Neurochemical System Overview

Neurochemical System

• Neurotransmitters are more diverse and their release is more complex than originally thought
• There are approximately 50 known neurotransmitters, potentially up to 100
• Neurotransmitters are often released from presynaptic sites, but can also be released from other sites
• Chemical signals are the primary mode of communication between neurons
• Neurotransmitter synthesis, release, and degradation are tightly controlled by various molecular mechanisms.

Criteria to define Neurotransmitters.

• Classical Neurotransmitters (NTs) are synthesized within the neuron from which they are released
• They are identifiable substances that can be characterized chemically or pharmacologically
• Exogenous application of the NT should elicit changes in the postsynaptic neuron(mimic the effects of stimulating the presynaptic neuron)
• NTs should act on specific receptors, whose action is blocked by antagonists or by genetic ablation
• There should be an active mechanism for terminating NT action

Major Classes of Neurotransmitters.

• Monoamines: Catecholamines, serotonin, melatonin
• Amino acids: Glutamate, GABA, glycine
• Peptide transmitters: Endorphins, somatostatin, oxytocin
• Acetylcholine: An organic compound released at the neuromuscular junction
• Gasotransmitters: Nitric oxide (NO), carbon monoxide (CO)
• Endocannabinoids: Lipid transmitters involved in retrograde neurotransmission

Regulation of the Monoamines (Catecholamines)

• Monoamine neurotransmitters contain an amino group connected to an aromatic ring via a two-carbon chain
• Monoaminergic systems are involved in regulating cognitive processes such as emotion, arousal, and memory
• Classical monoamines:
  • Imidazoleamines
  • Indolamines
  • Histamine
  • Serotonin (5-HT)
  • Catecholamines:
    • Dopamine (DA)
    • Epinephrine (Epi = adrenaline)
    • Norepinephrine (NE = noradrenaline)

Dopaminergic Projection System in the Brain.

• Major nuclei:
  • Substantia nigra → striatum
  • Ventral tegmental area → frontal and cingulate cortex, accumbens
  • Arcuate nucleus → pituitary

Norepinephrine and Epinephrine Projection Systems.

• Norepinephrine cells are located in the medulla and pons
• Epinephrine cells are located in two nuclei in the medulla

Serotonin Projections in the Brain.

• Neuroanatomical projection tracts exist from caudal raphe nuclei, rostral raphe nuclei, and the median raphe nucleus to various brain regions

Catecholamines (Dopamine, Norepinephrine, Epinephrine)

• Catecholamines are organic compounds derived from tyrosine
• Expression of specific biosynthetic enzymes defines neuronal identity (i.e., neurons only express enzymes needed for the neurotransmitter they release)

In Situ Hybridization (ISH)

• ISH is used to detect and localize specific DNA or RNA fragments within chromosome preparations, fixed cells, or tissue sections
• The technique involves hybridizing probes to target RNA in a series.
• Probes attach to complementary sequences within the target DNA or mRNA.

Immunohistochemistry/Immunofluorescence/Immunostaining

• Techniques to examine tissues with antibodies
• Methods for visualizing proteins and other molecules in tissue samples

Immunohistochemistry Combined with Fluorescent Reporter

• Techniques used to visualize specific proteins and molecules in brain tissue
• Methods include using antibodies that stain for the proteins being investigated

Distribution of TH and GAD1 mRNA in the Mouse Brain

• In situ hybridization using probes for TH and GAD1 mRNA shows distinct distributions indicating different cell types in the brain

"Birth and Death" of Classical Neurotransmitters (NTs)

• NTs, from synthesis to degradation, have tightly regulated steps via molecular mechanisms and signaling pathways

Regulation of Catecholamines

• 1. Biosynthesis: The amount and activity of tyrosine hydroxylase (TH) determine the rate of dopamine synthesis; TH expression and activity increase in response to demand for catecholamines
• 2. Storage: Catecholamines are stored in vesicles; transported into vesicles by the vesicular monoamine transporter (VMAT2).
• 3. Release and activation of postsynaptic receptors: Action potentials trigger calcium influx, which triggers synaptic vesicle fusion and neurotransmitter release, activating postsynaptic receptors. Molecular changes in postsynaptic receptors affect their activation and response
• 4. Inactivation: Released NTs are rapidly removed through re-uptake by transporters/enzymes (e.g., monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT))
• 5. Autoreceptors and feedback loops: Autoreceptors on presynaptic neurons provide feedback mechanisms to regulate NT synthesis, release, and firing.

Regulation of Serotonin

• The synthesis and regulation of serotonin are similar to catecholamines
• Serotonin is stored in vesicles with VMAT2.
• Autoreceptors, including 5-HT1A and 5-HT1B, play critical roles in regulating serotonin synthesis and release and signal pathways are involved in the pathophysiology of depression

Serotonin and Antidepressant Drugs

• Reuptake of serotonin (5-HT) by serotonin transporter (SERT) is a major mechanism for terminating 5-HT action
• Selective serotonin reuptake inhibitors (SSRIs) are a common type of antidepressant that target SERT
• Similar to catecholamines, serotonin can be inactivated by MAO; therefore, MAO inhibitors may indirectly have effects on serotonin regulation

How Ecstasy (MDMA) Works

• Ecstasy disrupts the transport of serotonin into vesicles within presynaptic neurons
• Serotonin is released into the synapse and its reuptake is blocked, causing elevated levels of serotonin in the synaptic cleft.

Amino Acid Transporters: GABA and Glutamate

• GABA and glutamate are the most prevalent amino acid neurotransmitters in the brain
• Their biosynthetic pathways are interlinked
• These amino acids are inactivated by reuptake

GABA and Glutamate Reuptake

• GABA is inactivated by reuptake via GABA transporters (GAT1, GAT3) and the vesicular GABA transporter (VGAT).
• Glutamate is inactivated by glutamate transporters (EAAT1–EAAT5)

Glutamate Transporters

• EAATs/VGLUTs are critical for glutamate regulation

Excitatory-Inhibitory Balance

• The balance of excitatory (glutamate) and inhibitory (GABA) neurotransmission is vital for normal brain function

Antibody labeling for Inhibitory Synapses

• VGAT is the appropriate antibody to label inhibitory synapses

Fusion Proteins for Synapse Quantification

• Fusion proteins (e.g., GFP-tagged proteins) are useful tools for precise quantification of synapses in animal models, but not in humans