0
1.0
1
0.3
D7
8.4
2
3
3.3
4.0
0.1
0.3
1.7
0.0
Contextual discrimination in place cells
1
2
4.4
D1
3
3.6
2.8
2.6
2.1
Fields initially similar, then over
time cells develop
discriminatory firing (slow
remapping)
5.1
1
4
2
5
6
7
8
3.1
2.6
2.9
8.1
5.4
1.5
2.1
0.5
0.0
0.2
0.2
0.6
1
2
3
5
D3
6.2
9
1.0
10
3.2
D5
2.3
5.3
2.0
0.6
3.9
3.1
0.2
0.7
4
5
1.1
3.1
1.7
0.0
1.0
1
0.3
D7
8.4
2
3
3.3
4.0
0.1
0.3
0.2
0.0
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Contextual discrimination in place cells
1
2
4.4
D1
3
3.6
2.8
2.6
2.1
Fields initially similar, then over
time cells develop
discriminatory firing (slow
remapping):
5.1
1
4
2
5
6
7
8
3.1
2.6
2.9
8.1
5.4
1.5
2.1
0.5
0.0
0.2
0.2
0.6
1
2
3
5
Cell fires in one environment,
but not in another
D3
6.2
9
1.0
10
3.2
D5
2.3
5.3
2.0
0.6
3.9
3.1
0.2
0.7
4
5
1.1
3.1
1.7
0.0
1.0
1
0.3
D7
8.4
2
3
3.3
4.0
0.1
0.3
0.2
0.0
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Contextual discrimination in place cells
1
2
4.4
D1
3
3.6
2.8
2.6
2.1
Fields initially similar, then over
time cells develop
discriminatory firing (slow
remapping):
5.1
1
4
2
5
6
7
8
3.1
2.6
2.9
8.1
5.4
1.5
2.1
0.5
0.0
0.2
0.2
0.6
1
2
3
5
D3
6.2
9
1.0
10
Cell fires in one environment,
but not in another, or
Cell fires in different locations
in each environment (less
common)
3.2
D5
2.3
5.3
2.0
0.6
3.9
3.1
0.2
0.7
4
5
1.1
3.1
1.7
0.0
1.0
1
0.3
D7
8.4
2
3
3.3
4.0
0.1
0.3
0.2
0.0
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Contextual discrimination in place cells
1
2
4.4
D1
3
3.6
2.8
2.6
2.1
Fields initially similar, then over
time cells develop
discriminatory firing (slow
remapping)
5.1
Day 1: 3/3 similar
1
4
2
5
6
7
3.1
2.6
2.9
8.1
5.4
1.5
8
2.1
Day 5: 1/7 similar
D3
6.2
9
1.0
10
0.5
0.0
0.2
0.2
1
2
3
5
3.2
D5
Day 3: 2/7 similar
2.3
5.3
2.0
0.6
3.9
3.1
0.2
0.7
4
5
1.1
3.1
0.6
Day 7: 0/5 similar
Observe development of
learning!
1.7
0.0
1.0
1
0.3
D7
8.4
2
3
3.3
4.0
0.1
0.3
0.2
0.0
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Memory for what has been learned?
28 days
17 days
Day 1:
First
Exposur es
Day 1:
Series
start
Day 21:
Series
End
Day 71:
2nd Delay
test
Day 21:
Series
End
1st Dela y
test
Day 71:
2nd Del ay
test
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Representations
initially similar
Over time, cells
learn to discriminate
the 2 shapes
Long-term
memory
Memory for what has been learned? YES!
