Non Invasive Measurement Of Neurotransmitters Biology Essay

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The nervous system is the control center for the human body. It is tasked with the integration and processing of information gathered from sensory organs located throughout the body. In response to the data gathered, the nervous system relays regulatory commands to the rest of the organs modifying their function as needed [1]. For example in cases of intense exercise where elevated levels of blood oxygenation are necessary, the nervous system issues commands that increase the heartbeat and therefore the blood circulation [1]. From an anatomical point of view the human nervous system can be divided into two main components: the Central Nervous System (CNS) and the Peripheral Nervous System (PNS). The Central Nervous System's main organs are the brain and the spinal cord. The Peripheral Nervous System includes the remaining neural tissue and can be further divided into an afferent division and an efferent division [3].

The elementary blocks of the nervous system are the neurons (or neural cells) and their supporting cells, the neuroglia (or glial cells) [3]. There are three classes of neurons depending on their function: the sensory (or afferent) neurons, the motor (or efferent) neurons and the interneurons which act as communication bridges between neurons [1]. Sensory neurons carry information concerning the body's internal conditions and external environment from a variety of sensory organs towards the CNS while the motor neurons carry commands away from the CNS [3]. Neurons can also be classified according to their shape, into four types: the anaxonic, the bipolar, the unipolar and the multipolar. The multipolar is the most abundant inside the CNS and it is mostly associated with motor function [1].

Figure The Anatomy of a multipolar neuron, illustrating its main regions and structural components (from Martini et al.) [1].

The main regions of a multipolar neuron are the soma, the dendrites, the axon and the telodendria [2]. The soma (from the Greek word "σώμα" for body) is the metabolic factory of the cell and includes the nucleus and the perikaryon. The perikaryon houses organelles with the ability to synthesize neurotransmitter chemicals [1]. The dendrites (from the Greek word "δέντρο" (dendro) for tree) are utilized by the nerve cells to receive incoming signals from other neurons while axons transport electrical signals through the neuron towards the telodendria [2]. At the end of the neuron the axon terminals house the synaptic vesicles, which contain and release the neurotransmitter chemicals upon receipt of a nerve impulse [3]. Neuron conductivity is attributed to a fairly simple interaction between the incoming stimulus and the neuron which we will describe briefly.

Inside a non - conducting neuron, a resting potential exists across the plasma membrane [3] at a value of approximately -70mV [1]. The resting potential is uniform throughout the neuron [2] and is created by the innate polarity of the plasma membrane. That is to say, the net electrical charge of the membrane's exterior is positive whereas the interior surface is negative [1]. This stems from the fact that the extracellular space has a high concentration of sodium ions (Na+) while the intracellular space exhibits a high concentration of potassium (K+) [2]. Moreover the membrane exhibits higher permeability towards potassium ions rather than sodium ions and for every pair of potassium ions that exit the cell three sodium ions enter to maintain this balance [3].Upon experiencing an external stimulus, the voltage gated sodium ion channels open [2], the membrane's permeability to Na+ increases and the cell's interior experiences a rapid influx of sodium. The additional positive charge causes the membrane to depolarize (at around -55mV [2]) and as the sodium influx continues the membrane's polarity is reversed [3]. It is precisely this reverse polarization of the neuron membrane that creates the action potential which propagates through the axon and triggers the neurotransmitter release. Eventually, when the transmembrane potential reaches a value of approximately +30 mV the inactivation begins. The voltage controlled sodium channels deactivate and the sodium - potassium levels return to normal levels [1].

Figure The generation of the action potential (from P.B. Wilhelm [3])

Note that according to the all-or-none principle, every stimulus will produce the exact same action potential (in amplitude) or not produce one at all [2].

1.1.2 Synaptic Transmission and Neurotransmitters

The signal exchange between neurons can only occur at specific sites, the synapses [6]. Neurons use two types of messengers to communicate: either chemical (neurotransmitters) or electrical pulses (nerve impulses). Therefore, we get two types of synapses: the electrical and the chemical. The later is by far more abundant in the human body [1] and this type of synaptic transmission is going to be our focus in this chapter.

In chemical synapses, unlike electrical, there is no physical contact between neurons and the interaction is far more complicated [1]. Generally speaking, every synapse incorporates a pre-synaptic neuron which is the one releasing the neurotransmitter as an output signal and a postsynaptic cell on the receiving end [3]. The "empty" area that exists between the presynaptic neuron and the postsynaptic cell is termed the synaptic left [6].

