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Monitoring Dopamine Ex Vivo During Electrical Stimulation Using Liquid-Microjunction Surface Sampling Emily L Gill, Megan Marks, Richard A Yost, Vinata Vedam-Mai, and Timothy Garrett Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04463 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017
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Analytical Chemistry
Monitoring Dopamine Ex Vivo During Electrical Stimulation Using Liquid-Microjunction Surface Sampling Emily L. Gill, ¶ Megan Marks, ¶ Richard A. Yost, ¶, § Vinata Vedam-Mai, ¥, ‡ and Timothy J. Garrett *, ‡, § ¶
Department of Chemistry, University of Florida, Gainesville, Florida, 32611 USA Department of Neurosurgery, University of Florida, Gainesville, Florida, 32610 USA § Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, 32610 USA ¥
ABSTRACT: Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ micro-extraction device, in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization (ESI) source. However, unlike traditional mass spectrometry (MS) techniques it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. PD is a neurological disorder, which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid (aCSF) to create near-physiological conditions, before direct sampling using LMJ-SS. A selection of metabolites including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database, mzCloud, to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post DBS, this is the first study to do so using MS methodologies.
English surgeon Dr. James Parkinson (1755-1824) can be credited with formal recognition and documentation of Parkinson’s disease (PD) in his essay entitled, An Essay on the Shaking Palsy (published in 1817).1 PD is a neurodegenerative disorder wherein the dopaminergic neurons of the ventrolateral compartment of the substantia nigra pars compacta specifically undergo degeneration, and the ensuing loss of dopamine results in the disruption in function of striatal circuits.2 This causes an imbalance of the direct and indirect pathways through the basal ganglia. The Parkinson’s Disease Foundation estimates that more than 10 million people are living with PD worldwide, and to date there are no definitive biomarkers for the disease, or treatments available to slows disease progression.3,4 The gold standard drug therapy for PD is levodopa, a dopamine replacement drug. For medication refractory patients, surgical procedures such as deep brain stimulation (DBS) are available. DBS is a surgical procedure that involves implanting an electrode in a specific target nucleus of the brain, delivering electrical impulses to this target via a pacemaker like device, helping to alleviate the symptoms of PD.5 It is important to note, that DBS does not replace or reverse the death of dopaminergic cells, rather it only offers symptomatic benefits. Owing to the fact that the etiopathogenesis of PD is not fully understood it is not surprising that the therapeutic mechanism of DBS is yet
to be fully elucidated. Understanding the therapeutic mechanism of DBS may provide further insight into the pathogenesis of the disorder. Mass spectrometry (MS) methodologies have been applied to the study of PD including, liquid chromatographymass spectrometry (LC-MS) and matrix assisted laser desorption ionization (MALDI).6,7,8 MS has typically been applied to postmortem or homogenized tissue samples, however, LC-MS coupled to in vivo microdialysis for the detection of neurotransmitters has been reported.9,10 Monitoring temporal changes using in vivo or ex vivo tissue can provide information regarding the relationship between neurotransmitters and other molecules and disease state and/or therapeutic intervention.11 Furthermore, microdialysis has proven a promising technique for monitoring metabolites during electrical stimulation events.12 Gu et al. report an electrically evoked release of dopamine using microdialysis coupled to liquid chromatography (LC) and an electrochemical detector.13 Furthermore, cyclic voltammetry (CV) has also been applied to in vivo electrical stimulation, successfully detecting a release of catecholamine’s, such as dopamine in rat striatum during a stimulation event.14 Dr. Kendall H. Lee from the Mayo Clinic (Rochester, NY) developed the Wireless Instantaneous Neurotransmitter Concentration Sensing System (WINCS) to monitor in vivo neurotransmitters in
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real-time.15,16 WINCS was able to detect subsecond striatal dopamine release in rats following electrical stimulation of dopaminergic fibers.17 Furthermore, Tanaka et al. measured the changes in extracellular dopamine levels in the rat striatum using in vivo microdialysis; they observe an increase in dopamine following transcranial direct current stimulation (tDCS), a non-invasive neurostimulation technique.18 An emerging technique is the liquid-microjunction surface sampling probe (LMJ-SSP) developed under Dr. Gary Van Berkel at Oak Ridge National Laboratory (Oak Ridge, TN).19,20 By applying the venturi effect which relates to the reduction in pressure when a solvent flows through narrow pipes, this method utilizes the continuous flow of extraction solvent along a pair of coaxial capillaries towards the sample surface acting as an in situ micro-extraction device. The inner capillary then pumps the extraction solvent containing the analyte(s) of interest away from the sample surface using a pneumatically assisted electrospray ionization (ESI). The result is a liquid-junction forming between the probe and the sample surface, which draws in metabolites for ionization using ESI and detection by MS (Figure 1).
