신경전달물질 탐구!!

[문형철]

https://www.youtube.com/watch?v=_ADYQgL6g_Y

신경전달물질 대분류

1) 아미노산 : 글루타메이트, 가바, 글리신, aspartate

2) monoamine : 카테콜아민(NE, Dopamine, serotonin)

3) neuropeptide

4) purine

5) gastransmitter 

https://www.nature.com/articles/s41392-021-00553-z

퓨린과 그 유도체, 특히 아데노신과 ATP는 세포 내 에너지 항상성과 뉴클레오타이드 합성을 조절하는 핵심 분자입니다. 또한 이러한 퓨린은 화학적 메신저로서 조직과 종에 걸쳐 퓨린 전달을 지원합니다. 퓨린은 “퓨린성 신호 전달”이라고 하는 세포 외 통신을 매개하는 플라스말렘말 퓨리노 수용체에 결합하고 활성화하는 내인성 리간드로서 작용합니다. 퓨린성 신호는 다른 전달체 네트워크와 교차 연결되어 증식, 분화, 이동, 세포 사멸 및 유기체의 적절한 기능에 중요한 기타 생리적 과정과 같은 세포 행동의 다양한 측면을 조정합니다. 퓨린성 신호의 병리적 조절 저하는 신경 퇴화, 류마티스 면역 질환, 염증 및 암을 포함한 다양한 질병에 기여합니다. 특히 통풍은 퓨린 대사 장애와 그에 따른 고요산혈증으로 인해 발생하는 가장 흔한 퓨린 관련 질환 중 하나입니다. 퓨린 수용체가 잠재적인 치료 표적이라는 강력한 증거가 있으며, 특정 퓨린 작용제와 길항제가 두드러진 치료 잠재력을 보여줍니다. 또한, 식이 요법과 약초 요법은 퓨린 대사를 회복하고 균형을 유지하는 데 도움이 되므로 인간 질환의 예방과 완화에 있어 건강한 라이프스타일의 중요성을 다룹니다. 퓨린 신호의 분자 메커니즘에 대한 깊은 이해는 인간 질병 치료에 대한 새롭고 흥미로운 통찰력을 제공합니다.

Purinoceptors mediates neurotransmission in nervus system. In presynaptic terminal, ATP is enriched in vesicles by transporter VNUT and then released in a Ca 2+ -dependent manner, or exported by other channels such as Panx1 independent of Ca 2+ . On one hand, extracellular ATP and its degradation product adenosine in turn activate purinoceptors in presynaptic terminal. This effect may regulate the release of other transmitters such as glutamate. On the other hand, extracellular ATP and adenosine can interact with postsynaptic purinoceptors, thereby regulating the excitability of neural cells. This event might also lead to the internalisation of AMPAR and NMDAR on postsynaptic membrane, leading to a decrease of glutamate-induced current. VNUT vesicular nucleotide transporter, Panx1 pannexin 1, AD adenosine, AMPAR AMPA receptor, NMDAR N-methyl- d -aspartate receptor 퓨리노수용체는 신경계에서 신경전달을 매개합니다. 시냅스 전단부에서 ATP는 수송체 VNUT에 의해 소포에서 농축된 다음 Ca2+ 의존적으로 방출되거나 Ca2+와 무관하게 Panx1과 같은 다른 채널에 의해 방출됩니다. 한편으로 세포 외 ATP와 그 분해 산물인 아데노신은 시냅스 전 말단에서 퓨리노 수용체를 활성화합니다. 이 효과는 글루타메이트와 같은 다른 전달 물질의 방출을 조절할 수 있습니다. 반면에 세포 외 ATP와 아데노신은 시냅스 후 퓨리노 수용체와 상호 작용하여 신경 세포의 흥분성을 조절할 수 있습니다. 또한 시냅스 후 막에서 AMPAR과 NMDAR의 내재화로 이어져 글루타메이트 유발 전류가 감소할 수 있습니다. VNUT 소포 뉴클레오타이드 수송체, Panx1 판넥신 1, AD 아데노신, AMPAR AMPA 수용체, NMDAR N-메틸-d-아스파르트산염 수용체

참고) Endogenous gasotransmitters nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S), and potential candidates sulfur dioxide (SO2), methane (CH4), hydrogen gas (H2), ammonia (NH3) and carbon dioxide (CO2), are generated within the human body.

Open AccessReview

A Review of Neurotransmitters Sensing Methods for Neuro-Engineering Research

by 

Shimwe Dominique Niyonambaza

*

Author to whom correspondence should be addressed.

Appl. Sci. 2019, 9(21), 4719; https://doi.org/10.3390/app9214719

Submission received: 1 May 2019 / Revised: 11 June 2019 / Accepted: 11 October 2019 / Published: 5 November 2019

Abstract

Neurotransmitters as electrochemical signaling molecules are essential for proper brain function and their dysfunction is involved in several mental disorders. Therefore, the accurate detection and monitoring of these substances are crucial in brain studies. Neurotransmitters are present in the nervous system at very low concentrations, and they mixed with many other biochemical molecules and minerals, thus making their selective detection and measurement difficult. Although numerous techniques to do so have been proposed in the literature, neurotransmitter monitoring in the brain is still a challenge and the subject of ongoing research. This article reviews the current advances and trends in neurotransmitters detection techniques, including in vivo sampling and imaging techniques, electrochemical and nano-object sensing techniques for in vitro and in vivo detection, as well as spectrometric, analytical and derivatization-based methods mainly used for in vitro research. The document analyzes the strengths and weaknesses of each method, with the aim to offer selection guidelines for neuro-engineering research.

요약

전기 화학적 신호 분자인 신경전달물질은 

적절한 뇌 기능에 필수적이며 

그 기능 장애는 여러 정신 장애에 관여합니다. 

따라서 

이러한 물질을 정확하게 감지하고 모니터링하는 것은 

뇌 연구에서 매우 중요합니다. 

신경전달물질은 

신경계에 매우 낮은 농도로 존재하며 

다른 많은 생화학 분자 및 미네랄과 혼합되어 있어 

선택적 검출 및 측정이 어렵습니다. 

이를 위한 수많은 기술이 문헌에서 제안되었지만, 뇌의 신경전달물질 모니터링은 여전히 어려운 과제이며 지속적인 연구의 대상입니다. 이 문서에서는 생체 내 샘플링 및 이미징 기술, 체외 및 생체 내 검출을 위한 전기화학 및 나노 물체 감지 기술, 체외 연구에 주로 사용되는 분광, 분석 및 유도체화 기반 방법을 포함하여 신경전달물질 검출 기술의 현재 발전과 동향에 대해 살펴봅니다. 이 문서는 신경 공학 연구를 위한 선택 가이드라인을 제공하기 위해 각 방법의 장단점을 분석합니다.

Keywords: 

neurotransmitters; sensing methods; nuclear medicine tomographic imaging; optical sensing techniques; electrochemistry; high performance liquid chromatography; microdialysis

1. Introduction

Most human body functions such as perception, motor control and cognitive behaviour are linked to the nervous system [1,2,3,4,5,6,7,8,9,10,11,12,13,14]. They are controlled by neural cell networks that generate and propagate electrochemical signals, in great part through the release and diffusion across synaptic junctions of neurotransmitters that mediate the exchange of those signals between the neural cells. A synapse involves a presynaptic neuron and a postsynaptic neuron that release and bind neurotransmitters, respectively.

As neurotransmitters are essential components in the nervous system, and in order to understand how the brain works and identify neurological diseases, multiple approaches have been proposed for neurotransmitter detection and monitoring. Depending on the used technology, the regularly used detection methods can be classified into five categories: (1) Nuclear medicine tomographic imaging, including positron emission tomography and single-photon emission computed tomography; (2) Optical sensing, including surface-enhanced Raman spectroscopy, fluorescence, chemiluminescence, optical fiber based biosensors and colorimetry; (3) Electrochemical detection, including voltammetry and amperometry; (4) Analytical chemistry techniques, including high performance liquid chromatography; and (5) Microdialysis. In practice, two or more techniques may be combined for better detection accuracy. However, the detection of neurotransmitter is still challenging due to their sparsity in the brain and their mixture with other molecules. Thus, for accurate detection and monitoring, a neurotransmitter has to be localized and identified among other biomolecules and other neurotransmitters, as they can have similar behaviours.

In order to qualify as a neurotransmitter, a molecule must meet the following criteria, according to Kandel et al. [15]:

1. 소개

지각, 운동 조절, 인지 행동과 같은 대부분의 인체 기능은 신경계와 연결되어 있습니다[1,2,3,4,5,6,7,8,9,10,11,12,13,14]. 이러한 행동은 전기화학 신호를 생성하고 전파하는 신경 세포 네트워크에 의해 제어되며, 대부분 신경 세포 간의 신호 교환을 매개하는 신경전달물질의 시냅스 접합부를 통한 방출과 확산을 통해 이루어집니다.

시냅스에는

각각 신경전달물질을 방출하고 결합하는

시냅스 전 뉴런과 시냅스 후 뉴런이 있습니다.

신경전달물질은

신경계의 필수 구성 요소이며,

뇌의 작동 방식을 이해하고 신경 질환을 파악하기 위해

신경전달물질 검출 및 모니터링을 위한 다양한 접근 방식이 제안되었습니다.

사용되는 기술에 따라 일반적으로 사용되는 감지 방법은 5가지로 분류할 수 있습니다:

(1) 양전자 방출 단층 촬영 및 단일 광자 방출 컴퓨터 단층 촬영을 포함한 핵의학 단층 촬영,

(2) 표면 강화 라만 분광법, 형광, 화학 발광, 광섬유 기반 바이오센서 및 비색법을 포함한 광학 감지,

(3) 전압 측정 및 전류 측정을 포함한 전기 화학 감지,

(4) 고성능 액체 크로마토그래피를 포함한 분석 화학 기술 및

(5) 미세투석을 포함한 분석 화학 기법.

실제로는 탐지 정확도를 높이기 위해 두 가지 이상의 기술을 결합할 수 있습니다.

그러나

신경전달물질은

뇌에 희소하고 다른 분자와 혼합되어 있기 때문에

검출이 여전히 어렵습니다.

따라서 정확한 검출과 모니터링을 위해서는 유사한 행동을 보일 수 있는 다른 생체 분자와 다른 신경전달물질 중에서 신경전달물질을 찾아내어 식별해야 합니다.

캔델 외[15]에 따르면 신경전달물질로 인정받기 위해서는 분자가 다음 기준을 충족해야 합니다:

•  
•  
•  
•  

The number of known neurotransmitters molecules exceeds 100 [16] and, based on their chemical structure, they are generally classified as amino acids, monoamines, neuropeptides, purines and gasotransmitters among others. Figure 1 shows the main locations of the brain where representative neurotransmitters are found in the highest concentrations.

• 이는 동일한 뉴런에서 합성 및 방출되어 시냅스 전 단자에 저장되어야 합니다;
• 방출이 시냅스 후 뉴런에서 특정 행동을 유도합니다;
• 외인성 투여도 동일한 효과를 일으켜야 합니다;
• 시냅스 후 세포에 대한 유도된 작용은 특정 메커니즘에 의해 중단될 수 있습니다.

