Comprehensive Study Guide: Neurotransmission, Brain Development, and Neurotrophic Signaling

Review of Synaptic Machinery and Neurotransmission (Lecture 4)

In considering the immediate consequences of a selective block of fast axonal transport in the absence of stimulation, the primary effect on the neurotransmission machinery is the block of the transport of neuropeptide precursors, such as endorphins, to the presynapse. Other processes, such as acetylcholine synthesis, occur locally in the presynaptic terminal and would not be immediately inhibited. Large dense-core vesicles existing prior to the transport block would remain, though their replenishment would eventually cease.

Calcium influx (${Ca^{2+}}$) plays a critical role in the process of exocytosis. It is essential for the synaptotagmin-mediated fusion of the vesicular and plasma membranes. This fusion allows for the quantal release of neurotransmitters into the synaptic cleft. It is important to distinguish this from the depolarization of the presynaptic terminal, which is the cause of the ${Ca^{2+}}$ influx (via voltage-gated channels) rather than the result of it.

Criteria for excitatory neurotransmission via ionotropic receptors: A neurotransmitter is considered excitatory if the reversal potential (${E_{rev}}$) for the current passing through the receptor-channel (typically cation-selective and permeable for ${Na^+}$ and ${K^+}$) is significantly above the threshold for the generation of an action potential (${AP}$). In mature neurons with a resting membrane potential of approximately $\text{-70} \text{\,mV}$, the activation of ${GABA_A}$ receptors leads to an influx of ${Cl^-}$, resulting in inhibitory postsynaptic potentials (${IPSPs}$).

Inhibitory Neurotransmission: GABA and Glycine Characteristics

GABA (${\gamma}$-aminobutyric acid) is the most common inhibitory neurotransmitter in the brain. It is primarily found in the interneurons of local circuits and functions to reduce the probability of action potential firing in mature neurons.

Glycine is a common inhibitory neurotransmitter found specifically in the spinal cord and the brainstem. In the spinal cord, approximately $1/2$ of all inhibitory synapses utilize glycine. Like GABA, glycine evokes inhibitory postsynaptic potentials (${IPSPs}$) in the mature nervous system.

Inhibitory Receptor Types

1. GABA Receptors:

   • GABA_A: Ionotropic, heteropentameric ligand-gated ${Cl^-}$ channels.

   • GABA_B: Metabotropic receptors.

   • GABA_C: Ionotropic, heteropentameric ligand-gated ${Cl^-}$ channels.

2. Glycine Receptors (GlyRs):

   • Ionotropic: Heteropentameric ligand-gated ${Cl^-}$ channels.

   • Metabotropic: ${mGlyR}$ (specifically $GPR158$).

Biosynthesis and Clearance Mechanisms of Inhibitory Transmitters

GABA Biosynthesis and Cycle

• Precursor: Glutamate, which is derived from Glucose.

• Enzyme: Glutamic acid decarboxylase (${GAD}$) requires the cofactor pyridoxal phosphate.

• Pathway: Glucose $\rightarrow$ Glutamate $\rightarrow$ GABA ($via \text{ } GAD$).

• Transport: Loaded into vesicles by $VIAAT$ (Vesicular Inhibitory Amino Acid Transporter).

• Removal: Reuptake into the presynaptic terminal or glial cells (astrocytes) via ${GAT}$ (GABA transporter).

• Degradation: Mitochondrial enzymes GABA transaminase and Succinic semialdehyde dehydrogenase.

Glycine Biosynthesis

• Precursor: Serine, derived from Glucose.

• Enzyme: Serine-hydroxymethyltransferase.

• Removal: Glycine transporter removes excess neurotransmitter from the cleft. It can also be processed by the Glycine cleavage system.

Developmental Shifts in Ionic Gradients and GABA/Glycine Polarity

The effect of GABA and glycine depends entirely on the intracellular and extracellular concentration of chloride (${Cl^-}$).

1. Mature Neurons:

   • ${[Cl^-]_{out} = 110 \text{\,mM}}$

   • ${[Cl^-]_{in} = 4 - 30 \text{\,mM}}$

   • Result: ${Cl^-}$ influx occurs when channels open, leading to hyperpolarization and $IPSPs$.

