Comonitoring of adenosine and dopamine using the wireless instantaneous neurotransmitter concentration system : proof of principle : laboratory investigation

Object

The authors of previous studies have demonstrated that local adenosine efflux may contribute to the therapeutic mechanism of action of thalamic deep brain stimulation (DBS) for essential tremor. Real-time monitoring of the neurochemical output of DBS-targeted regions may thus advance functional neurosurgical procedures by identifying candidate neurotransmitters and neuromodulators involved in the physiological effects of DBS. This would in turn permit the development of a method of chemically guided placement of DBS electrodes in vivo. Designed in compliance with FDA-recognized standards for medical electrical device safety, the authors report on the utility of the Wireless Instantaneous Neurotransmitter Concentration System (WINCS) for real-time comonitoring of electrical stimulation–evoked adenosine and dopamine efflux in vivo, utilizing fast-scan cyclic voltammetry (FSCV) at a polyacrylonitrile-based (T-650) carbon fiber microelectrode (CFM).
Methods

The WINCS was used for FSCV, which consisted of a triangle wave scanned between −0.4 and +1.5 V at a rate of 400 V/second and applied at 10 Hz. All voltages applied to the CFM were with respect to an Ag/AgCl reference electrode. The CFM was constructed by aspirating a single T-650 carbon fiber (r = 2.5 μm) into a glass capillary and pulling to a microscopic tip using a pipette puller. The exposed carbon fiber (the sensing region) extended beyond the glass insulation by ~ 50 μm. Proof of principle tests included in vitro measurements of adenosine and dopamine, as well as in vivo measurements in urethane-anesthetized rats by monitoring adenosine and dopamine efflux in the dorsomedial caudate putamen evoked by high-frequency electrical stimulation of the ventral tegmental area and substantia nigra.
Results

The WINCS provided reliable, high-fidelity measurements of adenosine efflux. Peak oxidative currents appeared at +1.5 V and at +1.0 V for adenosine, separate from the peak oxidative current at +0.6 V for dopamine. The WINCS detected subsecond adenosine and dopamine efflux in the caudate putamen at an implanted CFM during high-frequency stimulation of the ventral tegmental area and substantia nigra. Both in vitro and in vivo testing demonstrated that WINCS can detect adenosine in the presence of other easily oxidizable neurochemicals such as dopamine comparable to the detection abilities of a conventional hardwired electrochemical system for FSCV.
Conclusions

Altogether, these results demonstrate that WINCS is well suited for wireless monitoring of high-frequency stimulation-evoked changes in brain extracellular concentrations of adenosine. Clinical applications of selective adenosine measurements may prove important to the future development of DBS technology.

Wireless instantaneous neurotransmitter concentration system-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring - laboratory investigation

Object  In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time.

Methods  The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig.

Results   The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA.

Conclusions  By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

The taq IA and Ser311Cys polymorphisms in the dopamine D2 receptor gene and obesity

Dopamine D2 receptors (DRD2) in the central nervous system are involved in the regulation of feeding. It remains to be elucidated if mutations in the DRD2 gene contribute to the development of obesity. The aim of the present study was to investigate whether the Taq IA and Ser311Cys polymorphisms in the DRD2 gene are associated with obesity in Nauruan and Australian subjects. Subjects were selected based on extremes of the body mass index (BMI) distribution. Two groups of Australian women were selected. The leanest group had a mean BMI of 22.5 kg/m2 (range: 20.3-24.3) and the heaviest group had a mean of 36.1 kg/m2 (32.5-44.1). Four groups of Nauruan subjects were selected. Leanest men had a mean BMI of 33.0 kg/m2 (28.4-36.9), heaviest men had a mean of 52.8 kg/m2 (46.5-69.2), leanest women had a mean of 34.8 kg/m2 (28.2-41.8) and heaviest women had a mean of 55.1 kg/m2 (49.3-73.8). Subjects were genotyped for the Taq IA and Ser311Cys polymorphisms using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and allelic discrimination TaqmanTM PCR respectively. Leanest and heaviest groups were examined for differences in genotype frequency. Taq IA and Ser311Cys genotype frequencies did not differ significantly between leanest and heaviest Nauruan groups, or between leanest and heaviest Australians. Haplotype frequencies of these polymorphisms did not differ between leanest and heaviest groups. The Taq IA and Ser311Cys polymorphisms in the DRD2 gene are unlikely to be common causes of obesity in these populations.

Modeling gene-environment interaction in longitudinal data : risk for neuroticism due to interaction between maternal care and the Dopamine 4 Receptor gene (DRD4)

The purpose of this study was to investigate risk for neuroticism due to the joint action of low maternal care and compromised mesocorticolimbic ‘reward’ system function linked to a variable number tandem repeat (VNTR) in the dopamine 4 receptor gene (DRD4). Data were drawn from the Victorian Adolescent Health Cohort Study, a longitudinal study of the health and well-being of 2,000 young Australians followed from adolescence to young adulthood across 8 waves from 14- to 28-years. Genetic risk was defined by carriage of at least one copy of the 7-repeat allele or derivative alleles 5, 6, and 8 (labeled 7R+). Neuroticism was assessed in adolescence and young adulthood. We observed an approximately fourfold increase in the odds of reporting neurotic symptoms in carriers of the 7R+ disposition who reported low maternal care compared with non-carriers who reported high maternal care. The percentage of risk attributable to mechanisms in which both factors played a role was 35%. Findings are discussed in terms of implications for prevention.

Purity and enrichment of laser-microdissected midbrain dopamine neurons

The ability to microdissect individual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected sample. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected sample relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected samples. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells.

Hypothalamo-pituitary adrenal axis and sympatho-adrenal medullary system responses to psychological stress were not attenuated in women with elevated physical fitness levels

It is not clear if higher levels of cardiorespiratory fitness are associated with lower hypothalamo-pituitary adrenal (HPA) axis and sympatho-adrenal medullary (SAM) system reactivity to psychological stress in women. The association between cardio-metabolic risk markers and acute physiological responses to psychological stress in women who differ in their cardiorespiratory fitness status has also not been investigated. Women with high (n = 22) and low (n = 22) levels of fitness aged 30-50 years (in the mid-follicular phase of the menstrual cycle) were subjected to a Trier Social Stress Test (TSST) at 1500 h. Plasma concentrations of cortisol, adrenaline (Adr), noradrenaline (NA), and dopamine (DA) were measured in samples collected every 7-15 min from 1400 to 1700 h. Heart rate and blood pressure were measured at the same time points. Low-fit women had elevated serum triglyceride, cholesterol/HDL ratio, fasting glucose, and HOMA-IR levels compared with high-fit women. While cortisol, Adr, NA, HR, and blood pressure all demonstrated a significant response to the TSST, the responses of these variables did not differ significantly between high- and low-fit women in response to the TSST. Dopamine reactivity was significantly higher in the low-fit women compared with high-fit women. There was also a significant negative correlation between VO2 max and DA reactivity. These findings suggest that, for low-fit women aged 30-50 years, the response of HPA axis and SAM system to a potent acute psychological stressor is not compromised compared to that in high-fit women.