알려진

신경전달물질 분자의 수는

100개를 넘으며[16],

화학 구조에 따라 일반적으로

아미노산,

모노아민,

신경펩타이드,

퓨린,

가스전달물질 gasotransmitters 등으로 분류됩니다.

amino acids,

monoamines,

neuropeptides,

purines and

gasotransmitters 

그림 1은 대표적인 신경전달물질이 가장 높은 농도로 존재하는 뇌의 주요 위치를 보여줍니다.

Figure 1. Regions of the brain where neurotransmitters are mainly produced.

Although all neurotransmitters are essential to the good functioning of the central and peripheral nervous system, only a small group is involved in the majority of known neuropathies. As a result, the existing detection techniques are usually optimized for specific neurotransmitters, of which those of the monoamine and amino-acid groups and others such as acetylcholine. A summary description of those neurotransmitters follows:

그림 1. 신경전달물질이 주로 생성되는 뇌의 영역.
모든 신경전달물질은

중추 및 말초 신경계의 원활한 기능에 필수적이지만,

알려진 대부분의 신경병증에는 소수의 신경전달물질만 관여합니다.

따라서

기존의 검출 기술은 일반적으로

모노아민 및 아미노산 그룹과 아세틸콜린과 같은

특정 신경전달물질에 최적화되어 있습니다.

이러한 신경전달물질에 대한 간략한 설명은 다음과 같습니다:

1.1. Amino-Acids Neurotransmitters

1.1.1. Glutamate

Glutamate is the major excitatory neurotransmitter in the central nervous system [15]. It is present in 90% of the excitatory synapses [17] and plays a key role in the plasticity of the nervous system (defined as the ability of neuronal circuits to change their connectivity) [15,18,19,20]. Recent studies have shown that glutamate also plays a role in glia-neuron signalling, along with two other neurotransmitters: D-serine and glycine [21,22].

Glutamate (and gamma-aminobutyric acid (GABA), defined further below) dysfunctions may be involved in brain disorders such as epilepsy [23,24,25]. Indeed, seizure was observed in mice lacking the glutamate transporter GLT-1 [24]. Besides that, the use of microdialysis and electroencephalography to measure extracellular glutamate in patients with epilepsy has shown a hippocampal increase of this neurotransmitter [25]. Recently, a glutamate dysfunction model of schizophrenia has emerged in addition to the widely known hypothesis involving dopamine. Schizophrenia is characterized by the disruption of sensory processing, leading to hallucination, paranoia, cognitive deficits in attention, short-term memory and movement abnormality [26].

1.1. 아미노산 신경전달물질

1.1.1. 글루타메이트

글루타메이트는

중추신경계의 주요 흥분성 신경전달물질입니다[15].

흥분성 시냅스의 90%에 존재하며[17]

신경계의 가소성(신경 회로의 연결성을 변화시키는 능력으로 정의됨)에 중요한 역할을 합니다[15,18,19,20].

최근 연구에 따르면

글루타메이트는

다른 두 가지 신경전달물질( D-세린과 글리신)과 함께

신경교세포 신호 전달에도 중요한 역할을 하는 것으로 밝혀졌습니다: [21,22].

 Recent studies have shown that glutamate also plays a role in glia-neuron signalling, along with two other neurotransmitters: D-serine and glycine

글루타메이트(및 감마 아미노부티르산(GABA, 아래에서 자세히 설명)) 기능 장애는

간질과 같은 뇌 질환에 관여할 수 있습니다[23,24,25].

글루타메이트 매개 신경세포 과흥분이 발작을 유발하는 원인으로 작용한다는 사실은 널리 알려져 있습니다. 글루타메이트 수용체 중 생리적 및 병리학적 조건에서 N-메틸-D-아스파르트산염(NMDA)과 α-아미노-3-하이드록시-5-메틸이소옥사졸-4-프로피온산(AMPA) 수용체의 역할은 주요 임상 연구 대상이 되고 있습니다. NMDA 또는 AMPA 수용체의 작용제는 동물 또는 인간 대상에서 발작을 유발할 수 있고, 길항제는 동물 모델에서 발작을 억제하는 것으로 알려져 있어 항발작 약물 개발에서 NMDA 및 AMPA 수용체 길항제의 잠재적 역할을 시사하고 있습니다. 이러한 약물 중 몇 가지가 임상 연구에서 평가되었지만, 대부분 NMDA 수용체 길항제는 치료용으로 사용하기에 적절한 효능과 안전성을 입증하지 못했으며, 일부 형태의 뇌전증 치료에는 AMPA 수용체 길항제인 페람파넬만이 승인되었습니다. 이러한 결과는 발작 과정에서 각 글루타메이트 수용체의 역할에 대한 오해가 이러한 약물이 임상적 효능과 안전성을 입증하지 못하는 근본적인 원인일 수 있음을 시사합니다. 병리학적인 유전자 돌연변이, 자가면역성 간질에서의 역할, 약물 발견 연구 및 약리학 연구의 증거를 포함하여 NMDA와 AMPA 수용체에 대한 지식을 축적하면 발작 발생에서 두 수용체의 역할을 재고할 수 있는 귀중한 정보를 제공할 수 있습니다. 이 리뷰는 뇌전증에서 NMDA와 AMPA 수용체의 구체적인 역할을 더 명확히 밝히기 위해 여러 연구의 정보를 통합하는 것을 목표로 했습니다.

참고) 글루타메이트 -- 파킨슨 병

알파-시누클레인(aSyn)은 뇌의 전형적인 단백질 응집체에 축적되기 때문에 시누클레인 병증의 발병에 핵심적인 역할을 합니다. 그러나 어떻게 신경 퇴행에 기여하는지는 아직 명확하지 않습니다. 제2형 당뇨병은 파킨슨병(PD)의 위험 요인입니다. 흥미롭게도 이러한 질환의 공통적인 분자적 변화는 노화와 관련된 단백질 당화 증가입니다. 우리는 당화로 인한 신경세포 기능 장애가 시뉴클레인 병증의 원인이라는 가설을 세웠습니다. 여기서 우리는 뇌에서 aSyn을 과발현하는 마우스에서 메틸글리옥살(MGO, 당화제)의 영향을 분석했습니다. 뇌실 내 주사를 통해 단일 용량의 MGO를 투여한 aSyn 형질전환(Thy1-aSyn) 마우스에서 MGO 당화가 운동, 인지, 후각 및 대장 기능 장애를 강화하는 것을 발견했습니다. aSyn은 중뇌, 선조체 및 전전두피질에 축적되고 단백질 당화는 소뇌와 중뇌에서 증가합니다. 뇌 프로테옴의 변화를 정량화하는 데 사용되는 SWATH 질량 분석 분석에 따르면 MGO는 주로 중뇌에서 글루탐산 관련 단백질(NMDA, AMPA, 글루타미나제, VGLUT 및 EAAT1)을 증가시키지만 전자 수송 사슬에 주로 영향을 미치는 전전두피질에는 영향을 주지 않는 것으로 밝혀졌습니다. MGO를 주입한 Thy1-aSyn 생쥐의 중뇌에서 당화 단백질은 PD 및 도파민 경로와 밀접한 상관관계가 있습니다. 전반적으로, 우리는 MGO에 의한 당화가 PD와 유사한 감각 운동 및 인지 변화를 가속화하며 글루탐산 신호의 증가가 이러한 사건의 기저에 있을 수 있음을 입증했습니다. 우리의 연구는 PD 관련 시냅스 기능 장애에서 중뇌의 취약성 강화에 대한 새로운 빛을 비추고 당화 억제제와 항 글루타머성 약물이 시누클레인 병증의 질병 수정 치료제로서 가능성을 제시합니다.

글루탐산 신경전달계 조절 장애는 알츠하이머병(AD)의 병태 생리학에서 중요한 역할을 할 수 있습니다. 그러나 뇌 영역 전반의 글루타메이터 성분에 대한 보고된 결과는 서로 상반됩니다. 여기에서는 메타분석을 통해 체계적인 검토를 수행하여 인간의 AD 뇌에 일관된 글루타메이트 이상이 있는지 조사했습니다. 우리는 PubMed와 Web of Science(데이터베이스 출처-2023년 10월)에서 AD 인간 뇌의 글루타메이트, 글루타민, 글루타미나제, 글루타민 합성효소, 글루타메이트 재흡수, 아스파르트산염, 흥분성 아미노산 수송체, 소포성 글루타메이트 수송체, 글리신, D-세린, 대사성 및 이온성 글루타메이트 수용체를 평가하는 보고서를 검색했습니다(PROSPERO #CDRD42022299518). 연구 결과는 결과와 뇌 영역별로 종합되었습니다. 피질 영역, 전체 뇌(피질 및 피질하 영역 결합), 내측 피질 및 해마가 포함되었습니다. 풀링된 효과 크기는 표준화된 평균 차이(SMD)와 오탐률로 조정된 무작위 효과로 결정되었으며, 이질성은 I2 통계로 조사되었습니다. 검색 결과 6,936개의 논문 중 63개가 포함 기준을 충족했습니다(N = 709CN/786AD, 평균 연령 75/79세). 그 결과 알츠하이머병 환자의 뇌에서 글루타메이트(SMD = -0.82; I2= 74.54%; P<0.001) 및 아스파르트산염 수치(SMD = -0.64; I2= 89.71%; P= 0.006), 재흡수(SMD = -0.75; I2= 83.04%; P<0.001)가 감소한 것으로 나타났습니다. 또한 α- 아미노 -3- 하이드 록시 -5- 메틸 -4- 이소 사졸 프로피온산 (AMPAR)-GluA2/3 수준 감소 (SMD = -0.63; I2= 95.55 %; P= 0. 046), 기능 저하 N-메틸-D-아스파르트산염 수용체(NMDAR)(SMD = -0.60; I2= 91.47%; P<0.001) 및 NMDAR-GluN2B 서브유닛 수준의 선택적 감소(SMD = -1.07; I2= 41.81%; P<0.001)를 확인했습니다. 지역적 차이로는 피질 영역의 글루타메이트 수치와 피질 영역과 해마의 아스파르트산염 수치 감소, 글루타메이트 재흡수 감소, 내측 피질의 AMPAR-GluA2/3 감소, 피질 영역의 NMDAR 기능 저하, 내측 피질과 해마의 NMDAR-GluN2B 하위 단위 수치 감소 등이 있습니다. 연구된 다른 매개변수는 변경되지 않았습니다. 우리의 연구 결과는 글루타메이트 시스템의 고갈을 보여 주며 AD에서 글루타메이트 매개 신경 독성을 이해하는 것이 중요하다는 것을 강조합니다. 이 연구는 알츠하이머병의 치료법과 바이오마커 개발에 시사하는 바가 있습니다.

실제로

글루타메이트 수송체 GLT-1이 결핍된

마우스에서 발작이 관찰되었습니다[24].

그 외에도

뇌전증 환자의 세포 외 글루타메이트를 측정하기 위해

미세 투석과 뇌파 검사를 사용하면

이 신경전달물질의 해마가 증가하는 것으로 나타났습니다 [25].

최근에는

도파민과 관련된 널리 알려진 가설에 더해

조현병의 글루타메이트 기능 장애 모델이 등장했습니다.

조현병은

감각 처리가 중단되어

환각, 편집증, 주의력 결핍, 단기 기억력 및 운동 이상을

초래하는 것이 특징입니다 [26].