2. Immature/Developing Neurons:

   • ${[Cl^-]_{in}}$ is significantly higher due to different expression levels of chloride transporters.

   • ${[Cl^-]_{out}}$ remains relatively low or standard.

   • Result: ${Cl^-}$ efflux occurs when channels open, leading to a depolarizing effect and Excitatory Postsynaptic Potentials (${EPSPs}$).

Broad Classification of Neurotransmitters: Size and Function

Neurotransmitters are categorized by size and the potential they evoke:

• Small-molecule Neurotransmitters: Includes amino acids (Glutamate, GABA), biogenic amines, and Acetylcholine.

• Large-molecule Neurotransmitters (Neuropeptides): Contain between $3 - 36$ amino acids. Examples include opioid peptides (endorphins), Neuropeptide-Y, ${CGRP}$, ${PACAP}$, Substance P, and Galanin. These are often stored in large dense-core vesicles.

Biogenic Amines: Catecholamines, Serotonin, and Histamine

Catecholamines

All catecholamines are derived from the common precursor Tyrosine. They typically bind to metabotropic receptors and regulate complex behaviors:

• Dopamine: Involved in motor behavior, motivation, and reward.

• Noradrenaline / Adrenaline: Involved in stress, sleep/wakefulness, and attention.

Other Biogenic Amines

• Serotonin (5-HT): Regulates sleep, wakefulness, depression, anxiety, and food consumption. It acts on both ionotropic ($5HT_3$) and various metabotropic receptors.

• Histamine: Involved in arousal, attention, and balance. Acts via metabotropic receptors.

Overview of Nervous System Morphogenesis and Neurulation (Lecture 5)

Morphogenesis refers to the formation of structures and regions through cell proliferation, accompanied by bending, folding, constricting, segmentation, and patterning.

Early Events

• Cell Fate: Determined earlier as the fertilized egg divides through the asymmetric distribution of transcription factors.

• Neural Plate: The area within the ectoderm containing neural progenitor cells. It is the earliest event in nervous system development.

• Induction: The molecular mechanism where a cell or tissue influences the fate of nearby cells via chemical signals (inductive molecules). This depends on the presence of factors, receptors, and their respective concentrations.

Neurulation Stages

• Notochord: Of mesodermal origin, appearing at $18 - 21$ days (Carnegie stages). It defines body symmetry and the position of the nervous system. Significant for sending inductive signals (chordin, noggin) to the ectoderm to inhibit Bone Morphogenic Protein (${BMP}$), causing differentiation into neuroectodermal precursor cells.

• Neural Groove: Appears around $23$ days.

• Neural Folds: Prominent by $24 - 26$ days. The rostral growth dominates (rostro-caudal patterning), leading to the five major subdivisions of the brain.

• Neural Tube Closure: Occurs by $28 - 30$ days, marking the obvious progression of dorso-ventral patterning.

Molecular Patterning and Segmentation

1. Rostro-Caudal Patterning: Guided by signaling centers expressing inductive factors like Sonic hedgehog (${Shh}$) and ${FGF8}$.

2. Dorso-Ventral Patterning:

   • Floorplate: Sends ${Shh}$ signals to the ventral portion of the neural tube, inducing differentiation into motor neurons and interneurons (ventralization).

   • Roofplate: Provides signals such as ${TGF\beta}$ family proteins for the development of the dorsal portion of the nervous system.

3. Homeobox Genes (Hox genes):

   • In Drosophila, they code for DNA-binding transcription factors that regulate morphogenesis.

   • In humans, homologs regulate regionalization and segmentation. Their expression is regulated by inductive molecules (${Shh}$, ${RA}$, ${FGF8}$).

Neurogenesis, Gliogenesis, and Cell Fate Regulation

Origins

• Neurons, Oligodendrocytes, and Astrocytes: Originate from the neuroectoderm (neural precursor cells).

• Microglia: The exception. They originate from myeloid precursors in the yolk sac and enter the brain at early developmental stages (Human Gestational Week $5$).

Proliferation and Differentiation

• Ventricular-Subventricular Zone (V/SVZ): The innermost layer surrounding the lumen of the neural tube where neural precursor cells (stem cells) undergo mitosis.