28 days
17 days
Day 1:
First
Exposur es
Day 1:
Series
start
Day 21:
Series
End
Day 71:
2nd Delay
test
Day 21:
Series
End
1st Dela y
test
Day 71:
2nd Del ay
test
Lever, Wills, Cacucci,
Burgess, O’Keefe, Nature,
2002
Representations
initially similar
Over time, cells
learn to discriminate
the 2 shapes
Long-term
memory
Summary: CA1 neurons ‘learn’ to discriminate
Individual CA1 neurons show ‘long-term plasticity’
Discrimination is observed to increase with more experience of contexts
Once learned, the discrimination is remembered after month-long delay
Context-specific firing can develop rapidly if
contexts are significantly different
Trial Sequence
Environment
Days 1 to 5
Standard
Day 6, 8, 10
1st
Altered (3rd, 4th)
Door
Door
Shelves
Shelves
2nd
Black
Curtains
HP
HP
Holding platform
Cue card
HP
Recording
system
3rd
Recording
system
Cue card
4th
Both walled environments:
5th
6th
Intentionally very different
spatial contexts
Context-specific firing can develop rapidly if
Rat 1
Rat 2
Rat 3
contexts
are significantly
different
Cell 1
Cell 2
Cell 1
Cell 2
Cell 1
Cell 2
13 Hz
2 Hz
3 Hz
2 Hz
6 Hz
16
2
6
3
13
5
2
4
2
8
4
6
3
5
3
12
3
5
7
5
16
Lever et al,
unpublished
data
9
6
In this experiment, place cells have ‘remapped’ the different contexts
already within the 10-15 minute total trial time in each context
Context-specific firing can develop rapidly if
Rat 1
Rat 2
Rat 3
contexts
are significantly
different
Cell 1
Cell 2
Cell 1
Cell 2
Cell 1
Cell 2
13 Hz
2 Hz
3 Hz
2 Hz
6 Hz
16
2
6
3
13
5
2
4
2
8
4
6
3
5
3
12
3
5
7
5
16
9
6
As with slow discrimination for subtly-differing context, a) a place cell
can discriminate by firing in one context but not another, or by firing in
both contexts but in different locations b) it’s incidental learning
The hippocampal theta oscillation is sensitive to
novel contexts
Theta Phase and Memory states
Hippocampal LTP protocols are optimal using stimulation at theta frequency
Theta phase determines whether LTP is achieved, e.g. in CA1 stimulate at theta
peak -> strongest LTP
LTP
Wellestablished
result
LTD or no
change results
Model (Hasselmo et al, 2002) links these plasticity results to memory
states. In novelty-elicited encoding there should be:
a bias -> information from entorhinal cortex, presumed to arrive near
peak of principal-cell layer theta
Vs in retrieval, a bias -> predictive CA3 input (arriving at trough)
Every spike is assigned a theta phase of firing
We then aggregate all the spikes’ theta phases from:
a) CA1 b) Subiculum
Later CA1 mean theta phase in novelty
Highly familiar
environment
Very different Novel
environment
 = circular concentration
m = mean phase
Each polar plot represents all recorded CA1 spikes in that trial.
Mean spike phase normalised such that mean phase of all CA1 spikes in last
trial in familiar environment (‘Baseline’) is 0°.
Conclusion:
Theta phase may separate encoding and retrieval
If we can assume:
More Encoding during Novelty trials than in Familiar
trials
Then our results suggest that theta phase could play
a role in plasticity in the hippocampal memory
system, and the balance between encoding and
retrieval
Likely a general coding strategy in the brain?
Novel environments elicit theta frequency reduction
Novel environments elicit theta frequency reduction
Novel environments elicit theta frequency reduction
Decrease in theta
frequency of up to 1
Hz recorded
in each rat
in the novel
environment.
Novel environments elicit theta frequency reduction:
Summary
Familiar
Envt.
Novel
Envts.
Jeewajee, Lever et al (2008)
Hippocampus
Summary: Hippocampal theta and novelty
Novel environments elicit:
1) Later theta phase of firing in CA1 neurons (Lever et al,
2010, Hippocampus)
2) Lower theta frequency in hippocampal theta (Jeewajee,
Lever et al, 2008, Hippocampus)
This second finding is (relatively) easy to study.
This could be explored in rodent AD models without
needing to record hippocampal neurons.
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Villette et al (2010)
Amyloid-b pathology in the rat
J Neurosci
Basic idea:
a) Inject long-lasting Ab aggregates (Ab40 & Ab42 in 2:1 ratio) bilaterally into 4
injection sites in the dorsal hippocampus. [Ab40 20 mg/ml & Ab42 10 mg/ml,
Bachem, 0.25 ml per injection site]
b) Implant electrodes to record local field potentials from the hippocampus (a
little posterior to injection sites)
c) Give rats recognition memory task every two days for 3 weeks (first formal
test one day after injection), evaluate progressive impairment
d) Test theta power over course of experiment
e) Detailed analysis of theta oscillations and behaviour on key days
(D1, D7, D15, D21)
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Amyloid-b pathology in the rat
Ab rats show similar investigative
repertoire to controls
Empty Position
New Stimuli
Long term
No change
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Amyloid-b pathology in the rat
Ab rats show similar investigative
repertoire to controls
Empty Position
New Stimuli
Long term
No change
Ab rats overexplore the familiar items,
& underexplore the novel items
Classic memory test in rodents. Rats
should explore new/changed items more.
