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Colin LeverInstitute of Psychological SciencesUniversity of Leeds
ART PhD student Day, 15th March 2011
Studying cognitive processes in freely behaving rodents: neurons, oscillations, and behaviour
(focusing on hippocampal formation)
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
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
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
Group 1 = ‘normal aged’, Groups 2 & 3 = ‘possible AD’,
Group 4, 5, & 6 = ‘probable AD’
Group 7 = ‘definite AD’
Groups 1, 2, 3, 4, 5, 6, 7
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
Kordower et al, 2001, Annals of Neurology 49: 202-213
No cog impairment
Mild cog impairment
Alzheimer’s disease
Layer 2 ‘islands’
Layer 2 ‘islands’
Layer II entorhinal cells are critical
Kordower et al, 2001, Annals of Neurology 49: 202-213
No cog impairment
Mild cog impairment
Alzheimer’s disease
Layer 2 ‘islands’
Layer 2 ‘islands’
Very few layer 2 neurons
Layer II entorhinal cells are critical
Kordower et al, 2001, Annals of Neurology 49: 202-213
No cog impairment
Mild cog impairment
Alzheimer’s disease
Layer 2 ‘islands’
Layer 2 ‘islands’
Very few layer 2 neurons
Very few layer 2 neurons
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
Histology confirms the recording sites
of the electrodes
Electrodes gradually lowered to target site over days/weeks
e.g. one site is CA1 pyramidal layer
Example configuration of 1 drive
e.g. other site is Hpc fissure
Multi-site dual-drive extracellular recording (64ch)
Extracellular recording in freely moving rodent
Histology confirms the recording sites
of the electrodes
Electrodes gradually lowered to target site over days/weeks
e.g. one site is CA1 pyramidal layer
Example configuration of 1 drive
e.g. other site is Hpc fissure
Multi-site dual-drive extracellular recording (64ch) camera
Spikes& LFP
Track Head position & orientation: LEDs on front & back of head
HP
RecordingEnvironment(bird’s eye view)
Extracellular recording in freely moving rodent
Histology confirms the recording sites
of the electrodes
Electrodes gradually lowered to target site over days/weeks
e.g. one site is CA1 pyramidal layer
Example configuration of 1 drive
e.g. other site is Hpc fissure
Multi-site dual-drive extracellular recording (64ch) camera
Spikes& LFP
Track Head position & orientation: LEDs on front & back of head
10.1 peak rate (Hz)
Place cell Firing rate mapPlace cell Spike location plot
Extracellular recording in freely moving rodent
Histology confirms the recording sites
of the electrodes
Electrodes gradually lowered to target site over days/weeks
e.g. one site is CA1 pyramidal layer
Example configuration of 1 drive
e.g. other site is Hpc fissure
Multi-site dual-drive extracellular recording (64ch) camera
Spikes& LFP
Track Head position & orientation: LEDs on front & back of head
LFP showing theta oscillation
Am
plit
ud
e (m
V)
Time (seconds)
‘Raw’ theta (broad low-pass filter)
Analytic theta (apply offline 6-12 Hz filter, then Hilbert transform)
Dashed Lines indicate theta peak
Extracellular spike waveform on each of 4 tetrode tips
‘Place cells’ in CA1
Coloured square indicateswhere rat was when cell fired
Firing rate maps
(taking dwell timeinto account)
HP
Bird’s eye view of recording environment
All spikes Averaged spike
Extracellular recording in freely moving rodent:Recording many neurons simultaneously
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)
13.2 Hz
9.7
17.5
5.8
Large scaleLong distance between peaks~ 100 cm
Intermediate scale
Small scaleShort distance between peaks~30 cm
13.2 Hz
Mammalian brain divides the environment into triangular grids(broadly equilateral)
Each grid cell has a characteristic spatial scale
17.5
5.8
Large scaleLong distance between peaks~ 100 cm
Intermediate scale
Small scaleShort distance between peaks~30 cm
9.7
Grid cells (found in Entorhinal Ctx, presubiculum, parasubiculum, and subiculum)
Theta frequency & gain of movement-speed signal
13.2 Hz
Spatial scale related to systematic variation in the gain of a movement-speed signal (theta frequency changes)
Lower theta frequency MPOs in ventral Entorhinal grids, where grids have large spatial scale
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?