1.1.2. L-Aspartate

L-aspartate is a conjugate base of the aspartic acid, whose role as a neurotransmitter in the central nervous system has been subject to controversy [27,28]. Some researchers have proposed that L-aspartate may be a neurotransmitter in the visual cortex and in the cerebellum [29,30,31,32], and others have shown that, like L-glutamate, L-aspartate may be an excitatory neurotransmitter and a neuropeptide-like modulator in the hippocampus [27,33].

The synaptic terminations that contain aspartate vesicles are co-localized with neurons containing glutamate and GABA vesicles. Thus, it seems that L-aspartate can play a role in both excitatory and inhibitory pathways. Synaptic termination with only aspartate vesicles has not been discovered in the hippocampus. A role for aspartate as co-neurotransmitter with glutamate was previously reported in the rat corticostriatal neurons and its release may be modulated by dopamine [34,35,36,37].

1.1.2. L-아스파르트산염

L-아스파르트산염은

중추 신경계에서 신경 전달 물질로서의 역할이

논란의 대상이 되고 있는

아스파르트산의 공액 염기입니다[27,28].

L-aspartate is a conjugate base of the aspartic acid

일부 연구자들은

L-아스파르트산염이

시각 피질과 소뇌의 신경 전달 물질일 수 있다고 제안했으며[29,30,31,32],

다른 연구자들은 L-글루타메이트와 마찬가지로

L-아스파르트산염이

해마에서 흥분성 신경 전달 물질이자

신경 펩티드 유사 조절제일 수 있음을 보여주었습니다[27,33].

역사적으로 글루타메이트와 아스파르트산염이 신경전달물질로 기능한다는 가장 강력한 증거는 낮은 농도에서 CNS의 거의 모든 뉴런을 흥분시킨다는 관찰에서 나왔습니다. 성체 CNS에서 흥분성 아미노산 수용체에서 신경전달물질로 작용할 가능성이 가장 높은 후보로는 l-글루타메이트와 l-아스파르트산염이 있으며, 이 아미노산은 가장 널리 분포하는 일부 신경세포 유형에서 사용됩니다. 글루타메이트와 아스파르트산염은 중추신경계에 고농도로 존재하며 시험관 내에서 전기 자극을 받으면 Ca2+ 의존적인 방식으로 방출됩니다 . 두 가지 모두 생체 내에서 이온토포레즈될 때 뉴런에 강력한 흥분 효과를 나타냅니다 . 고친화성 흡수 시스템은 복제되었으며 많은 신경 경로와 관련된 신경 말단 및 신경교세포에 위치합니다(아래 참조). 선택적 결합 부위는 시험관 내에서 자가 방사선 면역 조직 화학 및 약리학 기법을 통해 입증할 수 있습니다 . 신경전달물질 후보로서 글루타메이트와 아스파르트산염의 명확한 규명은 다른 많은 기능에 관여하기 때문에 방해를 받았지만, 이 역할에 대해서는 보편적으로 인정되고 있습니다. 글루타메이트와 아스파르트산염은 혈액-뇌 장벽을 통과하지 않는 비필수 아미노산으로 포도당과 다양한 다른 전구체에서 합성됩니다. 글루타메이트와 아스파르트산염의 합성 및 대사 효소는 뇌의 두 가지 주요 구획인 뉴런과 신경교세포에 국한되어 있습니다( 그림 15-7 ). 글루탐산은 α-케토글루타르산 및 글루타민과 함께 대사 풀에 있습니다. 신경 말단에서 방출된 글루타메이트의 상당 부분은 아마도 신경교 세포로 흡수되어 글루타민으로 전환될 것입니다. 그런 다음 글루타민은 신경 말단으로 다시 순환하여 글루타메이트와 가바의 전달 물질 풀을 보충하는 데 참여합니다. 현재 중추신경계에서 글루타메이트의 세포 외 대사에 대한 증거는 거의 없습니다.

아스파르트산염 소포가 포함된 시냅스 종말은

글루타메이트 및 GABA 소포가 포함된 뉴런과

공동 국소화되어 있습니다.

따라서

L-아스파르트산염은

흥분성 및 억제성 경로 모두에서 역할을 할 수 있는 것으로 보입니다.

해마에서 아스파르테이트 소포만을 이용한 시냅스 종결은 발견되지 않았습니다. 아스파르트산염이 글루타메이트와 공동 신경 전달 물질로서의 역할은 이전에 쥐의 피질 피질 뉴런에서 보고되었으며, 그 방출은 도파민에 의해 조절될 수 있습니다 [34,35,36,37].

1.1.3. Gamma-Aminobutyric Acid (GABA)

GABA is the major inhibitory neurotransmitter in the central nervous system [15]. It is synthesized from glutamic acid [15].

Studies have shown that GABA is excitatory in early development [38]. Indeed, it induces a depolarization instead of hyperpolarization in various area of the nervous system, including the neocortex, hippocampus, hypothalamus, cerebellum and spinal cord. This is due to the higher chloride concentration in neurons during the early development of the human body, leading to an outward instead of inward chloride flux [38]. Therefore, the role of GABA during the first years of youth is not the same as in adults. The change of expression‎ of two categories of chloride transporters, the sodium potassium chloride co-transporters and the potassium chloride co-transporters mediate the change of GABA action from excitatory to inhibitory [38]. Ben-Ari proposed that this GABA action switch promotes synapse formation [38]. He proposed the following three-stage mechanism [38]:

•  
•  
•  

Poo et al. also showed that the excitatory action of GABA plays a major role in the morphological maturation of cortical neurons [39].

1.1.3. 감마-아미노부티르산(GABA)

GABA는

중추신경계의 주요 억제 신경전달물질입니다[15].

글루탐산에서 합성됩니다 [15].

연구에 따르면 GABA는 발달 초기에 흥분 작용을 하는 것으로 나타났습니다 [38].

실제로

신피질, 해마, 시상하부, 소뇌 및 척수를 포함한 신경계의 다양한 영역에서

과분극 대신 탈분극을 유도합니다.

이는 인체의 초기 발달 과정에서

뉴런의 염화물 농도가 높아져 염화물 플럭스가 안쪽이 아닌 바깥쪽으로 흐르기 때문입니다 [38].

따라서

청소년의 첫해 동안 GABA의 역할은 성인과 동일하지 않습니다.

두 종류의 염화물 수송체,

즉 염화칼륨 나트륨 공동 수송체와 염화칼륨 칼륨 공동 수송체의 발현 변화는

GABA 작용이 흥분성에서 억제성으로 변화하는 것을 매개합니다 [38].

Ben-Ari는 이 GABA 작용 스위치가 시냅스 형성을 촉진한다고 제안했습니다 [38]. 그는 다음과 같은 3단계 메커니즘을 제안했습니다 [38]:

• GABA는 처음에 수송체의 발현으로 인해 뉴런에 흥분 효과를 일으킵니다.
• 글루타메이트 시냅스는 GABAergic 뉴런 이후에 형성됩니다.
• 가바 및 글루타메이트 흥분 작용은 시냅스 형성을 조절하는 세포 내 칼슘의 진동을 가능하게 합니다.

Poo 등은 또한 GABA의 흥분성 작용이 피질 뉴런의 형태학적 성숙에 중요한 역할을 한다는 것을 보여주었습니다 [39].

1.1.4. Glycine

Glycine is the major inhibitory neurotransmitter in the spinal cord, with a higher concentration at the ventral horn [15,40,41,42]. Glycine also acts as a neurotransmitter in the brainstem and medulla, and is also a required co-agonist with glutamate for N-methyl-d-aspartate (NMDA) receptors [43]. It is involved in voluntary motor control and sensory processing, and also in auditory, cardiovascular and respiratory functions [41,42]. There are glycine receptors in the bipolar, ganglion and amacrine cells of the retina, with different subunit expressions [44]. As with GABA, Glycine acts as an excitatory neurotransmitter in early development, and may thus play a role in neuronal differentiation, proliferation, and connectivity [41,44].

The mutations in glycine receptors seem to be involved in motor control disorders like hypertonia and hyperekplexia [40,42]. Glycine transmission may also be involved in alcohol intoxication [42].

Studies have shown that GABA and glycine are co-released in certain neurons in the cerebellum, spinal cord and superior olive, but the interaction between the two neurotransmitters is not well understood [41]. Both glutamate and glycine sites must be occupied to lead to channel opening, with glutamate being released by the presynaptic neurons and glycine by glial cells [42].

1.1.4. 글리신

글리신은

척수의 주요 억제성 신경전달물질로,

배쪽 뿔에서 농도가 더 높습니다[15,40,41,42].

Glycine is the major inhibitory neurotransmitter in the spinal cord, with a higher concentration at the ventral horn

글리신은

또한 뇌간과 수질에서 신경전달물질로 작용하며,

N-메틸-D-아스파르트산염(NMDA) 수용체에 대한

글루타메이트와 필수적인 보조 작용제이기도 합니다[43].

자발적 운동 조절과

감각 처리, 청각, 심혈관 및 호흡 기능에도 관여합니다[41,42].

망막의 양극성, 신경절 및 아마크린 세포에는

서로 다른 서브유닛 발현을 가진

글리신 수용체가 있습니다 [44].

GABA와 마찬가지로

글리신은 초기 발달에서 흥분성 신경전달물질로 작용하므로

신경세포 분화, 증식 및 연결에 중요한 역할을 할 수 있습니다 [41,44].

글리신 수용체의 돌연변이는 하이퍼토니아 및 하이퍼플렉시아 같은 운동 조절 장애에 관여하는 것으로 보입니다 [40,42]. 글리신 전달은 알코올 중독에도 관여할 수 있습니다 [42].

연구에 따르면 GABA와 글리신은 소뇌, 척수 및 우수 올리브의 특정 뉴런에서 공동 방출되지만 두 신경전달물질 간의 상호 작용은 잘 알려져 있지 않습니다 [41]. 채널 개방으로 이어지려면 글루타메이트와 글리신 부위가 모두 점유되어야 하며, 글루타메이트는 시냅스 전 뉴런에 의해, 글리신은 신경교 세포에 의해 방출됩니다 [42].

1.1.5. D-Serine

D-serine is a molecule released by glial cells to act as a neurotransmitter or neuromodulator [45]. The concept of a functional role of D-configuration amino acids in higher organisms like mammals is relatively new, since they were thought to be only used in less complex living organism forms such as bacteria or insects [46,47]. It is present in various parts of the brain and immunohistochemical studies have shown its presence in the rostral cerebral cortex, hippocampus, anterior olfactory nuclei, olfactory tubercule, corpus striatum, and amygdala [46,47,48,49]. More precisely, D-serine is localized in the protoplasmic astrocytes of the grey matter that ensheath synapses [46,47].

Studies have shown that D-serine is involved in the glutamatergic hypothesis of schizophrenia [47]. Studies with schizophrenic patients showed that D-serine-based treatment seems to be effective for positive and negative symptoms [50,51].

1.1.5. D-세린

D-세린은

신경교세포에서 신경전달물질 또는 신경조절물질로 작용하기 위해

방출되는 분자입니다[45].

박테리아나 곤충과 같이 덜 복잡한 생물체 형태에서만 사용되는 것으로 생각되었기 때문에 포유류와 같은 고등 유기체에서 D-구성 아미노산의 기능적 역할에 대한 개념은 비교적 새로운 것입니다 [46,47].

