Microdialysis Sampling
High Temporal and Spatial Resolution Microdialysis to Identify Neurological Disease and CNS Efficacy Biomarkers Robert Kennedy, Woonghee Lee, Omar Mabrouk, Thomas Slaney, Non Thitaphat, Peng Song, Paige Malec Collaborators: George Rebec, Shelly Flagel, Huda Akil, Margaret Gnegy
Monitoring Neurotransmitters in the Living Brain
Goal: Identify chemical signals in behavior, learning, pharmacology, pathophysiology
Technical Challenge: Measure release and dynamics for all neurotransmitters in vivo at high spatial and temporal resolution
In Vivo Monitoring Methods PET
Some limits: $$$, immobilized
Implantable Sensors
Some limits: Few analytes, one at a time, no basal concentrations
Genetically Encoded Fluorescent Sensors •Peters, et al. (2002) Bioelectrochemistry 9: 461-482 •Okumoto S et al. PNAS 2005;102:8740-8745
Some limits: genetic, sensor development
Microdialysis for In Vivo Monitoring aCSF
Sample transfer tubing
Fractions Or On-line analysls
Pump
2.0
1.5
1.0
0.5
0
Microdialysis Probe
5
10
15
20
Microdialysis Sampling
aCSF 0.3 - 1 mL/min
Fractions Analyzed: HPLC, RIA, MS
-Highly Versatile but… -Typically only measure 1-2 compounds at a time -Low temporal resolution (10 min) because of assay requirements
1-3mm
(1 s = 16 nL sample)
-Low spatial resolution 200mm
Low Molecular Weight Neurotransmitters & Metabolites
Methods for Neurotransmitter Analysis
LC-EC for dopamine, NE, 5-HT LC-Fluorescence for Amino acids Enzyme assay for Ach LC-MS?
Recent Progress in LC-MS Methods for Neurotransmitter Analysis
Polar neurotransmitters hard to retain Ion-pairing Hydrophilic Interaction LC (HILIC) is promising but LOD problematic
Ji et al., Anal. Chem. 2008, 80. 9195-9203 Zhang et al., Rapid Commun. Mass Spectrom. 2007, 21. 3621-3628
Benzoyl Chloride as Derivatization Reagent
Polar compounds retained Improves MS sensitivity Easy to quantify Reacts with many functional groups
Multicomponent Neurochemical Detection by 400 Benzoylation with LC-MS 350 300 250
%
GABA GluTh Gly Ser 200 Glu Hist Asp 150 Carn Gln Tau 100 ACh 50
HVA
Oct
NM 5HIAA
0 2 4 6 Time (min)
8
10
12
Epi DA
3MT DOPAC AMPH
Ado 0
LDOPA 5HT
NE
14
16
Dialysate Plasma 18 Tissue 20 22 24
Neurochemical Phentotyping by Microdialysis: Drug Abuse Vulnerability bred High Responders (bHR) – – – – –
Increased cocaine self-administration Greater response to stressors Novelty seeking More aggressive “Impulsive”
bred Low Responders (bLR) – Less likely to self-administer – Avoid novelty – “Anxious”
Flagel et al., 2010 Neuropsychopharmacology
Neurochemical changes in response to cocaine
Norepinephrine differences
Norepinephrine – Involved in stress and mood – Thought to be involved in ADHD and addiction – Recently shown to modulate cocaine induced dopamine release and hyperactivity in the nucleus accumbens Mitrano et al., 2011 Neuropsychopharm
Neurotransmitteromics uncovered norepinephrine as a possible major contributor to bHR and bLR behavioral traits
Amphetamine • 2nd most abused drug • Approximately 15% of 10th graders have used amphetamines without a prescription.
Effects of Amphetamine • Amphetamine: – euphoria – hyperactivity – tachycardia – appetite suppression – addiction and craving
• Increases extracellular dopamine • No current universally accepted chemical treatment for abuse.