9.7
17.5
5.8
Grid cells
Large scaleLong distance between peaks~ 100 cm
Intermediate scale
Small scaleShort distance between peaks~30 cm
Burgess et al Hippocampus 2005
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
Head direction cells (presubiculum, entorhinal ctx)
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 HzSo 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 cellsAnchor 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.
4.43.6 2.8
5.12.6 2.1
0.33.3
4.03.11.1
8.4 0.1 0.3 0.00.2
3.1 2.9 8.12.6 5.4 1.5 2.1
6.2 0.5 0.01.0 0.2 0.2 0.6
0.63.2
3.1 1.7
3.9 0.2 0.7 0.0
2.3
1.0
5.3
2.0
1 2 3
1 2 54 6 7 8
109 1 2 3 5
1 2 3 4 5
D1
D3
D7
D5
Fields initially similar
Contextual discrimination in place cells
4.43.6 2.8
5.12.6 2.1
0.33.3
4.03.11.1
8.4 0.1 0.3 0.00.2
3.1 2.9 8.12.6 5.4 1.5 2.1
6.2 0.5 0.01.0 0.2 0.2 0.6
0.63.2
3.1 1.7
3.9 0.2 0.7 0.0
2.3
1.0
5.3
2.0
1 2 3
1 2 54 6 7 8
109 1 2 3 5
1 2 3 4 5
D1
D3
D7
D5
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Fields initially similar, then over time cells develop discriminatory firing (slow remapping)
Contextual discrimination in place cells
4.43.6 2.8
5.12.6 2.1
0.33.3
4.03.11.1
8.4 0.1 0.3 0.00.2
3.1 2.9 8.12.6 5.4 1.5 2.1
6.2 0.5 0.01.0 0.2 0.2 0.6
0.63.2
3.1 1.7
3.9 0.2 0.7 0.0
2.3
1.0
5.3
2.0
1 2 3
1 2 54 6 7 8
109 1 2 3 5
1 2 3 4 5
D1
D3
D7
D5
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Fields initially similar, then over time cells develop discriminatory firing (slow remapping):
Cell fires in one environment, but not in another
Contextual discrimination in place cells
4.43.6 2.8
5.12.6 2.1
0.33.3
4.03.11.1
8.4 0.1 0.3 0.00.2
3.1 2.9 8.12.6 5.4 1.5 2.1
6.2 0.5 0.01.0 0.2 0.2 0.6
0.63.2
3.1 1.7
3.9 0.2 0.7 0.0
2.3
1.0
5.3
2.0
1 2 3
1 2 54 6 7 8
109 1 2 3 5
1 2 3 4 5
D1
D3
D7
D5
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Fields initially similar, then over time cells develop discriminatory firing (slow remapping):
Cell fires in one environment, but not in another, or
Cell fires in different locations in each environment (less common)
Contextual discrimination in place cells
4.43.6 2.8
5.12.6 2.1
0.33.3
4.03.11.1
8.4 0.1 0.3 0.00.2
3.1 2.9 8.12.6 5.4 1.5 2.1
6.2 0.5 0.01.0 0.2 0.2 0.6
0.63.2
3.1 1.7
3.9 0.2 0.7 0.0
2.3
1.0
5.3
2.0
1 2 3
1 2 54 6 7 8
109 1 2 3 5
1 2 3 4 5
D1
D3
D7
D5
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Fields initially similar, then over time cells develop discriminatory firing (slow remapping)
Day 1: 3/3 similar
Day 3: 2/7 similar
Day 5: 1/7 similar
Day 7: 0/5 similar
Observe development of learning!
Contextual discrimination in place cells
D a y 1 :
S e r i e s
s t a r t
D a y 2 1 :
S e r i e s
E n d
D a y 7 1 :
2 n d D e l a y
t e s t
D a y 1 :
F i r s t
E x p o s u r e s
S e r i e s
E n d
D a y 2 1 :
1 7 d a y s
1 s t D e l a y
t e s t
2 8 d a y s
2 n d D e l a y
t e s t
D a y 7 1 :
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Long-term memory
Representations initially similar
Over time, cells learn to discriminate the 2 shapes
Memory for what has been learned?