뇌의 다양한 부위에 존재하며 면역 조직 화학적 연구에 따르면 복측 대뇌 피질, 해마, 전후각핵, 후각 결절, 선조체 및 편도체에 존재하는 것으로 나타났습니다 [46,47,48,49]. 더 정확하게 말하면, D-세린은 시냅스를 감싸고 있는 회백질의 원형질 성상세포에 국한되어 있습니다[46,47].

연구에 따르면 D-세린은

조현병의 글루탐산 가설에 관여하는 것으로 나타났습니다 [47].

조현병 환자를 대상으로 한 연구에 따르면

D-세린 기반 치료가 양성 및 음성 증상에

효과적인 것으로 나타났습니다 [50,51].

1.2. Monoamine Neurotransmitters

Monoamines contain one amino group connected to an aromatic ring by a two-carbon chain. They are classified into three groups: catecholamines, indolamines and imidazoleamines. The three known catecholamines (dopamine, norepinephrine and epinephrine) are all derived from the same precursor, L-DOPA, which is itself derived from the amino acid tyrosine [15]. Indolamines such as serotonin, melatonin and tryptamine all contain an indole compound ring. Imidazoleamines such as histamine contain an imidazole ring connected to an amino group.

1.2. 모노아민 신경전달물질

모노아민은

두 개의 탄소 사슬에 의해 방향족 고리에 연결된

하나의 아미노기를 포함합니다.

이들은

카테콜아민,

인돌아민,

이미다졸아민의

세 가지 그룹으로 분류됩니다.

알려진

세 가지 카테콜아민(도파민, 노르에피네프린, 에피네프린)은

모두 아미노산 티로신에서 파생된 동일한 전구체인

L-DOPA에서 파생됩니다[15].

세로토닌,

멜라토닌,

트립타민과 같은 인돌아민은

모두 인돌 화합물 고리를 포함합니다.

히스타민과 같은

이미다졸아민은

아미노기에 연결된 이미다졸 고리를 포함합니다.

1.2.1. Dopamine

Dopamine is one of the neurotransmitters of great clinical importance for motor functions and motivational behaviors. Its dysfunction is involved in many psychiatric disorders, including drug addiction, schizophrenia, Parkinson’s and Huntington’s disease. The dopaminergic neurons are localized in the subsantia nigra pars compacta and in the ventral tegmental aera [52].

Dopamine seems to also be involved in drug addiction. For instance, it is known that cocaine inhibits the dopamine transporter [53]. However, cocaine can also block the norepinephrine and serotonin transporters, which can also reuptake dopamine.

Schizophrenic behavior includes a disturbance of cognition and perception with symptoms including delusions, hallucination, antisocial behavior and lack of motivation [54]. The cognitive symptoms may be caused by a hypodopaminergic activity in the mesocortical pathway, while the psychotic symptoms may be caused by a hyperdopaminergic activity in the mesolimbic pathway [55]. The role of dopamine in schizophrenia is strongly supported by the fact that dopamine antagonists are efficient to treat patients, and positive symptoms are related to dopamine dysregulation [54].

1.2.1. 도파민

도파민은

운동 기능과 동기 부여 행동에

임상적으로 매우 중요한 신경전달물질 중 하나입니다.

도파민의 기능 장애는

약물 중독, 정신분열증, 파킨슨병, 헌팅턴병 등

많은 정신과 질환에 관여합니다.

도파민성 뉴런은

시상하 흑질과 복측 피질에 국한되어 있습니다 [52].

도파민은

약물 중독에도 관여하는 것으로 보입니다.

예를 들어,

코카인은

도파민 수송체를 억제하는 것으로 알려져 있습니다 [53].

그러나

코카인은

노르에피네프린과 세로토닌 수송체를 차단하여

도파민을 재흡수할 수도 있습니다.

조현병 행동에는 망상, 환각, 반사회적 행동, 동기 부여 부족 등의 증상과 함께 인지 및 지각 장애가 포함됩니다 [54]. 인지 증상은 중피질 경로의 저도파민성 활동으로 인해 발생할 수 있으며, 정신병적 증상은 중변연계 경로의 고도파민성 활동으로 인해 발생할 수 있습니다 [55]. 조현병에서 도파민의 역할은 도파민 길항제가 환자 치료에 효과적이며 긍정적 인 증상이 도파민 조절 장애와 관련이 있다는 사실에 의해 강력하게 뒷받침됩니다 [54].

1.2.2. Norepinephrine and Epinephrine

Norepinephrine and epinephrine, also known as noradrenaline and adrenaline respectively, are monoamine molecules which act as hormones and neurotransmitters in the human body. As neurotransmitters, they are involved in the autonomic nervous system [56], which is composed of the sympathetic and parasympathetic systems. The autonomic nervous system is also known as the “fight or flight” system.

In the brain, norepinehrine neurons are localized in the locus coerulus, a nucleus in the brainstem that projects to various regions of the brain, including the limbic system which is involved in the regulation of emotions and cognition [57,58]. In the hypothalamus, amygdala and locus coerulus, the increase of norepinephrine release had been associated with anxiety [59]. Adrenergic neurons are also present in different regions of the brain, including the lateral tegmental system and medulla, but the epinephrine function as a neurotransmitter is poorly understood [57]. It is believed that it plays a role in the fight-or-flight response by increasing the heart rate, vasodilatation, pupil dilation, and blood sugar [60,61].

One of the major hypotheses for depression involves a deficit in monoamines, more precisely in serotonin and/or norepinephrine [58], and the antidepressant drugs currently used act on mechanisms involving monoamines regulations. Those mechanisms are: inhibition of reuptake of serotonin/norepinephrine, antagonism of presynaptic inhibitory serotonin/norepinephrine receptors and inhibition of monoamine oxidase [58]. More evidence exists on the role of serotonin at the origin of depression, but the evidence also shows a role for epinephrine. For instance, the locus coerulus has projection to the limbic system, including the amygdala, hippocampus and hypothalamus, which are all involved in emotion and cognition. Studies on animals have also shown that increasing norepinephrine level induces a protection from depression, while norepinephrine depletion studies have shown increased risk for depression. Drugs that act specifically on norepinephrine are effective to treat depression [58].

1.2.2. 노르에피네프린과 에피네프린

노르아드레날린과 아드레날린으로도 알려진

노르에피네프린과 에피네프린은

인체에서 호르몬 및 신경전달물질로 작용하는

모노아민 분자입니다.

신경전달물질로서

교감신경계와 부교감신경계로 구성된 자율신경계[56]에 관여합니다.

자율 신경계는

"투쟁 또는 도피" 시스템이라고도 합니다.

뇌에서 노르에피네프린 뉴런은

감정과 인지 조절에 관여하는 변연계를 포함하여

뇌의 다양한 영역으로 돌출되는 뇌간의 핵인 소체핵에 국한되어 있습니다[57,58].

시상하부, 편도체 및 구상체에서

노르에피네프린 방출의 증가는

불안과 관련이 있는 것으로 나타났습니다 [59].

아드레날린성 뉴런은

측대상계와 수질을 포함한 뇌의 다른 영역에도 존재하지만

신경전달물질로서의 에피네프린 기능은 잘 알려져 있지 않습니다 [57].

심박수, 혈관 확장, 동공 확장, 혈당을 증가시켜

투쟁-도피 반응에 관여하는 것으로 알려져 있습니다[60,61].

우울증의 주요 가설 중 하나는

모노아민,

더 정확하게는 세로토닌 및/또는

노르에피네프린의 결핍과 관련이 있으며[58],

현재 사용되는 항우울제는

모노아민 조절과 관련된 메커니즘에 작용합니다.

이러한 메커니즘은

세로토닌/노르에피네프린의 재흡수 억제,

시냅스 전 억제 세로토닌/노르에피네프린 수용체의 길항작용,

모노아민 산화효소의 억제 등입니다 [58].

우울증의 기원에서 세로토닌의 역할에 대한 더 많은 증거가 존재하지만, 에피네프린의 역할에 대한 증거도 있습니다. 예를 들어, 편도체, 해마, 시상하부 등 감정과 인지에 관여하는 변연계로 투사되는 시교차상체(locus coerulus)는 감정과 인지에 모두 관여합니다. 동물 연구에서도 노르에피네프린 수치가 증가하면 우울증으로부터 보호되는 반면, 노르에피네프린이 고갈되면 우울증에 걸릴 위험이 높아진다는 사실이 밝혀졌습니다. 노르에피네프린에 특이적으로 작용하는 약물은 우울증 치료에 효과적입니다[58].

1.2.3. Serotonin

5-hydroxytryptamine (5-HT), most commonly known as serotonin, is a neurotransmitter that plays an important role in many behavioral functions [62]. It regulates sleep and wake states, feeding behavior, aggressive behavior and mood/depression among others [15,57,62]. Many currently used antidepressants act on the mechanism of serotonin or norepinephrine transmission. The use of selective serotonin reuptake inhibitors has given more insight on the role of serotonin in depression [63]. Serotonergic neurons are localized in the raphe nuclei and their axons project into the prefrontal cortex, basal ganglia, hippocampus, hypothalamus and spinal cord [15,64]. A serotonin dysfunction, along with the dopamine model of schizophrenia, was proposed by Meltzer [65].

1.2.3. 세로토닌

세로토닌으로 가장 일반적으로 알려진

5-하이드록시트립타민(5-HT)은

많은 행동 기능에 중요한 역할을 하는 신경전달물질입니다[62].

수면 및 각성 상태,

섭식 행동,

공격적 행동,

기분/우울증 등을 조절합니다[15,57,62].

현재 사용되는 많은 항우울제는

세로토닌 또는 노르에피네프린 전달 메커니즘에 작용합니다.

선택적 세로토닌 재흡수 억제제의 사용은

우울증에서 세로토닌의 역할에 대한

더 많은 통찰력을 제공했습니다 [63].

세로토닌 신경세포는

라페핵에 국한되어 있으며

축삭은 전전두엽 피질, 기저핵, 해마, 시상하부 및 척수로 돌출되어 있습니다 [15,64].

조현병의 도파민 모델과 함께 세로토닌 기능 장애는 Meltzer에 의해 제안되었습니다 [65].

1.2.4. Histamine

Histamine is a signaling molecule involved in different physiological functions, and it acts as a neurotransmitter in the central nervous system. Histaminergic neurons are localized in the tuberomammillary nucleus of the hypothalamus and project to various regions of the brain and spinal cord, including the amygdala, cerebral cortex, substantia nigra, striatum, thalamus and retina [57,66,67,68]. Studies have shown that this neurotransmitter is involved in various disorders including Alzheimer’s disease and schizophrenia [67]. Histamine may have an anticonvulsive role during development [68].

1.2.4. 히스타민

히스타민은 다양한 생리적 기능에 관여하는 신호 분자로

중추신경계에서 신경전달물질로 작용합니다.

히스타민성 뉴런은

시상하부의 결절 유선핵에 국한되어

편도체, 대뇌 피질, 흑질, 선조체, 시상 및 망막을 포함한

뇌와 척수의 다양한 부위에 투사됩니다 [57,66,67,68].

연구에 따르면 이 신경전달물질은

알츠하이머병과 정신분열증을 비롯한 다양한 질환에 관여하는 것으로 나타났습니다[67].

히스타민은 발달 과정에서

항경련제 역할을 할 수 있습니다 [68].

