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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, 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