Amphetamine
18
Amphetamine Promotes PKCb Activation & Enhances Outward DA Flux Through DAT
Amp
+
PKCβ
_
+ DAT
Gnegy M, Eur. J. Pharm. 2003, 479: 83
CCG215103
Inhibits Protein Kinase C (PKC) CNS Permeable
20
Subcutaneous Administration of 6 mg /kg CCG215103 Decreases AMPH Effects Locomotion
Effect on endogenous levels of neurochemicals
1-way RM ANOVA 22 p < 0.0001
Effect on endogenous levels of neurochemicals
1-way RM ANOVA
23 p < 0.0001
Extending the Method: 70 Compounds
Classification of 70 BzCl Labeled Metabolites Misc
Di/Tripeptides
Neurotransmitters
Polyamines Trace Amines
Amino Acid Metabolites
Neurotransmitter Metabolites
Amino Acids
Microdialysis for In Vivo Monitoring: Temporal Resolution Issues aCSF Pump
Microdialysis Probe
Sample transfer tubing
Fractions
Fraction volume will decrease with increasing temporal resolution
Analytical Challenge of High Temporal Resolution Sampling 4000
1.2x10 1.0
Sample Per h
3000 0.8
2500 2000
0.6
1500
0.4
1000 0.2
500
Higher Throughput
0
0.0 0
2 4 6 8 Temporal resolution (min)
10
Moles of analyte per sample
3500
-11
Use Segmented Flow to Collect Nanoliter Fractions from Dialysis Probes Sample
Immiscible Fluid (fluorinated oil/ gas)
On-Board Droplet Generator for Awake Animal Experiments with Reagent Addition Dialysis Probe
Droplet Generator
Coupling to Analysis On-line analysis
MS Enzyme CE
PDMS Headpiece aCSF 4 cm Volume ~2 nL
Off-line Analysis Collection Tubing
Pump
MS Enzyme CE
Microdialysis Interfaced to Segmented Flow ESI-MS
“MS-Based Sensor”
MS Sensing In Vivo: ACh Response
Recording Rapid Chemical Changes in the Brain
50 25 0
Aspartate (% of Basal)
0
200
400
600
800
1000
Aspartate 10 s
400
300B. K.; Pomerleau, F.; Burmeister, Day, J. J.; Huettl, P.; Gerhardt, G. A. J. 200 Neurochem. 2006, 96, 1626-1635. 100 0 0
200
400
600
Glutamate 500
10 s
400 300 200 100 0 0
800
1000
Taurine (% of Basal)
GABA mM)
10 s
Glutamate (% of Basal)
GABA
200
200
400
Taurine
600
800
1000
10 s
150 100 50 0 0
200
400
600
800
1000
Huntington’s Disease • “CAG Repeat” disease • Neurodegenerative • Cognitive and motor impairment • Death within 15 years • ~30k cases in USA • Little treatment available
Glutamate Neurotransmission Decrease of Glt-1 expression (glutamate toxicity?)
HD Mice Have Impaired Glu Uptake Measured by Microdialysis with Electrophoresis Percent Basal (Glu)
220
WT
160
100
40 80 s
HD
Drugs to Affect Glutamate Transporter
“beta-Lactam antibiotics offer neuroprotection by increasing glutamate transporter expression” by Rothstein JD, Patel S, Regan MR, et al. NATURE, 2005, 433: 73-77
ceftriaxone
Increase of Glt-1 with Ceftriaxone Treatment
Ceftriaxone Improves Motor Performance
Recovery of Normal Glu Uptake in HD Mice with Cef Percent Basal (Glu)
220
Control 160
HD (R6/2)
HD + Cef
S S S
100
40 80 s
Spatial Resolution Issues
mm
Ventral Tegmental Area (VTA)
Miniaturized Alternative to Microdialysis Sampling Microfabricated “Push-Pull” Sampling Probe
Improved Spatial Resolution
Pull
Push
1 mm
Microdialysis PushPull
50 mm 50 nL/min flow
Rat Brain “Atlas”
Microfabricated Probes
Channel ID: 19 µm
Inside channel view 50 mm
Evolution of Sampling Probes: Better Spatial Resolution Dialysis Mini Push-pull Microfab Push-pull
Tyrosine Hydroxylase is Localized
Red Nucleus
Ventral Tegmental Area
0.5 mm
Ikemoto, S. Neuroscience & Biobehavioral Reviews 2010, 35, 129-150.
