Sorting the connections with multi-electrode neuronal ensemble recording techniques:

Sorting the connections with multi-electrode neuronal ensemble recording

techniques:

Single-unit and local field potential activity from rat to man

Dr Rob Mason

Institute of Neuroscience

School of Biomedical Sciences

University of Nottingham Medical School

and the LAB TEAM

Neuronal Networks Laboratory

LAB TEAM

epilepsy Ben Coomber [Dr Mike O’Donoghue ~ Dept

Neurology, QMC]

Clare Roe schizophrenia Dr Jill Suckling [Prof CA Marsden]

Dr Dissanayake anxiety Dr Carl Stevenson [Prof CA Marsden]

pain Dr Steve Elmes [Dr V Chapman]

rodent USVs Beth Tunstall [Dr S Beckett]

data analysis Margarita Zachariou [Prof S Coombes / Dr M Owen]

[Mathematics Dept]

Dr David Halliday (University of York)Prof D Auer (fMRI / phMRI ~ QMC)


Seminar overview

• Multichannel Electrophysiological Recording Technologies

Illustrate with reference to 2 experimental rodent model projects:

• Hippocampus-mPFC ~ Epilepsy model – role of endocannabinoid system

• Sensory gating in hippocampus & mPFC ~ Schizophrenia model – PCP effects

• Amygdala-mPFC ~ Stress models – maternal separation & “drug stressors”• Left-Right mPFC ~

• Rat language ~ ultrasound vocalisations (USVs) & affective state


Sorting the connections with multi-electrode neuronal ensemble recording techniques

To & From ~ rat prefrontal cortex

Sorting signal from noise

Ensemble Neuronal Unit activity

LFP activity

DATA Analysis & Interpretation

Distribution of neurones contributing to signals recorded by tetrode array

after Busaki 2004

Bionics 100-channel (“hedgehog”) array

Michigan MEAprobe 16-channels

NBLabs 8-channel array

4-channel - independent manipulation

Examples of Electrode Arrays

MULTIPLE ELECTRODE ARRAY RECORDING in vivo

• Single-site recording

e.g. hippocampal sub regions (CA1 & CA3)

• Multiple-site recording

e.g. prefrontal cortex & hippocampus

NBLabs 16-channel micro-wire array

• CA1 & CA3

Multiple Electrode Recordings in vivosimultaneous multichannel spike & field potential

recording

• 64-channel MAP system• 32-channel MAP system – with CinePlex for behavioural studies • 64-chanel Recorder system• 16-chanel Recorder system used MAP & Recorder for in vitro MEA studies with brain slices & neuronal cultures

Extracellular Recording - signal filtering & discrimination

AP Spike discrimination: separate action potential (AP) signal from noise

• AP amplitude detection / AP waveform shape recognition

Signal filtering: separate unit activity and Local Field Potentials (LFPs)

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Units + EEG / LFP

Units

Multiple Neuronal (spike) Recording - two electrodes

micro-electrodes

Neurones

Amplifiernerve impulses

(action potential “spikes”)

Electrode 1

Electrode 2

Off Line Sorting of spike waveforms - single unit isolation

Movie: Sorting of unit spike data using Principal Component Analysis

• distinct AP spike waveforms represented as clusters in 3D space• 7 units isolated – each unit colour-coded

Multiple (ensemble) neurone recording

Advantages

• Investigating neuronal ensemble/network function - closer to working “brain”

• Good experimental design - fewer animals required (“3Rs” ~ Home Office)

• Masses of data

Disadvantages

• Masses of data? Data processing? Data interpretation

Data Visualisation Emergent Properties

Population Dynamics

Unit activity

Single units – spike rasters /FRH / ISIH / PSTHs burst analysis /

Unit pairs – cross-correlation / coherence /Unit ensembles – PCA / ICA / synchrony index / PDC

