LECTURE 5

Neurotransmission Machinery and Inhibitory Systems

Inhibitory Neurotransmitters: GABA and Glycine

In the mature human central nervous system (CNS), GABA (gamma-aminobutyric acid) and glycine serve as the primary inhibitory neurotransmitters. Their function is characterized by reducing the probability of action potential (AP) firing in neurons.

GABA (Gamma-Aminobutyric Acid)

• Occurrence: GABA is the most common inhibitory neurotransmitter in the brain. It is most frequently found in the interneurons of local circuits.

• Receptor Types:

  1. $GABA_A$: An ionotropic receptor. It is a heteropentameric ligand-gated channel permeable to chloride ($Cl^-$).

  2. $GABA_B$: A metabotropic (G-protein coupled) receptor.

  3. $GABA_C$: An ionotropic receptor, also a heteropentameric ligand-gated $Cl^-$ channel.

• Binding Sites: Ionotropic GABA receptors typically possess two binding sites for the neurotransmitter.

Glycine

• Occurrence: Glycine is a common inhibitory neurotransmitter specifically in the spinal cord and the brainstem. In the spinal cord, approximately $\frac{1}{2}$ of all inhibitory synapses utilize glycine.

• Receptor Types ($GlyRs$):

  1. Ionotropic: Heteropentameric ligand-gated $Cl^-$ channels. These receptors typically have between $2$ to $5$ binding sites for glycine.

  2. Metabotropic ($mGlyR$): Specifically identified as $GPR158$.

Synthesis and Degradation Pathways

GABAergic Synapse Lifecycle

1. Precursor: Glucose is converted into Glutamate ($Glutamic \text{ } acid$).

2. Synthesis: The enzyme Glutamic acid decarboxylase ($GAD$), requiring the cofactor Pyridoxal phosphate, catalyzes the conversion of Glutamate into GABA.

3. Vesicular Packaging: GABA is loaded into vesicles by the Vesicular Inhibitory Amino Acid Transporter ($VIAAT$).

4. Reuptake and Metabolism: GABA is removed from the cleft via reuptake into the presynaptic terminal or glial cells (astrocytes) through GABA transporters ($GAT$). Inside astrocytes, it can be broken down by GABA transaminase and Succinic semialdehyde dehydrogenase.

Glycinergic Synapse Lifecycle

1. Synthesis: Glucose leads to the production of Serine. The enzyme Serine-hydroxymethyltransferase converts Serine into Glycine.

2. Vesicular Packaging: Glycine is also loaded into vesicles via $VIAAT$.

3. Removal: Glycine is removed from the synaptic cleft by Glycine transporters. It can be further processed by the Glycine cleavage system.

Ionic Mechanisms and Maturation of GABAergic Effects

In ionotropic GABA and glycine receptors, $Cl^-$ is the primary permeant ion. The direction of flow depends on the electrochemical gradient.

Mature Neurons

• Concentrations: External chloride concentrations ($[Cl^-]_{out}$) are approximately $110 \text{ } mM$, while internal concentrations ($[Cl^-]_{in}$) range from $4 - 30 \text{ } mM$.

• Effect: Upon ligand binding, $Cl^-$ flows into the cell ($Cl^- \text{ } influx$), causing hyperpolarization of the membrane (Inhibitory Postsynaptic Potential or IPSP).

Immature Neurons

• Concentrations: Internal chloride concentrations ($[Cl^-]_{in}$) are high during early development.

• Effect: Because $[Cl^-]_{in}$ is high, binding of GABA causes $Cl^-$ to move out of the cell ($Cl^- \text{ } efflux$). This results in a depolarizing effect (Excitatory Postsynaptic Potential or EPSP), which can trigger action potentials in the developing chick spinal cord and other embryonic systems.

Classification and Dynamics of Neurotransmitters

Classification by Size

1. Small-molecule neurotransmitters: Include molecules such as Acetylcholine, Glutamate, GABA, Glycine, and biogenic amines like Dopamine, Noradrenaline (Norepinephrine), and Adrenaline (Epinephrine).

2. Large-molecule neurotransmitters (Neuropeptides): These consist of chains of $3$ to $36 \text{ } amino \text{ } acids$. Named examples include opioid peptides (e.g., endorphins), Neuropeptide-Y, CGRP, PACAP, Substance P, and Galanin.

Biogenic Amines and Function

Catecholamines (Dopamine, Noradrenaline, Adrenaline)

• Biosynthesis: All are derived from the common precursor Tyrosine.

• Receptors: Primarily bind to metabotropic receptors.

• Functional Roles: Motor behavior, motivation, reward, stress, sleep/wakefulness, attention, and feeding behavior.

Serotonin and Histamine

• Serotonin: Binds to both ionotropic ($5HT_3$) and metabotropic receptors. Regulates sleep, wakefulness, depression, anxiety, arousal, and food consumption.

• Histamine: Binds to metabotropic receptors. Involved in arousal, attention, and balance.