Figure The anatomy of a synapse (from P. B. Wilhelm et al. [3])

The neurotransmitter trigger and release mechanisms depend principally on the action potential and voltage - gated ion channels of the presynaptic neuron respectively. Upon the arrival of an action potential at the axonal terminals (also known as synaptic terminals [1]), the voltage - controlled calcium (Ca2+) channels are activated and the presynaptic neuron experiences an influx of calcium cations at the synaptic knob [5] causing a rise in their local concentration. This localized increase lasts for a fraction of a millisecond [1] and causes the synaptic vesicles to merge with the presynaptic membrane releasing their neurotransmitter chemicals into the synaptic cleft [6] (see figure 1.2.1). The amount of neurotransmitter released by the synaptic vesicles depends on the action potential that triggers the reaction and it may not always be the same or even adequate to achieve communication [1]. Once the neurotransmitter had been released into the synaptic cleft, it diffused towards the postsynaptic cell where it binds to its neuroreceptors [3]. Once the two bind, the chemically gated ion channels of the receptor open and an influx of positive ions from the extracellular space depolarizes and in time re-polarizes the postsynaptic membrane creating an action potential at the postsynaptic neuron [6]. At this point the transneuronal communication has been achieved and the postsynaptic neuron activates enzymes, specific to individual neurotransmitters, which remove them from the synaptic cleft [1]. The postsynaptic cells generally implement two types of receptors: ionotropic and metabotropic. The first act directly onto the ion channels altering the ion concentration balance while the later produce secondary messengers that affect the metabolic processes of the postsynaptic cell and the ion channels, indirectly [6].

The neurotransmitters found inside the human central nervous system can be divided into two types according to their function: excitatory and inhibitory [1]. Excitatory neurotransmitters are depolarizing (reduce membrane potential) which means that they promote the production of action potentials in the postsynaptic neuron [2]. On the other hand, inhibitory neurotransmitters are hyperpolarizing (increase membrane potential) preventing neurons from activation [3]. Chemically, the classification of neurotransmitters is a lot more diverse and there are several categories. The major chemical groups of neurotransmitters are: biogenic amines (epinephrine, dopamine, etch), dissolved gases, neuropeptides and amino acids [4], [1]. During our investigation we will focus mainly on the properties of amino but we will also examine certain metabolites, significant to brain biochemistry.

To begin with, glutamate (GLU) and γ - aminobutyric acid (GABA) fall under the amino acid category and are very common neurotransmitters within the CNS [4], [1]. Glutamate is the most significant [18] and most abundant [2], [17] excitatory neurotransmitter found within the CNS. It is derived from a-ketoglutarate during the Krebs cycle [2] and plays a crucial part in higher human functions such as learning and memory [1]. The effect of glutamate on synaptic cells can be both direct and indirect depending on the neuroreceptor [1]. Note that its concentration in the healthy brain ranges from 8 to 12 mM [8]. GABA on the other hand, is a major inhibitory neurotransmitter of the central nervous system, and it can also bind with both ionotropic and metabotropic receptors [1]. It is produced from glutamate [2] and glutamine via decarboxylation [18] and accounts for the neurotransmitter used in over 20% of the CNS's synapses [1]. The concentration of GABA inside the brain is small, relative to the rest of the amino acids, and varies from 0.5 to 1.5 mM [8]. Finally, the category of biogenic amines includes the catecholamines dopamine, norepinephrine, epinephrine plus serotonin and histamine [1]. All of the catecholamine neurotransmitters are synthesized from the amino acid tyrosine as intermediary products of a series of chemical reactions [2], [33]. Amine neurotransmitters are usually present in CNS neurons associated with motor functions, visceral functions, mood, arousal and attention [1], [33], [34].

1.1.3 Brain Metabolites

In the previous section we discussed the general principles of brain neurotransmitters and the key functions of the most important among them. Neurotransmitters definitely play an important role in neuronal communications and other physiological processes inside brain but they are not alone. For the purposes of this investigation we have included a brief discussion on several brain metabolites that present great significance in the study of the brain's pathophysiology. First of all, the term metabolite refers to the intermediary and final products of the chemical reactions that constitute the human metabolism or to ingredients essential to the metabolism which enter the body through food [1]. The metabolites N acetylaspartate (NAA), myoinositol (M-INS), creatine (CR), glutamine (GLN) and choline (CHO) are very significant to the human body in both health and disease and will be discussed in this section.