1.3. Other Chemical Substances

Several other substances exist that are identified as neurotransmitters. Acetylcholine is the most studied as it is the first neurotransmitters to have been identified, due to its release at neuromuscular junctions and hence relative ease of identification and tracing [69]. It is responsible for muscle contraction at the neuromuscular junction and is also released by the post-ganglional neurons in the parasympathetic system [15,57]. In the central nervous system, the cholinergic system plays a major part in consciousness [70]. Cholinergic neurons are present in various parts of the brain and brain stem including the striatum, cranial nerves and vestibular nuclei.

An acetylcholine deficit in the cortical cholinergic system has been associated with hallucination in Lewy bodie’s dementia [71]. Hypocholinergic activity in the hippocampus and in the cortex has also been associated with memory dysfunction in Alzheimer’s disease [70]. Acetylcholine also plays a role in the sleep–wake cycle [72].

Other substances identified as neurotransmitters in the brain include purines such as adenosine triphosphate (ATP) [73]; some soluble gases known as gasotransmitters [74] including carbon monoxide [75], nitric oxide [76] and hydrogen sulfide [77]; and neuropeptide Y and substance P of the neuropeptide group [57].

1.3. 기타 화학 물질

신경전달물질로 확인된

다른 여러 물질이 존재합니다.

아세틸콜린은

신경근 접합부에서 방출되어

상대적으로 식별 및 추적이 용이하기 때문에

가장 먼저 확인된 신경전달물질이기 때문에 가장 많이 연구되고 있습니다[69].

신경근 접합부에서

근육 수축을 담당하며

부교감신경계의 신경절 후 신경세포에서도 방출됩니다 [15,57].

중추신경계에서

콜린성 신경계는

의식에 중요한 역할을 합니다[70]. 콜린성 뉴런은 선조체, 뇌신경, 전정핵을 포함한 뇌와 뇌간의 다양한 부위에 존재합니다.

대뇌 피질 콜린성 시스템의 아세틸콜린 결핍은 루이소체 치매의 환각과 관련이 있습니다 [71]. 해마와 피질의 콜린성 저활동은 알츠하이머병의 기억 기능 장애와도 관련이 있습니다 [70].

아세틸콜린은

수면-각성 주기에도 중요한 역할을 합니다[72].

뇌에서 신경전달물질로 확인된 다른 물질로는

아데노신 삼인산(ATP)[73]과 같은 퓨린,

일산화탄소[75],

산화질소[76],

황화수소[77] 등 가스전달물질[74]로 알려진

일부 가용성 가스,

신경펩타이드 Y 및 신경펩타이드 그룹의 물질 P[57] 등이 있습니다.

1.4. Concluding Remarks on Neurotransmitters

Several chemical substances have been identified as neurotransmitters and others have yet to be discovered. As reported in Table 1, the concentration of these substances in the brain is very low and they are often only produced in specific regions; for example, L-aspartate is mostly produced in the hippocampus while dopamine is produced in the hypothalamus and substantia nigra. Nevertheless, they are among the most important substances that the body produces. They are involved in many vital functions such as vision, learning, sleep and emotions among others, and their dysfunction can lead to serious health issues, including anxiety, depression, schizophrenia, Parkinson’s and Alzheimer’s diseases among others. Given their important roles in the central nervous system and the severity of disorders linked to their dysfunctions, the accurate detection and measurement of neurotransmitters are among the most important challenges in neuroscience.

1.4. 신경전달물질에 대한 마무리 발언

신경전달물질로 확인된 화학물질은

여러 가지가 있으며

아직 발견되지 않은 물질도 있습니다.

표 1에서 볼 수 있듯이 이러한 물질은

뇌에서 농도가 매우 낮고

특정 부위에서만 생성되는 경우가 많습니다

(예: L-아스파르트산염은 주로 해마에서 생성되는 반면

도파민은 시상하부와 흑질에서 생성됩니다).

그럼에도 불구하고

이들은 신체에서 생성되는

가장 중요한 물질 중 하나입니다.

도파민은

시각, 학습, 수면, 감정 등 여러 가지 중요한 기능에 관여하며

기능 장애는 불안, 우울증, 조현병, 파킨슨병, 알츠하이머병 등

심각한 건강 문제를 일으킬 수 있습니다.

중추신경계에서 신경전달물질의 중요한 역할과 기능 장애와 관련된 장애의 심각성을 고려할 때 신경전달물질의 정확한 검출과 측정은 신경과학에서 가장 중요한 과제 중 하나입니다.

Table 1. Summary of major neurotransmitters.

Given the importance of their detection, several articles reviewed the advances and challenges in neurotransmitter detection. Wu et al. [1] reviewed analytical and quantitative in vivo neurotransmitter monitoring techniques based on electrochemical and imaging approaches. Cross-species PET (positron-emission tomography) imaging advances and trends for neurotransmitter detection in brain have been surveyed by Finnema et al. [5]. Electrochemical sensing based on nanomaterial has been extensively surveyed by Sanghavi et al. [7]. The microelectronics interface design consideration was summarized in a review by Mirzaei and Sawan [9]. Advances in nanomaterials and polymer-based sensors and their potential for in situ optical neurotransmitters detection application were reported in a review by Soleymani [10]. Other works detailed the detection methodology for a specific neurotransmitters detection technique have been reported [8,11,12,14]. However, all those reviews lack a comparison of the strengths and weaknesses of the surveyed techniques. This work aims to provide such as comparison and highlight the guidelines for choosing a given neurotransmitter detection technique. The remaining of this article reviews the different methods used for their sensing and detection. It includes classical methods such as microdialysis and analytical chemistry-based methods, as well as more recent research techniques such as optical and in vivo electrochemical sensing for a faster response, instrument miniaturization, high sensitivity and selectivity. The classical techniques such as microdialysis are well studied and can be used for neurotransmitter sampling for clinical purposes in hospitals. On the other hand, the cytotoxicity of the materials used by the other techniques such as nano-object optical sensing methods is not fully studied. Thus, despite their high sensitivity and selectivity, these techniques are currently only used for in vitro research.

2. Neurotransmitter Detection Methods

2.1. Nuclear Medicine Tomographic Imaging

2.1.1. Positron Emission Tomography (PET)

PET is a non-invasive neuroimaging technique that indirectly measures neuronal activity or neurotransmitters release [91,92]. In nuclear medicine, it is used to measure the metabolic or molecular activity of an organ in three dimensions, after the intravenous injection of a radioactive tracer, usually based on an injected isotope with short half-life, whose disintegration results in positron emission. Then, when a positron encounters an electron, their annihilation produces two photons of 511 keV (gamma photons) that propagate in opposite directions towards a scintillator for detection, hence defining a line of response. Each annihilation that takes place is localized by measuring the respective times of flight of the two photons before detection, and the results contribute to the reconstruction of an image of the overall process by tomography.

For cardiac, brain and tumor imaging, the radioactive element is often incorporated in glucose molecules to form a tracer with similar biological activity. Once in the blood stream, the tracer binds to glucose-consuming tissues, and the ensuing metabolic activity is recorded via functional images that are acquired by a gamma camera and reconstructed by a computer algorithm. For neurotransmitter release, the measurement relies on the competition between radio labeled neurotransmitter receptor ligands and the already bounded neurotransmitter target to its receptors [93]. It is related to measuring the change of binding potential of the receptor. For example, drug injection induces a dopamine change that is often measured with the 1111C-raclopride tracer, radio labelled as a D2/D3 receptor antagonist [93,94,95,96]. Then, the dopamine release is inferred by the decrease in radioligand binding to the receptor sites.

PET offers the potential of accurately finding where a given neurotransmitter is concentrated in the brain, but since it is based on the tracking of the tracer location, and the emitted photons are attenuated differently throughout the body, it is often necessary to normalize the observed activity with sophisticated algorithms for improved detection. On the other hand, the technique is almost with no risk to the patient, since a very small dose of radioactive tracer with low half-life is used.

Before 2009, the so-called Novel Methods leading to New Medications in Depression and Schizophrenia (NEWMEDS) only validated radioligands for endogenous dopamine detection. Since then, the development of new radioligands permit the detection of extracellular concentrations of many neurotransmitters including dopamine, serotonin and noradrenaline in human, monkey and rat brain [5]. Indeed, the combination of PET, MRI and EEG are now used in clinical domain for increased spatial and temporal resolution measurements of neurotransmitters such as serotonin [6].

2.1.2. Single-Photon Emission Computed Tomography (SPECT)

SPECT is another nuclear tomography neuroimaging technique that can be used to observe neurotransmitter release in the human brain [97,98]. To perform a SPECT, a radioactive product that emits gamma radiation is injected into the patient bloodstream. This radiotracer is chosen according to its chemical properties to selectively bind to some tissues and thus highlight certain biological processes. Then, a gamma camera is rotated around the patient to create an image of the radiopharmaceutical distribution. The camera consists essentially of a collimator that allows the angular selection of photons and gamma-ray detectors, and a tomographic reconstruction algorithm estimates the three-dimensional mapping of the radioactive activity, hence giving the distribution of the radiotracer in the body.

Like PET, SPECT is based on the in vivo competition between the endogenous neurotransmitter and the radiotracer to bind to a receptor. For example, the stimulation of dopamine release by amphetamine reduces the binding of 123123I-iodobenzamide (123123I-IBZM), which is a dopamine antagonist. However, SPECT differs from PET in other aspects: (1) the single gamma photon emitted in SPECT comes directly from the radioisotope atom, as opposed to the two photons created by annihilation in PET [99], hence allowing PET to offer higher spatial resolution (as it uses two photons to localize each point of interest); (2) More massive isotopes, such as 123123I-IBZM, can be used to label other molecules. For instance, 123123I-IBZM is a used in SPECT to study dopamine neurotransmission in the human brain [97,98]; (3) SPECT exhibits a much lower sensitivity (approximately two to three orders of magnitude) than PET, but it is also less costly.

While SPECT and PET are successful techniques for the spatial visualization of molecules such as neurotransmitters, they are limited by the complexity involved in label development, due to the signal overlapping between the labeled compound and the target metabolite [2]. The matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was developed to provide a high resolution multiplexed spectra at near-cell spatial resolution [3]. However, applying MALDI-MS requires the modification of most neurotransmitters due poor ionization efficiency. Shariatgorji et al. used pyrylium salts to ionize several neurotransmitters and, in combination multiplexed data acquisition, several neurotransmitters including GABA, glutamate, serotonin and dopamine, among others, could be accurately detected [2].

2.2. Optical Sensing Techniques2.2.1. Surface-Enhanced Raman Spectroscopy (SERS)

SERS uses photon interactions with matter to detect and identify molecules. This Raman spectroscopy technique enhances the Raman scattering by molecules adsorbed on an irregular metal surface, with an amplification factor that can reach 10 to 100 billion [100]. The method requires a functionalized surface for each molecule, and it can be made sensitive enough to detect single molecules [101]. The signal is enhanced by two main effects: electromagnetic and chemical enhancements. The enhancing materials are usually made of noble metals, e.g., Au/Ag nanorods and Au/Ag nanoparticles, and they are used as substrates to generate high electromagnetic fields. However, the SERS signals derived from individual nanoparticles are often too weak for detection; for high SERS signals, nanoparticle assemblies with tunable gaps can improve the electromagnetic fields. In general, the smaller the gap, the stronger the signal [102].