Spatially Resolved Measurements in VTA/RN
Microinjection into the Brain to Evoke Local Changes in Neurochemistry Microinjector
2 mm Dialysis Probe
Push-pull
500
10 s
400 300 200 100 0
Taurine (% of Basal)
0
200
400
600
800
10 s
150 100 50
2 mm probe
0 0
200
400
600
800
Glutamate
1500
1000
10 s
1000 500 0 0
1000
Taurine
200
Glutamate (% of Basal)
Glutamate
Taurine (% of Basal)
Glutamate (% of Basal)
Effect of Probe Size on Measuring Local 2 mm Probe Changes 0.5 mm Probe
200
50 100 150 Relative Time (s) Taurine
10 s
100
0
Small probe
Less Invasive Sampling Track Dialysis Probe Track
mm
Photograph of Probe with Droplet Generator
R1
Push Oil
1 cm
R2
PDMS chip Pull Collection Tubing
Probe
Combining High Temporal and Spatial Resolution R2
R1
Plugs Oil
Vacuum
PDMS chip Holder
Inlet
500 µM Probe
Sample
In Vitro Sampling of Glutamate at 150 nL/min 120000
R² = 0.996
120000 13 µM 60000
9 µM
80000
0
RFU
0
5 10 [Glu], µM
15
6 µM
40000
4 µM 2 µM
0 220
270
320
• Rise time = 5 s
370 Time (s)
420
470
520
Microfabricated Dialysis Probe
Summary • LC-MS Method for 70 neurochemicals • Used to assess drug effects, disease states, and • •
behavior Microfabricated probes for better spatial resolution Droplet technology for high temporal resolution
Acknowledgements
Monitoring Neurotransmitters in the Living Brain
Goal: Identify chemical signals in behavior, learning, pharmacology, pathophysiology
Technical Challenge: Measure release and dynamics for all neurotransmitters in vivo at high spatial and temporal resolution
In Vivo Monitoring Methods PET
Some limits: $$$, immobilized
Implantable Sensors
Some limits: Few analytes, one at a time, no basal concentrations
Genetically Encoded Fluorescent Sensors •Peters, et al. (2002) Bioelectrochemistry 9: 461-482 •Okumoto S et al. PNAS 2005;102:8740-8745
Some limits: genetic, sensor development
Microdialysis for In Vivo Monitoring aCSF
Sample transfer tubing
Fractions Or On-line analysls
Pump
2.0
1.5
1.0
0.5
0
Microdialysis Probe
5
10
15
20
Microdialysis Sampling
aCSF 0.3 - 1 mL/min
Fractions Analyzed: HPLC, RIA, MS
-Highly Versatile but… -Typically only measure 1-2 compounds at a time -Low temporal resolution (10 min) because of assay requirements
1-3mm
(1 s = 16 nL sample)
-Low spatial resolution 200mm
Low Molecular Weight Neurotransmitters & Metabolites
Methods for Neurotransmitter Analysis
LC-EC for dopamine, NE, 5-HT LC-Fluorescence for Amino acids Enzyme assay for Ach LC-MS?