NAA is an amino acid derivative that originates from the neurotransmitter N - acetyl - aspartyl - glutamate (NAAG) that coexists with NAA inside neurons and is transported down the axons [10], [12], [20]. The fact that NAA can be found only in neurons and axons combined with documented changes in its concentration in neurodegenerative disorders have established it as the principal marker for neurons in both brain physiology and pathology [12-14], [17-19]. The normal concentration of NAA in the healthy brain ranges from 6-10 mM [8]. Myoinositol (M-INS) is a pentose sugar [10] produced primarily by the neuroglia, astrocytes [17] and it is often used as an astrocyte marker in pathophysiological studies of the human brain [18], [19]. Myoinositol is also associated with the breakdown of the myelin sheath surrounding axons and abnormalities in its concentration have been used to indentify demyelinating disorders [13], [17]. Creatine and phosphocreatine are also important because they contribute to the production of Adenosine Triphosphate (ATP), the "fuel" source of every human cell [12]. Creatine originates in the liver and is then transported to the brain and rest of the body [10]. Total creatine concentration inside the brain is almost constant at 8.6 mM and it is believed that it remains unaffected by the majority of diseases [12], [17], [37]. Therefore, creatine makes up for an efficient point of reference when determining changes in metabolite ratios. Glutamine is another significant metabolite in the brain's metabolism and chemically belongs to amino acids [17]. It is produced inside astrocytes which use glutamate as feedstock [2]. Its close connection to astrocytes and glutamate turnover metabolism, render it diagnostically useful in range of diseases. The concentration of glutamine in the normal brain is approximately 2-6 mM [8].

We will end this discussion with choline and choline containing compounds. The name choline is in fact a collective term that represents small amounts of free choline, phosphocholine and glycerophosphocoline [10], [12], [17]. These compounds participate a great deal to membrane synthesis and breakdown and thus make up for efficient markers of diseases with significant membrane turnover [10]. Choline cannot be produced by the human body [2], and it is derived from our diet [20]. It is the basic compound for acetylcholine (ACh) [20], a neurotransmitter of great importance for the motor neurons of the spinal cord and is commonly encountered in neuromuscular junctions. In addition, ACh is believed to play an important part in high level functions such as memory, cognition and emotions [2]. Note that, the normal concentration of choline in human white matter is approximately 1.6 mM [17] whereas in gray matter it is slightly reduced to 1.4 mM [19].

1.2 Quantification of Brain Neurotransmitters and Metabolites

Magnetic Resonance Spectroscopy began to find human applications in the fields of biology and medicine in the 1980s [11]. It had previously served the scientific community (especially organic chemists) for years in the study of molecules and complex chemical compounds. Ever since its introduction in the biomedical sciences it has been used to study several human structure and predominantly the brain. The application of magnetic resonance spectroscopy on the study of the brain received the term "neurospectroscopy" [12] and experienced the biggest growth. Thanks to the rapid technological evolution of MRS hardware, signal acquisition methods and signal processing, the molecular structures inside the brain and their biochemical processes are now better understood [23],. The milestones in the evolution of MRS into an established clinical modality were the introduction of superconducting magnets in 1965 and the implementation of the Fourier transform into NMR [10]. Superconducting magnets offered higher field strengths that allowed for better spectroscopic resolution and improved signal to noise ratio [12] while Fourier transform revolutionized the area of signal processing.

MRS is now common in clinical practice and has become an essential tool in diagnosis, treatment verification and monitoring of many types of pathology [12]. Emphasis must be given to the fact that MRS was the first and currently is the only non - invasive technique with the capacity to measure the concentration of human neurotransmitters and brain metabolites in - vivo. In addition magnetic resonance spectroscopy provides a high degree of accuracy and carries minimal risk. Compared to other diagnostic modalities used in clinical practice it has the ultimate advantage of being a non ionizing examination thus the risk involved is as we said minimal for both patients and staff. Before the evolution of MRS, the only available quantitative data originated from studies of postmortem brain homogenate or invasive animal studies [31-32]. In vitro techniques were used to study neurotransmitters via fluorescence, immunohistochemical assays and invasive cerebral probes [31]. In vivo techniques included the invasive methods of cortical cup, push-pull cannula, microdialysis, spectrophotometry and voltametry [31], [32]. These methods have been reviewed by Hutchison et al. [31] and Benveniste [32] and will not be discussed more extensively in this investigation.