Local Field Potentials

FFT / spectrogramsLFP/EEG signal bandsLFP-unit coherenceLFP PDC

ENSEMBLE DATA - DISPLAY & ANALYSIS

MASSIVE data sets

• Dual/Triple site recordings ~ 64 channels simultaneous units & LFPs

VTA - mPFC hippocampus - mPFCamygdala - mPFCmPFC – mPFCspinal cord – thalamus – cerebral cortex

• Systemic pharmacological manipulation

• Local pharmacological manipulation

“injectrode” integrated microiontophoresis with recording array

• Electrical stimulation – periphery / CNS structures

• Independent electrode array microdrive – 8 channel drive

• Anaesthetised & awake-behaving preparations


LAB EXPERIMENTAL DIRECTIONS

mPFCx interconnectivity & functional context – in vivo studies

maternal separation - Depression- Stress

circadian - affective states

- Sensory Gating- Schizophrenia- Epilepsy

USVs- affective states

medial Pre-Frontal Cortex

Amygdalanucleus AccumbensHippocampusSCN

Contralateral

mPFC

VTA

schizophreniadrugs of abuse

PVt

bladder

University of Nottingham Medical SchoolNeuronal Networks Laboratory

Project #1: Epilepsy

• kainate-induced epileptiform activity ~ TLE

• functional network interactions ~ hippocampus mPFC

• role of endocannabinoid system ~ CB1R pharmacology

• perforant path stimulation-evoked seizure activity


FAAHMGL

Reuptake

Endo-CANNABINOID System

• Endogenous cannabinoids (eCBs) identified

e.g. anandamide (AEA) and 2-arachidonylglycerol (2-AG)

• Act at cannabinoid G protein coupled-Receptors: CB1 & CB2 - ? CB3

• eCBs are synthesised post-synaptically on-demand.

• eCBs act at pre-synaptic CB1 receptors.

• eCB reuptake occurs via a transporter.

• Metabolism:– AEA by fatty acid amide

hydrolase (FAAH)– 2-AG by monoacylglycerol

lipase (MGL).Figure taken from Wilson & Nicoll (2002) Science.

• Kainic acid (KA): established convulsive agent producing seizures in awake rats ~ targeting temporal lobe

Model uses KA, administered systemically (10mg/kg, i.p.) anaesthetized rats ~ ensemble neuronal unit and LFP activity

• Study aims to establish whether URB597, selective inhibitor of FAAH enzyme (eCB levels), attenuates KA-evoked neuronal activity

• Role of CB1 cannabinoid receptors assessed using selective CB1 antagonist AM251

Epilepsy Study: AIMS

Anandamide Metabolism by FAAH

URB597

Inhibits anandamide metabolism

KA + Vehicle: Rat #1

Basal: Rat #1 Basal: Rat #2

KA + URB597: Rat #2

Unit and LFP activity in mPFC and hippocampus

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• Effects on hippocampal neural firing rate (~40 cells; n=5 rats)

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Spike-triggered averaging of LFP using 1a as ref cell

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• Effects on spike-triggered averaging of mPFC LFPs

Post-KA administration

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Cross-correlation analysis ~ PFC – PFC / PFC- Hippocampal neuronal pairs

# units

Cross-correlogram TF #1

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Time-Series cross-correlograms – TF #1-13

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Cross-correlation analysis of PFC neuronal pairs

• Effect of kainate (10mg/kg; i.p.) administration at epoch 4

Time Frames #1-13

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PDC Analysis – unit ensemble data

• PDC was applied to identify the direction of activity between hippocampusand mPFC - technique that has the potential to reveal the neuronal ensemble drives.

Note: the magnitude of the classical coherence gives no information about directional connectivity - but its phase may do so.


Project #2: Schizophrenia

• auditory-evoked sensory gating ~ hippocampus & mPFC

• effects of PCP / ketamine ~ model

• effects of social isolation ~ model


Sensory gating in hippocampus:

A model for schizophrenia ?