Suitable SERS substrates were designed to detect various neurotransmitters by adding silver colloids [103], silver nanoparticles [104] or hollow core photonic crystal fiber [105] to the cell culture medium. SERS is used to detect concentrations of choline (Limit Of Detection (LOD): 2 µM), acetylcholine (LOD: 4 µM), dopamine (LOD: 100 nM) and epinephrine (LOD: 700 µM) [104], glutamate (LOD: 100 nM) and GABA (LOD: 100 µM) [105]. In the case of dopamine, an LOD of 6 fM was achieved using Au@Ag nanorod dimers based on aptamer that are thiolated, which DNA specifically binds to dopamine [102].

Although noble metallic nanorods and nanoparticles coupled with aptamers permit a highly sensitive and selective SERS, some of them are toxic or cause irreversible aggregations. Thus, techniques such as the one described in [102,104,105] are usually not usable for in vivo diagnosis.

2.2.2. Fluorescence

Fluorescence is the emission of photons by a molecule or material such as metal nanoparticles or quantum dots, following light stimulation at one or more specific wavelengths. It reflects the returns of electrons to their ground state after shifting into a higher energy state by the incident light. The electrons dissipate part of the received energy through vibrations and return to their ground state by emitting a photon with the remaining energy, which is typically lower than the incident one, yielding a longer emitted wavelength. For example, neurotransmitters including serotonin, dopamine and norepinephrine have been detected by fluorescence measurements carried out at 320 nm, with excitation at 279 nm [106].

Although the fluorescence sensing method is sensitive, interfering compounds can seriously hinder its neurotransmitter detection efficiency. For example, 5-methoxytryptamine, N-acetylserotonin or melatonin all affect serotonin detection [107]. Solvent extraction with few interfering materials may be avoided with ion exchange resin (Amberlite CG-50), which acts as a suitable vehicle for the refinement and concentration of serotonin from biological samples [108]. In addition, high-sensitivity fluorescence sensing of serotonin was achieved with ninhydrin, which acts as a fluorescence catalyst [109]. Fluorescence efficiency was also improved through the use of O-pthaldehyde (OPT) under heat and strong acid conditions [110]. This method requires only 50 mg of tissue samples, but the extraction, concentration and purification methods, and the assay method as well, are time consuming and the fluorescence intensity is low. Serotonin can also be detected in biological fluids using ethanol extraction, which enhances fluorescence intensity without any time-consuming steps [111]. However, this technique is not appropriate for repetitive analysis as serotonin in blood is rapidly oxidized by oxyhaemoglobin [111]. Adding ascorbic acid with dry ice acetone can avoid this oxidation [112]. Finally, combining fluorometric detection with HPLC reached a high sensitivity and selectivity for serotonin [113] and dopamine [114] with an LOD of 1 pM concentration, and a signal to noise ratio of 5.

An in situ fluorescein-based neurotransmitter detection protocol was developed by Dey et al. for the rapid and low-cost rapid detection of histamine. Dye-coated paper strips were used to detect histamine in various biological fluids, including human plasma and urine. The spectral analysis of the samples revealed that this technique can be used to detect histamine at concentrations as low as 24 nM [4].

Fluorescent single-walled carbon nanotubes have been used to detect dopamine with an LOD of 11 nM [115]. The single-walled carbon nanotubes are modified with a DNA/RNA coronae that acts as a conformational switch, thus reversibly modulating the nanotube fluorescence. As reported by Kruss et al., by using a specific polymer wrapper, this technique can be used to detect other catecholamines with very high sensitivity and selectivity [115]. A similar work was reported by Mann et al. with an improved LOD of 2.3 nM [116].

2.2.3. Förster Resonance Energy Transfer (FRET) and Photoinduced Electron Transfer (PIET)

FRET is based on the radiation-free transfer of energy from a “donor” fluorophore to an “acceptor” fluorophore [117,118]. Upon excitation, the electrons at the valence band of the donor move to a higher energy or conduction band, and then immediately return to the lower energy state. Thus, an emission of light at different wavelengths is observed [119]. The quenchers (acceptors) absorb the energy from the donors in the excited state. Consequently, an energy transfer and subsequent quenching of the donor fluorophore is observed, while the acceptor fluorophore sees an increase in its emission intensity [120]. The quenchers are normally organic dyes, quantum dots (QDs), nanoparticles or biological molecules with an absorption spectrum close to the emission spectrum of the donor.

For instance, dopamine can quench the fluorescence of QDs [121,122,123]. Quinone, an oxidized form of dopamine, is considered as an electron acceptor. When the QDs are excited to a higher energy state, their electrons are pushed to the lowest vacant molecular orbital of quinone and, then, the electrons which are transferred back to the valence band of the quantum dot induce a quenched state of QDs. The rate of electron transfer depends on the pH and concentration of the oxidized dopamine in the solution [124]. QDs can transfer energy to molecules able to generate singlet oxygen [125]. Dopamine can thus behave as a photosensitizer by generating singlet oxygen through auto-oxidation. In alkaline solutions, functionalized quantum dots are conjugated to dopamine via hydrogen bonding which can be oxidized to quinone, leading to quenching the fluorescence of the quantum dots with a high LOD of 0.3 nM [122,126,127]. Hybrid quantum dots with natural thiol containing molecules on the surface have good solubility and high selectivity for dopamine in biological fluids, for a high LOD of 0.8 nM [128].

As cadmium-based quantum dots are expensive and toxic, they have been replaced by more affordable ZnO quantum dots that are biocompatible and environmentally-friendly for dopamine sensing [129]. Fluorescence detection techniques without heavy metal ions release were developed through the use of natural amino-acids as surface coating to show a high LOD of 875 pM [130]. Considering the heavy metal toxicity and environmental hazards [131], graphene quantum dots are preferred to semiconductor based quantum dots due to their environmentally-friendly properties [132], excellent water stability (neurotransmitters are always in aqueous solutions), stable photoluminescence and high electrical conductivity [133]. Polyprole microsphere–graphene core–shell hybrid systems for dopamine open a new prospect for sensing dopamine with LOD of 10 pM in real-time sensing [134].

Gold nanoparticles are also excellent acceptors to replace the traditional organic quenchers [135] for photoinduced electron transfer based dopamine sensing [136]. Gold nanoparticles can be functionalized with aptamers, which are single-stranded nucleic acids with a specific affinity to one molecule—for example, dopamine. They are screened by an in vitro selection technique named SELEX (systematic evolution of ligands by exponential enrichment) [137,138]. Dopamine binding aptamers prevent the aggregation of gold nanoparticles upon NaCl addition. Following the addition of dopamine, it is conjugated to the dopamine binding aptamer, leaving unprotected nanoparticles. The later aggregate, resulting in the fluorescence quenching of rhodamine B. The fluorescence intensity is proportional to dopamine concentration with an LOD of 2 nM [139].

2.2.4. Chemiluminescence

Chemiluminescence is the production of light during a chemical reaction: An energy donor molecule enters an excited state and transfers that energy to an acceptor (luminescence carrier), which releases a photon in order to return to its fundamental state, hence the luminescence. The technique was used for the identification of catecholamines in the culture of a clonal line of rat pheochromocytoma cells [140]. The chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and H2O2 was used in an HPLC (High Performance Liquid Chromatography, described below) detection system to determine fluorescamine-labeled catecholamines [141]. The technique was coupled with HPLC for the separation of various biological samples with high selectivity [142]. Other works used a flow injection inhibitory chemiluminescence to detect the concentration of dopamine hydrochloride [143,144]. The method relies on the ability of dopamine hydrochloride to inhibit the chemiluminescence of a luminol–potassium hexacyanoferrate(III) system [145,146] and a potassium permanganate–formaldehyde system in acidic medium [147]. A sensitivity of 6 nM was reported [143].

The real-time measurement of catecholamines in urine samples was achieved by combining HPLC and flow injection with the chemiluminescence generated through the Ca inhibition effect on the oxidation of luminol by iodine in alkaline solution [144]. Further advancement in flow injection systems with plant tissue-derived molecular probe polyphenol oxidase is also used [148,149], catalyzing the oxidation of dopamine by oxygen. The latter generates hydrogen peroxide to induce chemiluminescence with luminol [150].

2.2.5. Optical Fiber Biosensing

The principle of optical fiber biosensors is based on the optical transduction induced by the binding of the target molecule to an immobilized receptor on optical fibers acting as transduction elements [151]. The optical fiber core is doped with germanium for a higher refractive index than the surrounding cladding made of silica, which then induces light propagation through total internal reflection [152]. When light passes through the optical fiber, the generated electromagnetic field or evanescent wave is related to the distance-dependent, rapid light intensity decay that occurs between the interface and the medium of smaller refractive index. In the case of molecular recognition, the evanescent light excites a fluorophor and the resulting fluorescence emission is propagated through the fiber towards an optical sensor [153]. The optical fiber-based sensing technique was first applied to glutamate sensing by immobilizing glutamate dehydrogenase at the probe (core), which catalyzes the production of reduced nicotinamide-adenine dinucleotide (NADH) upon oxidation with glutamate. The obtained NADH produces fluorescent light that is proportional to the concentration of glutamate [154]. This technique is advantageous in monitoring glutamate release and continuous monitoring of neurophysiological responses, and an LOD of 0.22 µM and absolute mass detection limit of 44 fg were reported [155].

Fiber optic aptasensors have been used for dopamine detection around the physiological concentration (LOD: 37 nM) [156]. The sensor is composed of a recognition element made of a 57-mer dopamine binding aptamer and a probe made of a nonadiabatic tapered optical fiber. The aptamer has a specific affinity to dopamine and, in the presence of dopamine, it changes its structure from a random conformation to a rigid pocket-like tertiary conformation as shown in Figure 2. This change of tertiary conformation induces a variation of the refractive index around the tapered fiber, hence indirectly permitting the detection of dopamine.

Figure 2. Schematic representation of the fiber optic and aptamer based dopamine detection [156].

2.2.6. Colorimetry

This technique uses the interaction between a molecule of interest and other compounds to induce a colored reaction. The concentration of the target molecule then depends on the color and intensity of the resulting solution. Using this technique, dopamine was detected when binding with Ag-catechol [157], gold nanoparticles [158] and AHMT functionalized gold nanoparticles [159]. The reported LOD was 60 nM [157]. Other neurotransmitters were also detected using this technique, such as noradrenaline (LOD: 20 µM), adrenaline (LOD: 2.5 µM) and L-DOPA (LOD: 2.5 µM) [160].

The colorimetry technique can also use resonance light scattering. It then induces a collective oscillation of electrons on the surfaces of plasmonic nanoparticles and results in an absorption/scattering effect at different wavelengths upon aggregation. The resonance light scattering technique can be advantageous due to its sensitivity and selectivity. Plasmonic nanoparticles based on gold and silver are highly efficient at light absorption and scattering [161]. For example, dopamine has been detected though a shift in wavelength induced by the aggregation of gold nanoparticles synthesized primarily with dopamine as reducing agent [160,162,163]. The indirect aggregation of nanoparticles is caused by the hydrogen bond formation within the Au-dopamine-TGA complex, thereby inducing a shift from red—purple—gray—yellow when dopamine concentration increases. However, a reversible shift (blue to red) was induced by dopamine concentration in aggregated melamine functionalized gold nanoparticles [164,165]. Furthermore, aggregation induced by the selective binding between gold optical probes and dopamine through specific molecular bridges including aptamers avoids common interference with other neurotransmitters [159,166]. In some occurrences, dopamine sensitivity is affected by the medium because, it is oxidized to quinone which is unstable and quickly polymerized to polydopamine in alkaline conditions. However, polydopamine nanoparticles are intrinsically fluorescent in an acid medium, allowing dopamine sensing with an LOD of 40 nm [167]. The reliable detection of dopamine with the low-cost resorcinol, reaching an LOD of 1.8 nM, is a good alternative for the real-time detection of this neurotransmitter [168]. Although neurotransmitter detection in the cerebral system is still challenging, the colorimetric technique to detect dopamine with functionalized gold nanoparticles is promising [158].

