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Fluorescent Nano sensors as a use to measure Dopamine

Fluorescent Nanosensors are a specific type of nanosensor based on single walled carbon nanotubes (SWNTs) (SWCNT) that have been synthesized with a near infrared (nir) fluorescent polymers. This enables the nanosensor to luminesce in specific conditions, such as, when it encounters dopamine (DA). This type of nanosensor has also been able detect molecules such as, serotonin, norepinephrine, fibrinogen, and estradiol. Fluorescent nanosensors have been utilized in modern research to further the understanding of chemical signaling in the brain. Dopamine (DA) is a modulatory neurotransmitter, it is thought to play a large role in the learning and reward systems in the brain. A lack of optimum neurotransmission in dopaminergic pathways is thought to be linked to multiple neurodegenerative diseases such as Parkinson’s disease and schizophrenia.

Introduction

Cell to cell signaling is a fundamental biological process that negates the foundations of life. It is fundamental for the proper functioning of the central nervous system and physiological processes. One important aspect of cell signaling is the ability of neurons to transmit and process information. Despite the many advancements in neuroscience, and work on the Dopamine hypothesis of schizophrenia little is known about the neurotransmission of dopamine, and what mediates its release. There have been several analytical measures in the past that have set out to measure neurotransmission such as MRI methods with contrast agents or electrochemical approaches, however, Mann et al (2017) have acknowledged a need for greater tools with higher spatial and temporal resolution. Janasoff et al (2020) also acknowledges that there has been little work on neurochemical function as tools to study dopaminergic activity have not been readily available. The ability to measure neural interactions and circuits in the brain had been relatively unexplored with nanotechnology until the recent development of fluorescent nanotubes, which has been the primary method to directly look at neurotransmitter activity in the brain. Studies have shown that the nanosensor can activate and fluoresce in the presence of Dopamine

Techniques in studying chemical signaling have been implemented that can allow detection of nanomolecular neurotransmitters such as dopamine. Nanosensors have had applications and extensive use already in sectors such as healthcare and the environmental sector. In these circumstances the nanosensors involved were different categories of nanosensors such as DNA, enzyme, and temperature nanosensors. In current dopamine nanosensor research, the nanosensor of interest comes under the category known as a molecular nanosensor, as it can react in the presence of a specific molecule. Fluorescent nanosensors are implemented in the study of cell signaling because they can detect chemical changes at a nanomolecular level. There size can range from (~10-9 m diameter, ~10-6 m length) and they can react to specific molecules and luminesce within the infrared spectrum. At present the research using the fluorescent nanosensors has only been conducted on transcranial slices of mouse brain. In vivo deep tissue imaging is a hopeful application for future research.

Dopamine is a neuromodulator, which operates via modulatory neurotransmission. Unlike other neurotransmitters such as GABA and Glutamate which operate via classic neurotransmission. In classic neurotransmission, the concentrations of GABA and Glutamate rise in the presynaptic cleft to mediate communication between presynaptic and post synaptic neurons. Neuromodulators like dopamine however can diffuse beyond the synaptic cleft and form multiple types of release sites and even release from the somata and dendrites. It is because of this difference that dopaminergic release is described as ‘highly heterogenous across release sites’.

Materials & Methods Nanosensor Synthesis In all current methodology the fluorescent nanosensors had to be synthesized in the laboratory where research was conducted. All studies based there nanosensors from single walled carbon nanotubes (SWNTs).

In Beyene et al’s study (GT)6 oligonucleotides and single walled carbon nanotubes (SWNTs) were prepared and mixed in a solution of 100mM of NaCl. The solution was then sonicated and incubated. The solution was then centrifuged at 16,000g for 90 minutes to remove any unsuspended carbon nanotubes. In Elizarova et al’s research they used a suspension of heavy water before centrifuging the SWNTs and (GT)10 oligonucleotides as this helped to separate the non-functionalized SWNTs. Functionalized nanosensors were then measured for their infrared absorption with a UV-VIS-nIR Spectrometer.