• Sensory gating: mechanism(s) by which irrelevant sensory information is filtered ~ enables efficient information processing

• Auditory Conditioning-Test paradigm: measures reduction in auditory-evoked response produced by Test stimulus following a Conditioning stimulus

• Stimuli: 3kHz sine-wave / 10ms duration / presented 500ms apart / 80-90dB

• human P50 wave = rat N40 component

• Gating absent in - schizophrenic patients (& family)- normal volunteers given PCP /

amphetamine

- rats given PCP / amphetamine


1sLFP2 averaged 128 trials

Units

LFPs

Hippocampal CA3 - auditory-evoked unit & LFP activity

- 128

- 1

Trial #

sCs Ts

mV

N40N40

Averaged LFP

T/C ratio = 55%

eventtone

dentate gyrus CA3 region CA3 region

Single-unit PSTHs ‘ rasters histograms – “gating rats”

Unit 1

Unit 2

Unit 3

Unit 4

Spike raster

PSTH

LFP

LFP trial raster

Hippocampal CA3 auditory-evoked unit & LFP activity

Effects of PCP (1mg/kg i.p.) attenuates / abolishes sensory gating

Basal [128 trials]

T/C ratio = 32%i.e. exhibits gating

45mins after PCP

T/C ratio = 66%gating attenuated

1

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128

SUMMARY III

Sensory Gating Studies

LFP studies:

• Demonstrate sensory gating in isoflurane-anaesthetised rat : T/C ratio = 35 ± 15%

• SG is abolished / attenuated following PCP : T/C ratio = 65 ± 5%

• control rats SG is unaffected by clozapine : T/C ratio 40%

• Clozapine (5mg.kg-1) blocks action of PCP on SG : T/C ratio 35%

Unit studies:

• Similar observations

Project #3:

Rat ultrasound vocalisations (USVs) & affective state

• Sorting the pips from the squeaks

•Role of nuc Accumbens in USV-mediated Behaviours ~50 kHz (Reward) call(Brudzynski, 2001)

• nucleus Accumbens - mPFCx functional connectivity


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CURRENT APPROACHES & FUTURE DIRECTIONS

Human Studies

Neuro-Robotics & NeuroProsthetics

Hybrid Brain-Machine interfaces (HBMIs)

• Cochlear implants

• Monitoring & control of epileptic seizures

• Robotic limbs


(A) Seizure control

(B) Robotic arm control

Chips in the Brain ~ Brain-Machine Interfaces (BMIs) ~ Brain-Computer Interfaces

Current issue (May 2007) Scientific American MIND 18 (2): 65-69

Unit activity

vs

LFPs

Signal choice &

Algorithm

Brain-Computer Interfaces

Brain-Computer Interfaces

Recording WorkStation – Plexon IncElectrode array Implant

(1) ENSEMBLE RECORDINGS OF HUMAN SUBCORTICAL NEURONS AS A SOURCE OF MOTOR CONTROL SIGNALS FOR A BRAIN-MACHINE INTERFACE

Parag G. Patil et al - Neurosurgery (2004) – Duke University

~ 32-channel PtIr 40m wire array

~ 4 Deep Brain Stimulation electrodes

[Medtronics DBS]

Unit recordings – 4 microwires

Thalamic VOP/VIN STN

[Plexon Inc MAP recording system]

(2) ENSEMBLE RECORDINGS OF HUMAN SUBCORTICAL NEURONS AS A SOURCE OF MOTOR CONTROL SIGNALS FOR A BRAIN-MACHINE INTERFACE

Parag G. Patil et al - Neurosurgery (2004) – Duke University

STN recording ~ 24 units

Patient performance motor task

REFERENCES Human / Primate Recording Reviews

• LEARNING TO CONTROL A BRAIN–MACHINE INTERFACE FOR REACHING AND GRASPING BY PRIMATES

JM Carmena, et alPLoS Biology ~ http://biology.plosjournals.org 1(2): 193-208 (2003)