D a y 1 :
S e r i e s
s t a r t
D a y 2 1 :
S e r i e s
E n d
D a y 7 1 :
2 n d D e l a y
t e s t
D a y 1 :
F i r s t
E x p o s u r e s
S e r i e s
E n d
D a y 2 1 :
1 7 d a y s
1 s t D e l a y
t e s t
2 8 d a y s
2 n d D e l a y
t e s t
D a y 7 1 :
Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002
Long-term memory
Representations initially similar
Over time, cells learn to discriminate the 2 shapes
Memory for what has been learned? YES!
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
Intentionally very different spatial contexts
Days 1 to 5 Day 6, 8, 10 Standard Altered (3rd, 4th)
Both walled environments:
D oo r
H P
Re
cord
ing
syst
em
B lack C urtains
C ue card
D oo r
H P
Re
cord
ing
syst
em
H P
Shelves
C ue card
Shelves
Holding platform
1st
2nd
4th
6th
5th
3rd
Trial Sequence Environment
Context-specific firing can develop rapidly if contexts are significantly different
Rat 1 Rat 2 Rat 3
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
12 5 3 7 9
3 5 5 16 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 contexts are significantly different
Lever et al, unpublished data
Rat 1 Rat 2 Rat 3
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
12 5 3 7 9
3 5 5 16 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
Context-specific firing can develop rapidly if contexts are significantly different
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
LTPWell-establishedresult
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
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°.
k = circular concentration m = mean phase
Highly familiarenvironment
Very different Novelenvironment
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
Decrease in theta frequency of up to 1 Hz recorded in each rat in the novel environment.
Novel environments elicit theta frequency reduction
NovelEnvts.
Familiar Envt.
Novel environments elicit theta frequency reduction:Summary
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 Amyloid-b pathology in the rat
Basic idea:
a) Inject long-lasting A b aggregates (A 40 & b A 42 b in 2:1 ratio) bilaterally into 4 injection sites in the dorsal hippocampus. [A 40 20 b mg/ml & A 42 b10 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)
Villette et al (2010) J Neurosci
Decreased rhythmic GABAergic septal activity & memory-associated theta oscillations after hippocampal Amyloid-b pathology in the rat
New Stimuli
Empty Position
Long termNo change
Ab rats show similar investigativerepertoire to controls
Decreased rhythmic GABAergic septal activity & memory-associated theta oscillations after hippocampal Amyloid-b pathology in the rat
Ab rats overexplore the familiar items, & underexplore the novel items
New Stimuli
Empty Position
Long termNo change
Classic memory test in rodents. Rats should explore new/changed items more.Authors used rats’ investigative rearing.
Investigative behaviour is not selectivelyincreased for the new/changed items in Ab rats.
I.e. Ab rats show memory deficit
What about neurophysiological correlates?
Ab rats show similar investigativerepertoire to controls
Decreased rhythmic GABAergic septal activity & memory-associated theta oscillations after hippocampal Amyloid-b pathology in the rat
Ab rats develop reduced theta powerAb rats overexplore the familiar items, & underexplore the novel items
Ab rats show similar investigativerepertoire to controls
New Stimuli
Empty Position
Long termNo change
Decreased rhythmic GABAergic septal activity & memory-associated theta oscillations after hippocampal Amyloid-b pathology in the rat
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?
Ab rats overexplore the familiar items, & underexplore the novel items
Ab rats show similar investigativerepertoire to controls
New Stimuli
Empty Position
Long termNo change
“Loss of task-related theta frequency modulation after hippocampal Ab injection” Villette et al (2010)
J Neurosci
Ab rats
Controls
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.
Ab rats show reduced theta power
Ab rats do NOT show newvs old theta frequency difference
Decreased rhythmic GABAergic septal activity & memory-associated theta oscillations after hippocampal Amyloid-b pathology in the rat
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 A b 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.
Villette et al (2010) J Neurosci
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