• Radial Glia: Multipotent progenitors that facilitate both mitosis and migration.

• Lateral Inhibition: A process mediated by ${Delta-Notch}$ signaling. When a cell up-regulates the ${Delta}$ ligand, it binds to ${Notch}$ receptors on neighbors, inhibiting them from differentiating into the same cell type.

• Temporal Sequence: In the vertebrate CNS, neurons are generally generated first, followed by glial cells (astrogliogenesis and oligodendrogenesis).

Construction of Neural Circuits: Polarization and Axonal Guidance

Neuronal Polarization

• Neurite: An undifferentiated extension that will become an axon or dendrite.

• Polarity Scaffolding: Proteins like ${Par-3}$ regulate neurite elongation and axon formation.

• Marker Proteins: ${MAP-2}$ is targeted to dendrites; ${tau}$ is targeted to axons.

The Growth Cone

• A specialized motile structure at the tip of the extending neurite.

• Lamellipodium: Sheet-like expansion at the tip.

• Filopodium: Fine, finger-like extensions.

• Cytoskeleton: Actin regulates shape and motility; Tubulin (stable acetylated vs. dynamic tyrosinated) regulates elongation.

• Motility: Controlled by polymerization/depolymerization in response to environmental signals that affect ${Ca^{2+}}$ levels via ${TRP}$ channels.

Axon Guidance Molecules

• Chemoattractants (Tropic factors): Such as Netrin.

• Chemorepellents: Such as Slit and Semaphorins.

• Non-diffusible signals: Cell adhesion molecules and extracellular matrix.

• Fasciculation: Axons growing along each other's surfaces.

Example: Midline Crossing

1. At $t=1$, growth cones express Netrin receptors and are attracted to the ventral midline.

2. At $t=2$, Netrin receptors are down-regulated, and Slit receptors are up-regulated, causing the axon to grow away from the high concentration of Slit at the midline.

Neurotrophic Factors and Target-Dependent Survival

Discovery by Rita Levi Montalcini and Stanley Cohen (1986 Nobel Prize) of Nerve Growth Factor (${NGF}$). Once synapses are established, neurons depend on targets for survival via neurotrophins.

The Neurotrophin Family

• NGF (Nerve Growth Factor): Affects sympathetic and sensory neurons (DRG).

• BDNF (Brain-Derived Neurotrophic Factor): Affects various CNS neurons.

• NT-3 (Neurotrophin-3)

• NT-4/5

Neurotrophin Receptor Specificity and Retrograde Signaling

1. Trk Receptors (High Affinity for Mature Forms):

   • TrkA: Specific for ${NGF}$.

   • TrkB: Specific for ${BDNF}$, ${NT-4/5}$, and ${NT-3}$.

   • TrkC: Specific for ${NT-3}$.

2. p75 Receptor (Low Affinity for Mature, High Affinity for Precursors):

   • Binds to "pro-" forms (e.g., pro-NGF, pro-BDNF) and can induce complex signaling involving survival or death.

Signaling Mechanism: When ${NGF}$ binds to ${TrkA}$ at the axon terminal, it forms signaling endosomes. These are retrogradely transported to the cell body, where they activate pathways like ${MEK/ERK}$ to induce transcription factors for survival and extension.

Synaptic Pruning and Competitive Innervation Patterns

In early postnatal life, the number of synapses and innervation patterns are adjusted via trophic interactions.

• Polyneuronal Innervation: Immature state where multiple axons contact the same target.

• Synapse Elimination: Reduction of axonal inputs to ensure each target is innervated by the correct number of synapses and each axon contacts the right number of targets.

• Silent Synapses: The first glutamatergic synapses are often "silent" and become functional only after the insertion of ${AMPA}$ receptors.

Lecture Review Questions and Formative Assessment

1. Which structure sends inductive signals for neural plate formation?    The Notochord. It releases signals like chordin and noggin.

2. Two main mechanisms regulating early development?    Neural induction via signals from neighboring cells and early ${Homeobox}$ gene-dependent regionalization.

3. Requirements for guiding axons across the spinal cord midline?    Individual growth cones must express Netrin receptors to reach the midline, and then replace this interaction with chemorepellent signals (Slit) upon reaching the midline to ensure they cross and move away.