It should be obvious by now that MRS is indeed an efficient quantitative tool capable of neurotransmitter and brain metabolite quantification. In vivo quantification of the neurotransmitters glutamate, aspartate, GABA and glycine as well as the metabolites NAA, creatine, choline, glutamine and myoinositol have been reported in a wide range of MRS studies [8-9], [12-17], [19-20]. As we have discussed in the previous chapters, neurotransmitter chemicals and metabolites are directly connected to physiological processes inside the brain and members of both categories can function as markers for neurons, glial cells or other brain structures. Therefore, abnormalities in the normal concentration of such markers are very often indicative of disease. To exemplify, drops in NAA concentration have been attributed to the destruction of myelin or entire neurons and are usually encountered in neurodegenerative disorders such as Alzheimer's dementia, Multiple Sclerosis etch [8,12,13,17]. Decreased levels of NAA have also been reported in MRS studies of brain tumours, localized brain abscesses, epilepsy and stroke patients [8], [12] whereas increases have been documented in Canavan's disease [25] and amyotrophic lateral sclerosis (ALS), (also known as Lou Gehrig's disease) [20]. In Alzheimer's dementia, MRS presents a significant advantage in reference to diagnosis, since it has the capacity to detect AD associated abnormalities in myoinositol and creatine even in the early stages of the disease [13]. The amino acids glutamate and glutamine are also diagnostically relevant. Increases in their concentration have been reported in cases of hepatic encephalopathy, Reye's syndrome and hypoxia [8], [19] whereas drops in their normal concentration are often encountered in Alzheimer's disease [13], [17]. Myoinositol on the other hand is not very disease specific by itself but in combination with abnormalities in other metabolites can differentiate between various ailments [12]. Abnormal increases in its concentration have been reported in cases of brain inflammation [17] and Alzheimer's dementia [13], whereas decreases characterize hypo-osmotic patients [8]. Creatine has also diagnostic value and elevations in its concentration have been found in chronic liver disease [10]. Finally for choline, MRS studies have shown abnormalities in malignant brain tumours, demyelination, brain ischemia and inflammation [10], [19], [20].

1.3 Magnetic Resonance Spectroscopy

1.3.1 Principles of Nuclear Magnetic Resonance

Nuclear magnetic resonance is the basis of both magnetic resonance imaging (MRI) and MRS and in this chapter we will attempt a brief description of the principles behind it. MRI is principally based on the detection of the NMR signal arising from the hydrogen nuclei inside water molecules whereas MRS is more concerned with signals originating from the metabolites inside the body and more specifically for our purposes, the brain.

The NMR signal originates from atomic nuclei with unpaired nuclear spins (nonzero values of magnetic moment) [26]. Some of the nuclei that satisfy that physical mandate are hydrogen (1H), phosphorus (31P), carbon (13 C) and fluorine (19F) [30]. These nuclei are sufficiently abundant in the human body to yield a detectable NMR signal [26]. All of these isotopes have been used various studies with hydrogen being the most commonly used.

It is the constant spinning of said nuclei combined with their charge that causes them to behave like microscopic magnets and have magnetic moments. When a population of such nuclei is exposed to a static external magnetic field (B0) their magnetic moments interact with the field creating a torque that forces them into precession around B0. The precession takes place at a certain frequency, proportional to the magnitude of the external magnetic field, termed the Larmor (or resonance) frequency.

, equation

, γ is the gyro magnetic ratio, unique to each nucleus [26].

Nuclei with spin ½, like hydrogen, can assume one of two orientations, either with or against the external magnetic field (parallel or anti parallel). The parallel orientation is naturally preferred and in any given population of nuclei the majority aligns parallel to B0. From a quantum-mechanical point of view, the two orientations correspond to two distinct energy levels which can be occupied by the nuclei: a lower energy level corresponding to alignment parallel to the magnetic field and a higher energy level corresponding to the anti parallel orientation. The ratio of nuclei in the lower energy level to those on the higher level is governed by Boltzmann statistics:

, equation [26]

, where ΔΕ is the energy difference between the levels, k is the Boltzmann constant and T is the temperature. The energy difference between the two levels depends of course on the external magnetic field:

, equation [11]