Recent Progress in LC-MS Methods for Neurotransmitter Analysis
Polar neurotransmitters hard to retain Ion-pairing Hydrophilic Interaction LC (HILIC) is promising but LOD problematic
Ji et al., Anal. Chem. 2008, 80. 9195-9203 Zhang et al., Rapid Commun. Mass Spectrom. 2007, 21. 3621-3628
Benzoyl Chloride as Derivatization Reagent
Polar compounds retained Improves MS sensitivity Easy to quantify Reacts with many functional groups
Multicomponent Neurochemical Detection by 400 Benzoylation with LC-MS 350 300 250
%
GABA GluTh Gly Ser 200 Glu Hist Asp 150 Carn Gln Tau 100 ACh 50
HVA
Oct
NM 5HIAA
0 2 4 6 Time (min)
8
10
12
Epi DA
3MT DOPAC AMPH
Ado 0
LDOPA 5HT
NE
14
16
Dialysate Plasma 18 Tissue 20 22 24
Neurochemical Phentotyping by Microdialysis: Drug Abuse Vulnerability bred High Responders (bHR) – – – – –
Increased cocaine self-administration Greater response to stressors Novelty seeking More aggressive “Impulsive”
bred Low Responders (bLR) – Less likely to self-administer – Avoid novelty – “Anxious”
Flagel et al., 2010 Neuropsychopharmacology
Neurochemical changes in response to cocaine
Norepinephrine differences
Norepinephrine – Involved in stress and mood – Thought to be involved in ADHD and addiction – Recently shown to modulate cocaine induced dopamine release and hyperactivity in the nucleus accumbens Mitrano et al., 2011 Neuropsychopharm
Neurotransmitteromics uncovered norepinephrine as a possible major contributor to bHR and bLR behavioral traits
Amphetamine • 2nd most abused drug • Approximately 15% of 10th graders have used amphetamines without a prescription.
Effects of Amphetamine • Amphetamine: – euphoria – hyperactivity – tachycardia – appetite suppression – addiction and craving
• Increases extracellular dopamine • No current universally accepted chemical treatment for abuse.
Amphetamine
18
Amphetamine Promotes PKCb Activation & Enhances Outward DA Flux Through DAT
Amp
+
PKCβ
_
+ DAT
Gnegy M, Eur. J. Pharm. 2003, 479: 83
CCG215103
Inhibits Protein Kinase C (PKC) CNS Permeable
20
Subcutaneous Administration of 6 mg /kg CCG215103 Decreases AMPH Effects Locomotion
Effect on endogenous levels of neurochemicals
1-way RM ANOVA 22 p < 0.0001
Effect on endogenous levels of neurochemicals
1-way RM ANOVA
23 p < 0.0001
Extending the Method: 70 Compounds
Classification of 70 BzCl Labeled Metabolites Misc
Di/Tripeptides
Neurotransmitters
Polyamines Trace Amines
Amino Acid Metabolites
Neurotransmitter Metabolites
Amino Acids
Microdialysis for In Vivo Monitoring: Temporal Resolution Issues aCSF Pump
Microdialysis Probe
Sample transfer tubing
Fractions
Fraction volume will decrease with increasing temporal resolution
Analytical Challenge of High Temporal Resolution Sampling 4000
1.2x10 1.0
Sample Per h
3000 0.8
2500 2000
0.6
1500
0.4
1000 0.2
500
Higher Throughput
0
0.0 0
2 4 6 8 Temporal resolution (min)
10
Moles of analyte per sample
3500
-11
Use Segmented Flow to Collect Nanoliter Fractions from Dialysis Probes Sample
Immiscible Fluid (fluorinated oil/ gas)
On-Board Droplet Generator for Awake Animal Experiments with Reagent Addition Dialysis Probe
Droplet Generator
Coupling to Analysis On-line analysis
MS Enzyme CE
PDMS Headpiece aCSF 4 cm Volume ~2 nL
Off-line Analysis Collection Tubing
Pump
MS Enzyme CE
Microdialysis Interfaced to Segmented Flow ESI-MS
“MS-Based Sensor”
MS Sensing In Vivo: ACh Response
Recording Rapid Chemical Changes in the Brain
50 25 0
Aspartate (% of Basal)
0
200
400
600
800
1000
Aspartate 10 s
400
300B. K.; Pomerleau, F.; Burmeister, Day, J. J.; Huettl, P.; Gerhardt, G. A. J. 200 Neurochem. 2006, 96, 1626-1635. 100 0 0
200
400
600
Glutamate 500
10 s
400 300 200 100 0 0
800
1000
Taurine (% of Basal)
GABA mM)
10 s
Glutamate (% of Basal)
GABA
200
200
400
Taurine
600
800
1000
10 s
150 100 50 0 0
200
400
600
800
1000
Huntington’s Disease • “CAG Repeat” disease • Neurodegenerative • Cognitive and motor impairment • Death within 15 years • ~30k cases in USA • Little treatment available
Glutamate Neurotransmission Decrease of Glt-1 expression (glutamate toxicity?)