2.3. Electrochemistry

Electrochemistry-based detection relies on measuring electrical quantities such as current, potential or charge in relation to chemical parameters [169]. It has been extensively used for the study of catecholamines [42], due to their oxidation properties. In vivo measurement of catecholamines in extracellular fluid was achieved with carbon microelectrodes implanted into the brain of a rat [170].

The direct detection of neurotransmitters in the brain by this technique is very challenging given their very small concentrations, notwithstanding the presence of interfering compounds [171]. For example, the oxidation of ascorbic acid occurs at a voltage that is close to those of catecholamines, thus making their detection in the presence of ascorbate difficult [172,173]. Next, a description of the three main electrochemistry detection techniques is presented: fast-scan cyclic voltammetry, amperometry and coulometry.

2.3.1. Fast-Scan Cyclic Voltammetry (FSCV)

Voltammetry relies on measuring the current flowing between two electrodes, a working electrode and a counter electrode, subjected to a voltage difference during a redox chemical reaction. The working electrode is responsible for the redox current and the counter electrode maintains a constant potential while passing a current to counter the redox events at the working electrode. In practice, it is difficult to make this set up work and a third electrode, called the reference electrode, is added to relieve the counter electrode of maintaining the voltage difference.

In FSCV, the applied voltage is a repetitive ramp with a high slew rate [174]. During FSCV scans, an electroactive neurotransmitter near the working electrode rapidly oxidizes and transfers electrons to the electrode surface, thus generating a faradic current [175]. A reduction faradic current of approximately the same order as the oxidation current is also observed [174]. FSCV can be based on carbon-fiber electrodes to detect indolamines such as serotonin, and catecholamines such as adrenalin, noradrenalin and dopamine as illustrated in Figure 3 [176], since they can be oxidized at low voltage [177]. FSCV can be used for in vitro as well as in vivo experiments in single neurons, brain slices, and anesthetized or moving animals such as a rat with a small headstage fixed on the head [178].

Figure 3. Voltammetric detection of dopamine. Dopamine is oxidized to dopamine-o-quinone when a potential is applied to the electrode. The resulting electrons from this oxidation generate a current that is detected. When the potential is removed from the electrode, the dopamine-o-quinone is reduced back to dopamine, thus producing a current in the reverse direction [179].

When used for neurotransmitter sensing, FSCV consists first of maintaining the electrode potential beneath the oxidizing potential of the neurotransmitter of interest (e.g., –0.4 V for dopamine with an Ag/AgCl reference electrode). Then, the electrode potential is increased above the neurotransmitter oxidizing potential (around 1.0 V) at a high scan rate, usually 400 V/s. The duration of each cycle is 100 ms [177,179,180], and the measured current depends on the neurotransmitter concentration.

The main difference between FSCV and Cyclic Voltammetry (CV) is the very high scan rate used in FSCV. Indeed, the scan rate can be much higher than 400 V/s and the duration of each cycle can vary depending on application. It has been reported that FSCV is more convenient for neurotransmitter sensing in in vivo experiments because it has a better resolution than CV. However, both techniques should be able to detect the same molecules. If the molecule is not detectable with CV at high concentration, we should not expect it to be detected through FSCV either.

Each voltammogram (current vs. voltage curves) is unique to a substance and can be used to identify neurotransmitters as illustrated in Figure 4 [180]. Indeed, the voltammogram depends on the molecule’s properties such as its electron transfer rate and chemical stability [181]. Those factors determine the positions and shapes of the reduction and oxidation peaks, as well as their relative amplitudes [181]. As the current generated at the oxidation voltage corresponds to the concentration fluctuation versus time, it represents the changes in neurotransmitter concentration upon external stimulation [182]. FSCV based on in vivo dopamine sensing in the rat brain was demonstrated with Nafion-coated microelectrodes [183], tungsten-coated microelectrodes [184] and carbon fibre microelectrodes [183] to measure sub-micromolar changes in the concentration of dopamine upon stimulus. To enhance the sensitivity and selectivity of this technique for dopamine, electrode surface functionalization is needed such as modified carbon paste solid electrodes [185,186] and electrically-treated carbon micro-electrodes [187]. Electrode functionalization with polymers was also investigated, such as Nafion-coated electrode which is specific to cationic species such as dopamine, serotonin [188], clay-modified electrodes [189], electroactive polymer such as polypyrrole coated [190], poly (N,N-dimethylaniline)-modified electrodes [191], and uncoated carbon fiber microelectrodes [192]. A higher sensitivity is achieved by extending the anodic scan rate to +1400 V/s. Then, a brain tissue dopamine detection limit of 50 nM was achieved [193]. However, the detection of dopamine and serotonin is problematic because of the reactive species that are generated after oxidation and stick to the electrode surface, thus affecting the system sensitivity [194,195]. Therefore, when dopamine and serotonin are mixed together, selectivity becomes a challenge due to the similarities of the two molecules. That limitation can be improved by using carbon nanotube-modified microelectrodes [196]. Furthermore, selective dopamine detection in the presence of ascorbic acid was reported through electropolymerization of polyglycine on carbon paste electrodes, which improves the redox peak currents of dopamine [197].

Figure 4. Dopamine (DA) and serotonin (5-HT) voltammograms. The voltammogram of dopamine 1 µM shows one oxidation and one reduction peak. The voltammogram of serotonin 1 µM shows one oxidation and two reduction peaks [177].

2.3.2. Amperometry

Amperometry applies a fixed potential to a working electrode and measures the resulting oxido-reduction reaction current, which is proportional to the analyte concentration [198]. Like voltammetry, the current through the working electrode is measured with respect to a reference electrode, but, unlike FSCV, the temporal resolution of amperometry is not limited to the duration of the cycle. Thus, amperometry is more suitable to analyze the kinetics of neurotransmitter release.

The catecholamines and indolamines in a cell are efficiently detected with amperometry. A carbon-fiber microelectrode maintains a constant voltage above the oxidizing potential of the neurotransmitter (Figure 5), which is 650 mV and 800 mV for serotonin and catecholamine, respectively. The electrons released by the neurotransmitters are then transferred to the electrode [179,199]. The temporal resolution is limited by two factors: the diffusion of the neurotransmitter to the electrode and the kinetics of the electron transfer [180]. The current measured by the electrode is dependent on the concentration of neurotransmitter released by the cell [200], but the released neurotransmitter cannot be identified [179]. In this case, a complementary method such as high performance liquid chromatography is needed [200].

Figure 5. Detection of dopamine by amperometry. Experimental set-up for amperometric recording of vesicular catecholamine release from a secretory cell using a carbon fiber microelectrode; (A) schematic drawing of the electrochemical reaction; (B) amperometric recording from a PC12 cell; (C) example of an amperometric current transient on an expanded time scale [200].

2.4. High Performance Liquid Chromatography (HPLC)

HPLC separates the different molecules in a mixture called the mobile phase, which is injected into a separation column of a diameter of typically 3.0–4.6 mm, initially filled with a stationary phase made of porous particles of 2.5–10 µM diameter [201,202]. The molecules are introduced in the mobile phase under high pressure and then move through the stationary phase. The Van Deemter equation describes the relationship between linear velocity (flow rate) and column height [201]. Depending on the molecules’ properties, the interaction with the stationary phase is different: the molecules having a high affinity with the mobile phase migrate along the column faster, and are therefore the first to pass through it. The peak on the chromatograph is observed as shown in Figure 6, and the surface beneath the peak is proportional to the molecule’s quantity [201,203]. HPLC is a very sensitive technique that allows the detection of two enantiomers. it was used to detect D-serine in the brain [46]. It is also often coupled with other techniques such as chemiluminescence. When coupled with mass spectrometry, it has been used to detect several neurotransmitters including GABA, pyroglutamic acid, aspartic acid, asparagine, glutamic acid, glutamine, N-acetyl-L-aspartic acid, acetylcholine and its metabolite choline. The reported LOD for those neurotransmitters were 1.820, 0.046, 0.088, 1.930, 1.520, 1.050, 0.250, 0.012, and 0.620 nM, respectively.

Figure 6. HPLC analysis of some neurotransmitters; chromatogram of neurotransmitters separated by the microbore HPLC column. (A): standard DOPAC (50 pg), dopamine (150 pg), serotonin (100 pg) and 5-HIAA (50 pg); (B): DOPAC, dopamine, serotonin and 5-HIAA separated from zebrafish brain lysate [205].

Simultaneous detection of monoamine and amino acid neurotransmitters in rat endbrains was achieved by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification. Five molecules including L-glutamate, GABA, dopamine serotonine and 5-hydroxyindole acetic acid, which is a metabolite of serotonine, were separated and then detected with an LOD of 0.398–1.258 nM [204].

2.5. Microdialysis

Microdialysis is one of the well studied neurotransmitter detection techniques and its in vivo applications have been used for over three decades. For instance, it has been used for the measurement of neurotransmitters such as acetylcholine, neuropeptide, amino acids and amines in human brain [206]. It is usually used in conjunction with other detection and separation techniques such as liquid chromatography and mass spectrometry [207]. The technique consists of inserting into the brain a probe formed by a semi-permeable membrane of 1–4 mm length and 0.2–0.4 mm diameter, perfused at a constant flow between 0.1–5 µL/min as shown in Figure 7 [206,208,209]. Artificial cerebrospinal fluid is used to perfuse the probe to prevent ionic diffusion [208,209]. The membrane is semi-permeable so that molecules in the extracellular fluid will diffuse according to their gradient [206,208]. This principle can also be used to inject molecules into the extracellular fluid for reverse dialysis or retro-dialysis [208,209]. The micro-dialysis probe is placed outside the synaptic gap. Therefore, the neurotransmitter concentration measured by micro-dialysis is a balance between the molecules released into and removed from the extracellular space [208].

Figure 7. Microdialysis probe. A microdialysis probe is formed by a semipermeable membrane and perfused at a constant flow. Molecules will be able to diffuse across the membrane according to their gradient [209].

2.6. Other Detection TechniquesTandem Mass Spectrometry

Mass spectrometry ionizes analytes and sorts neurotransmitters according to their mass-to-charge ratio. The Tandem Mass Spectrometry is based on the same principle but uses multiple stages of detection and fragmentation. It is often combined with various types of chromatography [210,211,212] to detect over twenty-five types of neurotransmitters [213].

A detection method known as ultra-high performance liquid chromatography-tandem mass spectrometry, based on derivatization techniques developed by Zhao et al., can not only determine a variety of neurotransmitters but also can bring super high sensitivity, accuracy, selectivity, and the combination of high sensitivity and high time resolution of in vivo microdialysis [214]. This technique may play an important role in fast neuropathies diagnostics. In situ ultrasound-assisted derivatization dispersive liquid–liquid microex-traction coupled with ultra high-performance liquid chromatography tandem massspectrometry was demonstrated to be a potential method for sensitive, accurate and simultaneous monitoring of amino acids and monoamines neurotransmittes for neurodegenrative disordders such as Alzheimer’s disease [215].