Authors used rats’ investigative rearing.
Investigative behaviour is not selectively
increased for the new/changed items in Ab
rats.
I.e. Ab rats show memory deficit
What about neurophysiological correlates?
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Amyloid-b pathology in the rat
Ab rats show similar investigative
repertoire to controls
Empty Position
New Stimuli
Long term
No change
Ab rats overexplore the familiar items,
& underexplore the novel items
Ab rats develop reduced theta power
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Amyloid-b pathology in the rat
Ab rats show similar investigative
repertoire to controls
Empty Position
New Stimuli
Long term
No change
Ab rats overexplore the familiar items,
& underexplore the novel items
The reduced theta power Ab rats develop
is non-specific.
It occurs regardless of the task and
old/new space/object combinations.
e.g. Tested different group of Ab rats and
controls who are exposed to unchanging
stimuli in context. These Ab rats also show
reduced power.
Is there a neural correlate specific to the
old/new memory impairment?
“Loss of task-related theta frequency modulation after
Villette et al (2010)
hippocampal Ab injection”
J Neurosci
Controls
Ab rats
Ab rats show reduced
theta power
Ab rats do NOT show new
vs old theta frequency difference
On Days 15 & 21, control rats show behavioural discrimination of old vs
new items. Ab rats don’t. Thus, in parallel with memory deficits, Ab rats do
not show the novelty-elicited theta frequency reduction which emerges in
controls by D15 & D21.
Decreased rhythmic GABAergic septal activity &
memory-associated theta oscillations after hippocampal
Villette et al (2010)
Amyloid-b pathology in the rat
J Neurosci
Villette et al studied spatial/object associational novelty.
They replicate in their controls the Jeewajee, Lever et al
(2008) result based on environmental novelty:
New spatial/object combinations elicit higher levels of
investigation and lower-frequency theta oscillations in
controls.
Neither occurs in rats injected with Ab aggregates
Discovering neurophysiological correlates of
spatial/contextual representation and memory are useful
in building more precise animal models of dementia
That can provide a bridge between molecules and
behaviour.
Place cells can provide an intermediate
level of investigation between
molecules and behaviour
Research goals:
• study the network properties of hippocampal cells
in rodent models of Alzheimer’s disease.
• investigate relationships between physiological
and cognitive changes during the progression of the
disease.
One experimental model:
the Tg2576 mouse as a model of
‘Alzheimer-like’ dysfunction
• neuronal overexpression of a mutated
form of human amyloid (APP695SWE).
• develops elevated brain levels of soluble
amyloid by 6-8 months, and neuritic plaques
by 10-16 months.
• age-dependent impairment on spatial
navigation/memory tasks.
Lab Setup
Young mice: performance at different delays
1) Behaviour
2) HPC place cells
Aged mice: performance at different
delays
Delay p < 0.001
Genotype p < 0.005
Place cells in aged mice
Quantifying Spatial Characteristics
of
the Place Fields
Correlation between behaviour and Spatial
information
Basic Physiological Properties
Conclusions
• Place cell signalling is normal in young tg2576
mice but disrupted in some aged tg2576 mice.
• There is a correlation between place cell
disruption and spatial memory deficits.
• Combining place cell recording with spatial
memory testing will provide a powerful tool for
investigating molecular changes which lead to
the physiological alterations in Alzheimer’s
disease and for testing possible therapeutic
strategies.
Overall conclusion
Neurophysiology in behaving rodents linking
neurons and oscillations to behaviour
Is a useful and arguably necessary step
In creating good AD models in rodents
Thanks to:
LEEDS:
Christine Wells, Ali Jeewajee, Sarah Stewart, Vincent Douchamps,
UCL:
Ali Jeewajee, Stephen Burton, Francesca Cacucci, Tom Wills
Neil Burgess, John O’Keefe
And you for listening!
End