Figure 1. A schematic of the liquid-microjunction surface sampling probe (LMJ-SSP) (not to scale). Ambient ionization methodologies remove the vacuum constraints placed on the size and types of samples that can be analyzed and requires minimal sample preparation. Olson et al. have applied LMJ-SS to the direct analysis of cytological specimens.21 Furthermore, Hsu et al. took advantage of the real-time analytical potential of LMJ-SS when analyzing the metabolic signatures of living microorganisms such as yeast and pathogens.22 However, to the authors’ knowledge, the ex vivo analysis of brain tissue has not yet been reported using MS methodologies. In MS methodologies such as matrix assisted laser desorption ionization (MALDI), previously cryopreserved tissue is sectioned to a thickness of between 5 and 25 µm using a cryostat.23 This thickness range compensates for issues such as tears in thin sections and a lack of electrical conductivity, lengthily drying times and poor adhesion of the tissue to the glass slide for thicker sections.23 However, use of thicker tissue sections (>100 µm) are common in neuroscience and electrophysiology as the number of viable cells per-slice is greater whilst providing a larger, more intact neural network.24 Furthermore, freezing the tissue is often avoided to maintain tissue viability. Due to the fact that the
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brain is comprised of approximately 73% H2O, and freezing irreversibly expands H2O, thereby rupturing the cells.25,26 This rupture can cause the release of small molecules into the surrounding tissue disrupting the tissue environment. Therefore, alternative methods of sectioning brain tissue are used for brains which have not been frozen. A vibrotome, or a rodent brain matrix, both analogous to the cryostat, are often used to section fresh rodent brains which are not frozen at thicknesses of > 100 µm. Rodent brain matrices are precast metal sectioning blocks, used for precise and reproducible sectioning of fresh tissue (Figure 2A). This methodology allows for ex vivo real-time tissue measurements, whilst the tissue is bathed in artifical cerebrospinal fluid (aCSF) to maintain near-physiological conditions.27,28 Here, we apply LMJ-SS to ex vivo sections of mouse brain tissue, which have been subject to a short burst of acute electrical stimulation. Using this method, we were able to monitor the intensity of dopamine before and after electrical stimulation in the striatum region. Dopamine is a key neurotransmitter implicated in PD and has been shown to increase following electrical stimulation of the ventral tegmental and caudate nucleus, amoungst others.29,14 Our findings offer a new direction for real-time analysis of ex vivo tissue using mass spectrometric applications. Experimental Section Materials and Apparatus. A 1 mm coronal rodent brain matrix was purchased from ASI Instruments (Warren, MI), aCSF (pH 7.4) was purchased from Ecocyte Bioscience (Austin, TX) and Glucose-free aCSF (pH 7.1) was purchased from Tocris (Bristol, England). A dopamine standard (1 mg/ml in MeOH with 5% 1M HCl) and a norepinephrine standard (100 µg/mL in MeOH) were purchased from Cerilliant (Round Rock, TX, USA). A carnosine, glucose, phosphocreatine and 3,4-dihydroxyphenylacetic acid standard were purchased from Sigma-Aldrich (St Louis, MO). A Stanford Research System DS345 synthesized function generator and stainless steel monopolar electrode (with ground) with a 0.005” (127 µm) diameter tip (MS303/3-AIU/SPC) purchased from Plastics 1 (Roanoke, VA) was used for electrical stimulation. Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (#20148382). All experiments utilized adult C57BL/6 mice (N=4), which were housed with a 12h light-12h dark schedule, and were provided food and water ad libitum. Adult mice were sacrificed and the whole brain harvested immediately, without perfusion. Sectioning. Using a rodent brain matrix, the fresh whole mouse brain was sectioned immediately post sacrifice using a clean sharp razor blade (Figure 2A). The 1 mm sections were placed into a well plate containing aCSF, at pH 7.4 before being transferred onto glass slides for direct sampling (Figure 2B and 2C). Method validation and metabolite identification were performed on naive tissue slices prior to electrical stimulation. For the identification of time sensitive metabolites, a glucose-free aCSF was used. Sections were also frozen at -80 °C and sampled within 7 days to assess differences in signal intensity between fresh tissue maintained under near-physiological conditions and frozen tissue sections (Figure S-1 of the supporting information).
