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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, 15 th March 2011. Plan of the talk.

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slide1

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

slide2

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

slide3

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:

LTP (Bliss and Lomo, 1973)

Place cells (O’Keefe and Dostrovsky, 1971)

The Hippocampus is the first region to degenerate in ‘classic’ Alzheimer’s dementia

slide4

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

slide5

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

slide6

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, andBradley 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

slide7

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

slide8

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

slide9

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

slide10

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

slide11

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?

slide12

Extracellular recording in freely moving rodent

Example configuration of 1 drive

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

e.g. other site is Hpc fissure

Multi-site dual-drive extracellular recording (64ch)

slide13

Extracellular recording in freely moving rodent

Example configuration of 1 drive

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

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

slide14

Extracellular recording in freely moving rodent

Example configuration of 1 drive

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

e.g. other site is Hpc fissure

Place cell Firing rate map

10.1

peak rate (Hz)

HP

Multi-site dual-drive extracellular recording (64ch)

camera

Spikes

& LFP

Track Head position & orientation: LEDs on front & back of head

Place cell Spike location plot

Recording

Environment

(bird’s eye view)

slide15

Extracellular recording in freely moving rodent

Example configuration of 1 drive

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

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

Dashed Lines indicate theta peak

‘Raw’ theta (broad low-pass filter)

Amplitude (mV)

Analytic theta(apply offline 6-12 Hz filter, then Hilbert transform)

Time (seconds)

slide16

Extracellular recording in freely moving rodent:

Recording many neurons simultaneously

HP

Extracellular spike waveform on each of 4 tetrode tips

‘Place cells’ in CA1

Bird’s eye view of recording environment

Coloured square

indicates

where rat was

when cell fired

Firing rate maps

(taking dwell time

into account)

All spikes Averaged spike

slide17

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.

slide18

What do neurons do in region CA1?

CA1 pyramidal cells are ‘place cells’.

CA1 place cells show context-specific firing (later slides).

slide19

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

slide20

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.

slide21

Grid cells (found in EntorhinalCtx, 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

slide22

Grid cells (found in EntorhinalCtx, 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)

Each grid cell has a characteristic spatial scale

9.7

Intermediate scale

5.8

Small scale

Short distance between peaks

~30 cm

slide23

Theta frequency & gain of movement-speed signal

Grid cells

Spatial scale related to systematic variation in the gain of a movement-speed signal (theta frequency changes)

Lower theta frequency MPOs in ventral Entorhinalgrids, 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?

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

slide24

Head direction cells (presubiculum, entorhinalctx)

Code for Head Direction irrespective of location

e.g. the 4 quadrants of a cylinder

Burgess et al Hippocampus 2005

The brain’s compass

Parallel vectors

The four vectors do not converge on a point in the distance

slide25

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).

slide26

Boundary Vector cells in the Subiculum

(Lever et al, 2009, Journal of Neuroscience)

slide27

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

slide28

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

slide29

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

slide30

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

slide31

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?

slide32

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)

slide34

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

slide35

Do hippocampal neurons show learning?

What does it look like at the neuron level?

Contextual discrimination learning

Square vs Circle

slide36

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

slide37

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.

slide39

Contextual discrimination in place cells

Fields initially similar, then over time cells develop discriminatory firing (slow remapping)

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

slide40

Contextual discrimination in place cells

Fields initially similar, then over time cells develop discriminatory firing (slow remapping):

Cell fires in one environment, but not in another

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

slide41

Contextual discrimination in place cells

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)

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

slide42

Contextual discrimination in place cells

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!

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

slide43

Memory for what has been learned?

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

Representations initially similar

Over time, cells learn to discriminate the 2 shapes

Long-term memory

slide44

Memory for what has been learned? YES!

Lever, Wills, Cacucci, Burgess, O’Keefe, Nature, 2002

Representations initially similar

Over time, cells learn to discriminate the 2 shapes

Long-term memory

slide45

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

slide46

Context-specific firing can develop rapidly if contexts are significantly different

1st

2nd

3rd

4th

5th

6th

Trial Sequence Environment

Standard Altered (3rd, 4th)

Days 1 to 5 Day 6, 8, 10

Holding platform

Both walled environments:

Intentionally very different spatial contexts

slide47

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

Lever et al, unpublished data

In this experiment, place cells have ‘remapped’ the different contexts already within the 10-15 minute total trial time in each context

slide48

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

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

slide50

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

Well-established

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)

slide51

Every spike is assigned a theta phase of firing

We then aggregate all the spikes’ theta phases from:

a) CA1 b) Subiculum

slide52

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°.

slide53

Conclusion:

Theta phase may separate encoding and retrieval

If we can assume:

More Encoding during Novelty trials than in Familiar trials

Thenour 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?

slide56

Novel environments elicit theta frequency reduction

Decrease in theta frequency of up to 1 Hz recorded

in each rat

in the novel environment.

slide57

Novel environments elicit theta frequency reduction:

Summary

Familiar

Envt.

Novel

Envts.

Jeewajee, Lever et al (2008)

Hippocampus

slide58

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.

slide59

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-b pathology in the rat

Villette et al (2010)

J Neurosci

Basic idea:

Inject long-lasting Abaggregates (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 mlper injection site]

Implant electrodes to record local field potentials from the hippocampus (a little posterior to injection sites)

Give rats recognition memory task every two days for 3 weeks (first formal test one day after injection), evaluate progressive impairment

Test theta power over course of experiment

Detailed analysis of theta oscillations and behaviour on key days (D1, D7, D15, D21)

slide60

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-b pathology in the rat

Ab rats show similar investigative

repertoire to controls

Empty Position

New Stimuli

Long term

No change

slide61

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-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?

slide62

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-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

slide63

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-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?

slide64

“Loss of task-related theta frequency modulation after hippocampalAb injection”

Villette et al (2010)

J Neurosci

Controls

Ab rats

Ab rats do NOT show new

vs old theta frequency difference

Ab rats show reduced

theta power

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.

slide65

Decreased rhythmic GABAergicseptal activity & memory-associated theta oscillations after hippocampalAmyloid-b pathology in the rat

Villette et al (2010)

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 Abaggregates

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
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
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.

young mice performance at different delays
Young mice: performance at different delays

1) Behaviour

2) HPC place cells

aged mice performance at different delays
Aged mice: performance at different delays

Delay p < 0.001

Genotype p < 0.005

conclusions
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.
slide76

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

slide77

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!