• ENSEMBLE RECORDINGS OF HUMAN SUBCORTICAL NEURONS AS A SOURCE OF MOTOR CONTROL SIGNALS FOR A BRAIN-MACHINE INTERFACE

PG Patil, JM. Carmena, Miguel AL Nicolelis & DA TurnerNeurosurgery 55(1): 27-38 (2004)

• ASSISTIVE TECHNOLOGY & ROBITC CONTROL USING MOTOR CORTEX ENSEMBLE-BASED NEURAL INTERFACE SYSTEMS IN HUMANS WITH TETRAPLEGIA

JP Donoghue et alJ . Physiol 579(3) 603-611 (2007)


Lab web sitewww.nottingham.ac.uk/neuronal-networks


That’s all folks

in vivo Basal

Bicuculline (7.5 mg.kg-1 i.v.)

in vivo Basal

Kainate (10 mg.kg- I i.v.)


Project #4: Affective state

Cortico-limibic network interactions

• anxiety effects of maternal separationpharmacologically-induced (e.g. FG-7142)

anxiety

• behavioural sequalaerodent ultrasound vocalisations



Project #5:

Nociception & pain management

• dual spinal cord / supraspinal recording

Role of endocannabinoid system in normal physiology and pain (e.g. neuropathic) states


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Mechanically-evoked response in somatosensory thalamus (VPM)

• Cannabinoid receptor agonists are antinociceptive.

• CB1 predominantly expressed in the CNS but also present in the periphery.

• CB2 agonists inhibit:Acute pain [Zimmer et al.]

Inflammatory pain [Clayton et al.]

Neuropathic pain [Ibrahim et al.]

• CB2 receptors located on:Immune cellsNeuronal cells (?) [Griffin et al; Ross et al; Patel et al.]

• CB2 agonists lack CNS side effects.

Development of potent selective CB2 ligands:Agonist: JWH-133 Ki 3.4nM with a 200-fold selectivity over CB1 receptors.Antagonist: SR144528 Ki 0.67nM with a 50-fold selectivity over CB1 receptors.

Aim: To determine the involvement of the CB2 receptor in nociceptive processing.

The Role of the CB2 Receptor in Nociceptive Processing:

An in vivo electrophysiological studyNeuronal Networks Laboratory

Combined unit / LFP with USV / behavioural recording

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USV call start time

unit activity - nuc accumbens

USV Recorder input

Behavioural data

Local Field Potential (LFP)

Spectrogram of specific calls [AviSoft]

Behavioural Electrophysiology

Circular arena recording using CinePlex

Movie - rat HopScotch: 8-channel array in nuc. accumbens - 6 weeks post implant


Recorded video

Neural data

DISCUSSION • Following KA, hippocampal units (~80%) show an increase in firing; while mPFC units show either a decrease (~80%) or increase (~20%) in firing rate. mPFC units lose their characteristic bursting pattern after KA administration.

• CCH analysis shows that unit pair activity under basal conditions is more correlated within the mPFC compared to intra-hippocampal; mPFC appears to lead hippocampal firing. KA increased correlation within the hippocampus and mPFC; but the mPFC-hippocampal drive was lost.

• PDC of unit population activity also shows basal mPFC-hippocampal directionality (predominantly at low frequencies); this initially decreases after KA administration, then later increases at all frequencies.

• In basal conditions, PDC analysis of LFPs revealed evidence of information flow from CA3 to CA1 and reciprocal hippocampal-mPFC connectivity with predominant drive from mPFC to hippocampus. Following KA, there was increased drive from mPFC to hippocampus.

This alteration in functional connectivity in a seizure model has implications for memory and learning in epilepsy.

Caveat(s):

Need to consider possible influence of anaesthesia in directing “information flow” and/or (anaesthesia/KA-induced) short-term rewiring of neural circuitry. Other regions (not recorded) may be involved in communication between mPFC and hippocampus.

Sorting the connections with multi-electrode neuronal ensemble recording techniques:

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