HD Mice Have Impaired Glu Uptake Measured by Microdialysis with Electrophoresis Percent Basal (Glu)
220
WT
160
100
40 80 s
HD
Drugs to Affect Glutamate Transporter
“beta-Lactam antibiotics offer neuroprotection by increasing glutamate transporter expression” by Rothstein JD, Patel S, Regan MR, et al. NATURE, 2005, 433: 73-77
ceftriaxone
Increase of Glt-1 with Ceftriaxone Treatment
Ceftriaxone Improves Motor Performance
Recovery of Normal Glu Uptake in HD Mice with Cef Percent Basal (Glu)
220
Control 160
HD (R6/2)
HD + Cef
S S S
100
40 80 s
Spatial Resolution Issues
mm
Ventral Tegmental Area (VTA)
Miniaturized Alternative to Microdialysis Sampling Microfabricated “Push-Pull” Sampling Probe
Improved Spatial Resolution
Pull
Push
1 mm
Microdialysis PushPull
50 mm 50 nL/min flow
Rat Brain “Atlas”
Microfabricated Probes
Channel ID: 19 µm
Inside channel view 50 mm
Evolution of Sampling Probes: Better Spatial Resolution Dialysis Mini Push-pull Microfab Push-pull
Tyrosine Hydroxylase is Localized
Red Nucleus
Ventral Tegmental Area
0.5 mm
Ikemoto, S. Neuroscience & Biobehavioral Reviews 2010, 35, 129-150.
Spatially Resolved Measurements in VTA/RN
Microinjection into the Brain to Evoke Local Changes in Neurochemistry Microinjector
2 mm Dialysis Probe
Push-pull
500
10 s
400 300 200 100 0
Taurine (% of Basal)
0
200
400
600
800
10 s
150 100 50
2 mm probe
0 0
200
400
600
800
Glutamate
1500
1000
10 s
1000 500 0 0
1000
Taurine
200
Glutamate (% of Basal)
Glutamate
Taurine (% of Basal)
Glutamate (% of Basal)
Effect of Probe Size on Measuring Local 2 mm Probe Changes 0.5 mm Probe
200
50 100 150 Relative Time (s) Taurine
10 s
100
0
Small probe
Less Invasive Sampling Track Dialysis Probe Track
mm
Photograph of Probe with Droplet Generator
R1
Push Oil
1 cm
R2
PDMS chip Pull Collection Tubing
Probe
Combining High Temporal and Spatial Resolution R2
R1
Plugs Oil
Vacuum
PDMS chip Holder
Inlet
500 µM Probe
Sample
In Vitro Sampling of Glutamate at 150 nL/min 120000
R² = 0.996
120000 13 µM 60000
9 µM
80000
0
RFU
0
5 10 [Glu], µM
15
6 µM
40000
4 µM 2 µM
0 220
270
320
• Rise time = 5 s
370 Time (s)
420
470
520
Microfabricated Dialysis Probe
Summary • LC-MS Method for 70 neurochemicals • Used to assess drug effects, disease states, and • •
behavior Microfabricated probes for better spatial resolution Droplet technology for high temporal resolution
Acknowledgements