Microscope configuration A microscope was configured to capture the luminescence when the fluorescent nanotubes encountered dopamine in ex vivo imaging of brain slices. Elizarova et al used a custom-built Olympus IX53 microscope in their investigation, Beyene et al also used an Olympus microscope. This microscope was specialized to capture nIR and visible light. NIR imaging was captured and excited using a laser. Images were acquired with specialized cameras imaged at 15 frames per second on all cell cultures.

Brain slice acquisition Current research using fluorescent nanosensors has only investigated dopamine neurotransmission in ex vivo transcranial slices of mice brain. 60-day old male and female mice were were deeply anesthetized, and a brain extraction was performed, and coronal slices of the dorsal striatum were produced. Mouse brain slices were incubated in artificial cerebral spinal fluid and the nanosensors were allowed to diffuse in the brain tissue for 15 minutes. Neuromodulator release was stimulated with temporal control of tissue stimulation. A bipolar stimulating electrode was implemented to evoke terminal release within the dorsomedial striatum of the mouse brain slices. It was found that a single pulse could elicit a nIRCat signal and that increasing the strength of the stimulus led to larger evoked changes in nIRCat signal. .

Findings This new synthesized nanosensor was reported to be able to luminesce in DA concentrations ranging from 10 to 100 M DA concentration. nIRCats which were the name of the fluorescent nanosensor in Beyene et al’s study were also sensitive to norepinephrine (NE). However, there was a higher affinity for DA over NE and the investigation looked at the striatum. This was since the dorsal striatum contains many dopaminergic projections however lacks neurons that release NE. Although the investigation using these new nanosensors was performed in ex vivo brain slices, the findings revealed that the NirCats were successful at distinguishing fluctuations in DA concentration in the presence of ascorbic acid, which is present in cerebrospinal fluid. Elizarova et al used nanosensor layer capable of high spatiotemporal resolution known as AndromeDA. And investigated mixed cultures containing hippocampal and ventral midbrain neurons. Current research assessed the validity of the fluorescent nanosensors, and once it was deemed satisfactory in detecting dopamine, researchers investigated the effects of pharmaceutical drugs. There nanosensor with AndromeDA was used to examine the effect of L-DOPA, which increases DA levels. L-DOPA significantly increased peak AndromeDA activation at regions of interest, confirming the effectiveness of L-DOPA in pharmacological use. Findings in both studies showed the heterogeneity in DA release, as they found ‘hotspots’ where some varicosieties exhibited DA release in some areas and some hotspots exhibited no dopamine release, confirming that DA release sites do not all react in a similar way to certain DA drugs.

Beyene et al (2019) investigated the effects of nomifensine and ACSF. Both drugs are DA reuptake inhibitors that slows the clearance of DA by competitively binding to DA transporters. The findings showed that 10 M of nomifensine to the brain tissue solution with the nIRCat nanosensors yielded higher peak fluorescence modulation [(∆F/F)max = 0.108 ± 0.029 versus 0.189 ± 0.023 (means ± SD); n = 3; P = 0.0178] and a prolonged fluorescent signal compared to signals obtained in ACSF from the same field of view [decay time constant,  = 2.43 ± 0.24 s versus 10.95 ± 1.15 s (means ± SD); n = 3; P = 0.0002; Fig. 2, A (top versus bottom) and C]. DA receptor drugs were also tested, and it was found that nIRCat fluorescence intensity did not increase as the concentration of DA receptor drug concentration increased. Overall, the authors of the study highlight that ‘DA-induced nIRCat fluorescence signals were not altered in the presence of these same drugs.’

Application for future

The ability to detect dopamine using nanotechnology is a recent and major development in the field of neuroscience. The ability to functionalize SWCNT’s has brought interesting findings relating to pharmaceutical DA drugs and the inner workings of dopaminergic transmission. This approach provides scientists with new tools to detect DA transmission and gain further understanding into chemical signaling. Future expansion of this new methodology is highly encouraged since fluorescent nanosensors are easily functionalized and can be adjusted for colour specific responses and detection of other molecules. Furthermore, fluorescent nanosensors exhibit robust non photobleaching properties, meaning they can be used long-term in imaging experiments. In conclusion, there is application for future research as parallel developments may enable in vivo through cranium imaging.