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Analytical Chemistry
Figure 2. Fresh mouse brain tissue (A) inside the brain matrix, (B) 1 mm sections inside wells containing aCSF and, (C) mounted on a glass slide inside the ambient chamber. LMJ-SSP-ESI-MS. A Thermo Scientific Velos Pro Dual Pressure Linear Ion Trap Mass Spectrometer was used for all experiments. All data were collected in positive ion mode. The source voltage was 4 kV and the capillary temperature was 250 °C. For tandem mass spectrometry (MS/MS) a collision induced dissociation (CID) energy of 25 eV was used. The LMJ-SSP used was the commercially available Flowprobe by Prosolia (Indianapolis, IN). For tissue sections, the extraction solvent was 1:3 CHCl3: MeOH (identification) and 100% MeOH (electrical stimulation) at a flow rate of 40 µL/minute with matching gas pressure (high pressure nitrogen). For analysis of the standards, a 10 µL aliquot was pipetted directly into the liquid-junction and the
gas pressure was increased to facilitate over aspiration such that the standard was drawn in by the pneumatically assisted electrospray. To assess background interference, a 10 µL aliquot of aCSF was pipetted into the junction using the same method as the standard (Figure S-2 of the supporting information). Stimulation. The tissue was stimulated using a sinusoidal waveform at 150 Hz and 2 VP-P (AC) (Figure 3), which falls within the range of stimulation in PD patients.30,31 reconstructed ion chromatograms (XIC) represent data collected for 30 seconds with the electrode OFF and again for 30 seconds with the electrode ON (note a delay time associated with the waveform generator, Figure 6). A lower than typically used frequency of 50 Hz and a higher than usually used frequency of 350 Hz were used to see if a similar signal increase was observable. The times chosen was to account for the continuous extraction of metabolites by the LMJ-SSP, ensuring that over extraction had not occurred prior to electrical stimulation. To investigate the effect of duration of stimulation, the LMJ-SSP was turned off after 30 seconds and electrical stimulation was conducted for 1, 2.5 and 5 minutes before being turned back on for 30 seconds, showing an increase in the average intensity of TIC normalized dopamine with time. For all experiments the electrode was resting just beneath the tissue surface within the striatum region of the brain and the ground within the aCSF coating surrounding the tissue slice (Figure 3). Data Analysis. All data was analyzed using Excalibur (Thermo Scientific) and reconstructed in Microsoft Excel. The total ion count (TIC) was used to normalize the data and XIC were smoothed to maximize the signal to noise ratio.32
Figure 3. The experimental setup showing the electrode in position and the location of the striatum region (not to scale).
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Results and Discussion Sample Preparation. The application of rodent brain matrices for preparing 1 mm sections, allowed for maintaining tissue under near-physiological conditions for direct analysis using LMJ-SS (Figure 2A). Sectioning with the rodent brain matrix helped maintain the native structure of the tissue without deformation, which has been an issue in the past particularly with downstream applications utilizing mass spectrometry imaging (MSI) methodologies, as was demonstrated by Gill et al.33 Deformation often occurs after harvest the brain and while preparing it for analysis and does not allow for the maintenance of neural networks ex vivo. The use of aCSF, at pH 7.4 was to enable the tissue to remain “active” and achieve near-physiological condition. Care was taken to collect each biological dataset within a 30 minutes time frame. The methodology utilized is an over simplified adaptation of a perfusion chamber designed by Thomas et al., which allows tissue sections to be maintained under near-physiological conditions whilst preserving the ability to record from the tissue surface (i.e. electrophysiology).34 Similarly, the ambient nature of LMJ-SS provides potential for ex vivo surface sampling. Ex Vivo Analysis. In accordance with reports that axonal and dendritic activity can be maintained outside the body of a mammal for lengthily periods of time following the death of an organism, we sectioned fresh post sacrifice and maintained tissue in aCSF. Thereafter we identified key metabolites such as dopamine by comparison with standards (Figure 4A).35,36 The [dopamine + H]+ precursor ion at m/z 154.2 was isolated and fragmented to yield a variety of
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product ions (Figure 4B and 4C). Diagnostic ions include m/z 110.2 corresponding to the loss of CH2CH2NH2 and m/z 121.9 correspond to the loss of an OH and the NH2 group. Other metabolites identified include norepinephrine, a product of dopamine biosynthesis in the brain, carnosine known for its antioxidant properties and 3,4-dihydroxyphenylacetic acid a metabolic breakdown product of dopamine.37,38 Norepinephrine is thought to be associated with the non-motor symptoms of PD (data not shown for these metabolites).39 Additional confirmation was obtained using mzCloud, a spectra database, including gamma-aminobutyric acid (GABA) a neurotransmitter, and choline a precursor for the neurotransmitter acetylcholine. Strong signals were also observed for the phospholipid/glycerophospholipids region (700-900 Da) (Figure 4A). Rapid analysis is particularly important for timesensitive metabolites, which degrade quickly in active tissue, such as glycogen, phosphates and glucose. Glucose and phosphocreatine, both metabolites involved in energy regulation were identified and have the potential to be markers for metabolically active tissue (Figures S-3A, S-3B, S-4A and S4B of the supporting information). Identifications were made to simply demonstrate that the aCSF does not hinder the analytical capabilities of the LMJ-SSP, compared to the typical frozen tissue sections traditionally analyzed using surface sampling techniques (Figures S-2, of the supporting information) and moving forward dopamine was monitored during electrical stimulation events.
Figure 4. LMJ-SS-ESI mass spectra of, (A) ex vivo mouse brain tissue in aCSF (pH 7.4), (B) an MS/MS spectrum of the precursor ion at m/z 154.2 and, (C) an MS/MS spectrum of the precursor ion at m/z 154.2 corresponding to a dopamine standard.
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Analytical Chemistry
Ex Vivo Electrical Stimulation. In PD, the classic target area for DBS are the subthalamic nucleus and globus pallidus, which are located in the basal ganglia.40 These regions are difficult to target during ex vivo animal slice studies without stereotaxis. Therefore we chose to target the striatum region (Figure 3). The striatum is the largest nucleus in the basal ganglia and is part of the affected network in PD. We chose to study dopamine because it is the key neurotransmitter implicated in PD, with the death of dopaminergic neurons throughout the disease course.2 Using MS, our initial studies showed a significant (p