3. Neurotransmitter Detection Techniques’ Guidelines

The surveyed neurotransmitter detection techniques all have strengths and weaknesses as shown in Table 2. The suitability of a technique over the others depends on the intended application. For instance, in vivo usable techniques are better than those only usable in vitro for clinical applications, but this often comes with more complexity, lower sensitivity and/or selectivity, and higher cost. In addition, the methods with higher sensitivity and selectivity are often bulkier and require complex and time-consuming manipulations.

Table 2. Summary of neurotransmitters’ detection techniques.

Nuclear medicine tomographic imaging offers high spatial resolution, but the equipment is expensive, making these techniques only suitable for diagnostic purposes where the functional studies and accurate localization of neurotransmitters in the brain are needed. In addition, since they are not suitable for neurotransmitter concentration detection, they must be used with techniques that have higher concentration sensibility to get accurate neurotransmitter concentration measurements. In hospitals and research laboratories, they can be used with microdialysis or analytical chemistry techniques such as HPLC.

Analytical chemistry techniques may require costly equipment, but, as they are traditionally used in most bio-chemical laboratories for various chemical analyses, this equipment is available for most neuroscientists. In addition to cost, their inconvenience is that they require complex manipulations and are time consuming. On the other hand, microdialysis, sometimes used in conjunction with other methods such as HPLC, is one of the most used neurotransmitter detection methods for clinical use. It is well studied and can be used for continuously monitoring drug or metabolite concentrations in the extracellular fluid of virtually any tissue. However, as with all invasive detection techniques, it presents practical and ethical limitations for most researchers. Moreover, its temporal and spatial resolution is very limited when compared to nuclear medicine tomographic imaging.

With the rapid development of innovative technologies, and the emergence of multidisciplinary research outcomes in biomedical engineering, the current research tends to focus on more compact, portable and implantable neurotransmitter sensors. As a result, several techniques have been proposed or revisited. For instance, the electrochemical detection technique is reputed to be easy to implement in an implantable device, in addition to being cost effective. However, due to its low selectivity and the complexity of in-vivo chemical/molecular monitoring, its usage in real world applications still needs improvements in terms of reliability and selectivity. In addition, not all neurotransmitters can receive or yield electrons in a redox reaction on an electrode surface. Thus, only reactive neurotransmitters such as dopamine can be detected by using an electrochemical method without electrode functionalization. The electrochemical approach is also closely dependent on the electrode surface, which reacts with the targeted molecule. Thus, it is not possible to guarantee the stability and quality of the electrode for a long time; although it is possible to detect a good signal over a small number of detection cycles, the electrode degradation becomes significant as the number of cycles grows. This affects the number of electrons that can be trapped by the electrode, hence lowering the sensed current. Consequently, in terms of engineering, it is impossible to guarantee consistent results over time. That is the reason why, for instance, in the case of glucose sensors, the commercial products are based on one-time usage electrodes in order to guarantee result reliability.

If we compare the electrochemical research for glucose sensors and brain electrochemical sensing, the environment itself is very different. The glucose sensors are adapted to blood which is a very non-homogeneous liquid, but available in important quantities, while, in the case of the brain, the opposite is true: the cerebrospinal fluid volume is available in less volume, but it is more homogeneous. Moreover, brain volume samples are less reachable than blood because of the complexity to access the sampling zone, and when an electrode is implanted into the brain, the tissue can react with the implanted devices. All of these aspects make the use of electrochemical methods problematic for brain research. Still, some research uses this technique, whereas other techniques are being developed.

Optical sensing techniques are suitable for miniaturization, thanks to rapidly advancing optoelectronic and MEMS (micro-electromechanical systems ) micro-fabrication technologies, and the possibility to automate chemical reactions with micro-reactors. With adequately selected functionalized quantum dots (QDs), enhanced surfaces by noble metals particles or aptamers, this emerging technology is among the most promising for neurotransmitter detection with high accuracy. One of the known optical sensing weaknesses is the limitations due to Debye length. While aptamer modified surfaces provide high selectivity in SERS based biomolecule detection, Raman signal is only enhanced within the physiological Debye length, which is approximately 1 nm. Hence, this may complicate the detection of small molecules such as neurotransmitters. However, Nakatsuka et al. proposed using aptamer modified field-effect transistors (FET), which can overcome this limitation [218]. Their strategy of combining both high sensitive FET and high selective aptamer may open another branch of neurotransmitter detection based on electronics in parallel to the known optical techniques.

Another weakness of optical sensing techniques is that some of the sensing elements may be toxic and thus unusable for in vivo neurotransmitters detection. Furthermore, these techniques rely on the optical properties of neurotransmitter molecules. The approach is acceptable when experiments are performed in a laboratory environment and in vitro experiments. However, when molecules in the brain are targeted, the tissue interaction with and growth around the sensor should be considered. It is technically impossible to guarantee that the sensor environment will not be changed when in contact with the cerebral tissue. Knowing that most of optical sensors are based on optical stimulation of molecules, the optical path between the light source and the target molecule must remain clear, which is impossible to guarantee with actual technology, even if we use biocompatible materials. Indeed, when using such materials, we only ensure their non-toxicity, either directly or through secondary reactions. However, in many applications, the tissue growth helps to stabilize the system as is the case with pacemakers. Thus, we are confronted with controversial solutions: on one side, the tissue growth is important to stabilize the system in vivo, and, from the other side, it is not suitable to avoid optical path obstruction. In addition, unlike electrical techniques where electrical signals can pass through the tissue when the amplitude/frequency is adjusted adequately, optical stimulation is less adjustable. However, some researchers claim that, by increasing the intensity of visible light, they can go through some tissue, which is correct, with some limitations due to tissue density. If we compare this technology to pacemakers and deep brain stimulation which are successful technologies, brain optical stimulation still requires many validations before bringing it to a commercial level as a brain aid technology. Indeed, the electrical stimulation can be adjusted depending on the growing tissue, which makes electrical-based stimulation more practical. The growing tissue will change the electrode impedance and, consequently, by sampling the electrode impedance, it is possible to adjust the signal intensity as the tissue itself is electrically conductive.

In conclusion, considering today’s technologies, the optical sensors/stimulators are extremely sensitive to the biological environment, in contradistinction with the electrical sensors when applied in biomedical research. Electrical stimulations can also be adjusted more easily than optical stimulations. Finally, while some detection techniques such as SERS have been proven to be able to detect even a single molecule, noble metallic-based optical detection is relatively new and the cytotoxicity of the most used materials is currently unknown.

4. Conclusions

This work surveyed the most common neurotransmitters and their sensing techniques. These electrochemical messengers in the nervous system are synthesized and stored in presynaptic neurons, and they are released in the synaptic clef to act on postsynaptic neurons as inhibitors or exciters, thus affecting the transmission of information between neurons. More than 100 neurotransmitters have been identified and they are all essential for proper functioning of the nervous system. Given their vital role, their dysfunction can lead to serious malfunction of the central nervous system including psychiatric disorders such as schizophrenia, epilepsy, depression, Parkinson’s and Huntingdon’s disease, among others. Hence, for clinical or neuroscience research purposes, neurotransmitters have to be accurately detected and many techniques have been proposed to do so. However, given the low concentration of neurotransmitters and the large number of other biomolecules and minerals mixed with them, the accurate neurotransmitter detection in the brain is still a challenge. Some techniques such as microdialysis and HPLC have been clinically used for decades, but other methods such as optical fiber sensing and colorimetry are emerging with improved detection time, sensitivity and selectivity at lower cost. However, as neurotransmitters are generally locally produced in a specific area of the brain at a specific time, nuclear medicine tomographic imaging techniques are preferable where the kinetics of metabolism of the target molecule is required.

Author Contributions

All authors contributed to the overall review design, literature collection and analysis, and the writing of the manuscript. E.B., M.B., and A.M. contributed to the funding acquisition.

Funding

This work has been funded by the National Research Council of Canada (NSERC) and the Fonds de recherche du Québec–Nature et technologies (FRQNT)/Quebec Strategic Alliance for Microsystems (ReSMIQ).

Acknowledgments

The authors acknowledge the financial support from the National Research Council of Canada (NSERC) through the discovery grant program—the Fonds de recherche du Québec–Nature et technologies (FRQNT)/Quebec Strategic Alliance for Microsystems (ReSMIQ) through its complementary scholarship program. In addition, the authors are indebted to the Natural Sciences and Engineering Research Council of Canada (NSERC), the CHU de Québec Foundation and the Fonds de recherche du Québec – Nature et technologies (FRQNT)/Quebec Strategic Alliance for Microsystems (ReSMIQ) for their financial support. E.B. is a research scholar from the Fonds de recherche du Québec–Santé (FRQS) in partnership with the Antoine Turmel Foundation.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Wu, F.; Yu, P.; Mao, L. Analytical and Quantitative in Vivo Monitoring of Brain Neurochemistry by Electrochemical and Imaging Approaches. ACS Omega 2018, 3, 13267–13274. [Google Scholar] [CrossRef] [PubMed] [Green Version]
2. Shariatgorji, M.; Nilsson, A.; Goodwin, R.J.; Källback, P.; Schintu, N.; Zhang, X.; Crossman, A.R.; Bezard, E.; Svenningsson, P.; Andren, P.E. Direct targeted quantitative molecular imaging of neurotransmitters in brain tissue sections. Neuron 2014, 84, 697–707. [Google Scholar] [CrossRef] [PubMed]
3. Cornett, D.S.; Reyzer, M.L.; Chaurand, P.; Caprioli, R.M. MALDI imaging mass spectrometry: Molecular snapshots of biochemical systems. Nat. Methods 2007, 4, 828. [Google Scholar] [CrossRef] [PubMed]
4. Dey, N.; Ali, A.; Podder, S.; Majumdar, S.; Nandi, D.; Bhattacharya, S. Dual-Mode Optical Sensing of Histamine at Nanomolar Concentrations in Complex Biological Fluids and Living Cells. Chem.–A Eur. J. 2017, 23, 11891–11897. [Google Scholar] [CrossRef]
5. Finnema, S.J.; Scheinin, M.; Shahid, M.; Lehto, J.; Borroni, E.; Bang-Andersen, B.; Sallinen, J.; Wong, E.; Farde, L.; Halldin, C.; et al. Application of cross-species PET imaging to assess neurotransmitter release in brain. Psychopharmacology 2015, 232, 4129–4157. [Google Scholar] [CrossRef] [Green Version]
6. Bailey, D.; Pichler, B.; Gückel, B.; Barthel, H.; Beer, A.; Botnar, R.; Gillies, R.; Goh, V.; Gotthardt, M.; Hicks, R.; et al. Combined PET/MRI: From status quo to status go. summary report of the fifth international workshop on PET/MR imaging; February 15–19, 2016; Tübingen, Germany. Mol. Imaging Biol. 2016, 18, 637–650. [Google Scholar] [CrossRef]
7. Sanghavi, B.J.; Wolfbeis, O.S.; Hirsch, T.; Swami, N.S. Nanomaterial-based electrochemical sensing of neurological drugs and neurotransmitters. Microchim. Acta 2015, 182, 1–41. [Google Scholar] [CrossRef]