Transitions between energy levels are possible for the nuclei via absorption of photons. The energy of the photon must be exactly equal to the energy difference between levels otherwise the transition is impossible. That is to say, only photons with frequencies matching the resonance frequencies of the nuclei will successfully cause a transition between levels. From equations and we can see that the ratio between the number of spins in the higher and lower level depends on the energy difference between the two levels and the same applies for the net magnetization. Thus, since the energy difference between levels is very small, the net magnetization and consequently the magnetic resonance signal will also be small. The quantum mechanical description of NMR reveals the origins of the inherent insensitivity that characterizes MRS.

check references for everything green

Returning to the semi classical description, we have already explained how the difference between the opposing two states yields the net magnetization in the direction of the external magnetic field termed the longitudinal magnetization [26].

change 900 rf pulse to arbitrary angle and mention Ernst angle.

review and improve stuff on coherence and nmr signal generation.

Using a 90ÌŠ RF excitation pulse (alternating magnetic field, B1) we can force the longitudinal magnetization to get tipped away from its original axis and onto the transverse plane where it continues to rotate. The coherent rotation of the transverse magnetization will induce an alternating voltage to a receiver coil and give rise to the NMR signal. However, signal coherence does not last long and the signal slowly decays away. The returning of the system to its original state is termed relaxation and it describes the recovery of magnetization from the transverse back into longitudinal (equilibrium). Relaxation is affected by two types of processes: the first type of interaction is called spin - lattice or longitudinal relaxation, where nuclei shed their excess energy to their surrounding as they return to their ground state [29]. Longitudinal relaxation is described by the time constant T1 [28]. The second type of relaxation in the transverse and it is caused by spin-spin interactions that cause the MR signal to lose coherence and decay [29]. Transverse relaxation is described by the time constant T2 [11]. Relaxation can also be affected by inhomogeneities in the static magnetic field B0, which contribute to the loss of signal coherence. In such cases the transverse relaxation is no longer described by T2 and another time constant T2* is used instead:

, equation

The destruction of the MR signal due to static magnetic field inhomogeneities is commonly known as the free induction decay (FID) [29].

1.3.2 MRS data acquisition and the spectra

So far we have seen how the NMR signal is generated. But how does that signal carry useful information in magnetic resonance spectroscopy? Magnetic Resonance Imaging employs linear magnetic gradients, in addition to the B­­0 and B1 fields, to encode spatial information of the imaged anatomy. In contrast, MRS is used to encode chemical information from within the field of view rather than space. In Magnetic Resonance Spectroscopy an external magnetic field B0 is applied to a population of nuclei. B0 acts upon the atoms and molecules inducing small electrical currents [11], which in turn generate a small perturbation that opposes the external magnetic field [26], [28]. This microscopic magnetic field is proportional to B0 by a constant σ, termed the shielding constant [28]. Thus, the effective magnetic field is:

, equation [11]

This dimensionless constant is highly dependent upon the chemical environment of the nuclei and causes a discernable shift in their resonance frequency, called chemical shift [11], [26]. The resulting change in frequency is used to identify chemical composition, and is used to identify and measure neurotransmitters. Chemical shift values express the change in the resonance frequency of nuclei using an arbitrarily chosen frequency as a point of reference [11]. This changes are usually minuscule which is why they are expressed in parts per million (ppm) [10].

Add everything for J coupling and chemical shift

In the previous chapter we explained very simply the function of the 90ÌŠ RF pulse in NMR. However, the majority of NMR techniques use more than one RF pulse and more than one angle. There are hundreds of pulse sequences employing different pulses and timing diagrams to achieve the desired result. Depending on the contrast we wish to achieve we must select the appropriate sequence. For example functional MRI uses echo planar imaging

First explain what a pulse sequence is

Then include timing diagrams

then move on to specific sequences in mRS.

Explain TE, TR and etch. Including diagrams

Just mention steam and focus on PRESS. Say why you focus on it.

Move on to water suppression during acquisition and insert photos of spectra with and without CHESS.

The most common pulse sequences in magnetic resonance spectroscopy are the STEM and PRESS. STEAM stands for Stimulated Echo Acquisition Mode. PRESS = Point Resolved Spectroscopy

1.4 Spectral fitting

JMRUI mention

Explain what NMR scope and Quest do but not very much on how they do it.

Time domain fitting - why is it better?

Why do we say time domain fitting since we see the results and everything in the frequency domain (remember fourier transform has artifacts)

The actual fitting occurs in the time domain regardless of the fact that we see things in the frequency domain

Include things on water suppression during processing.