Chemical Synapse Sequence of Events

At a typical chemical synapse:

1. Action potential reaches the presynaptic terminal.

2. Depolarization opens voltage-gated calcium channels ($N, P/Q, R, \text{ and } T \text{ } types$).

3. Rise in presynaptic $Ca^{2+}$ triggers exocytosis.

4. Vesicles (including large dense-core vesicles for neuropeptides) release their contents into the cleft.

5. Neurotransmitters bind to postsynaptic receptors.

6. EPSPs (Excitatory) or IPSPs (Inhibitory) are generated.

7. Membrane is recovered via endocytosis.

Neural Development: Morphogenesis and Neurulation

Fundamental Concepts of Morphogenesis

Morphogenesis is the formation of structures and regions involving cell proliferation, bending, folding, constricting, segmentation, and patterning. The fate of cells is initially determined by the asymmetric distribution of transcription factors during early egg division.

• Induction: The molecular mechanism where a cell or tissue influences the fate of nearby cells/tissues via chemical signals (inductive molecules). This depends on the presence of specific factors, the concentration of those factors, and the availability of corresponding receptors.

• Neural Plate: The first event in nervous system development; an area within the ectoderm containing neural progenitor cells.

• Neurulation: The process of forming the neural tube from the neural plate.

Stages of Neurulation

• Carnegie Stages (Human Embryo):

  • $18 - 21 \text{ } days$: Appearance of the Notochord. The notochord is of mesodermal origin and defines body symmetry. It releases inductive signals like Chordin and Noggin to inhibit Bone Morphogenic Protein (BMP), causing ectodermal cells to differentiate into neuroectodermal precursor cells.

  • $23 \text{ } days$: Appearance of the neural groove.

  • $24 - 26 \text{ } days$: Prominent neural folds form. Rostro-caudal patterning begins. The growth of rostral (towards the head) folds dominates, and the five subdivisions of the brain emerge.

  • $28 - 30 \text{ } days$: Neural tube closure; dorso-ventral patterning becomes distinct.

Patterning and Segmentation

Dorso-ventral Patterning

• Floorplate: Secretes Sonic hedgehog ($Shh$), inducing "ventralization," leading to the differentiation of motor neurons and interneurons in the spinal cord.

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

Rostro-caudal Patterning

• Signaling centers utilize inductive factors like $Shh$ and $FGF8$.

• Segmentation: Regulated by Homeobox ($Hox$) genes. In Drosophila, these code for DNA binding proteins/transcription factors that regulate morphogenesis. In humans, their homologs regulate regionalization, though they require inductive molecules ($Shh, RA, FGF8$) for regulation.

Cell Lineage and Differentiation

Progenitors and Stem Cells

• Source: The neuroectoderm gives rise to neurons, oligodendrocytes, and astrocytes.

• Mitosis: Neural precursor cells (neural stem cells) undergo mitosis in the ventricular-subventricular zone ($V/SVZ$), the innermost layer surrounding the lumen of the neural tube.

• Radial Glia: Serve as multipotent progenitors and provide pathways for migration.

• Progenitor Division: Can be asymmetric to create a differentiated cell and another progenitor.

The Exception: Microglia

Microglia do not originate from the neuroectoderm. They derive from myeloid precursors in the yolk sac and enter the brain during early development (Human Gestational Week $5$).

Lateral Inhibition and Differentiation

• Mechanism: Cell-cell interaction regulates gradual differentiation.

• Signaling: The Delta ligand on one cell binds to Notch receptors on a neighbor. The cleavage product of Notch interacts with transcription factors to inhibit that neighboring cell from differentiating into the same type.

• Temporal Sequence: In the rodent cortex and vertebrate CNS, neurons are generally generated first, followed by glial cells.

Construction of Neural Circuits

Neuronal Polarization

This is the first step in circuit formation. It involves the differential distribution of cytoskeleton proteins.

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

• Proteins: Polarity scaffolding proteins like $Par-3$ regulate the elongation of neurites and axon formation. Microtubule-stabilizing proteins like $MAP-2$ are targeted to dendrites, while $tau$ is targeted to axons.

Growth Cone Motility

At the tip of an extending neurite is the growth cone, a motile structure containing:

• Lamellipodium: Guided by actin.

• Filopodium: Guided by actin.

• Cytoskeleton regulation: Actin regulates shape for directed growth; tubulin (both dynamic tyrosinated and stable acetylated microtubules) regulates neurite elongation.

• Ionic regulation: Cation-permeable $TRP$ channels (permeable to $Ca^{2+}, Na^+, Mg^{2+}$) respond to environmental signals to regulate polymerization and de-polymerization of actin and tubulin.

Axonal Guidance Molecules

• Fasciculation: Axons growing along each other's surfaces via cell adhesion molecules.

• Tropic Factors (Diffusible):

  • Chemoattractants: Netrin. Netrin receptors are expressed when an axon needs to be drawn toward the ventral midline.

  • Chemorepellents: Slit and Semaphorins. Once an axon crosses the midline, netrin receptors are down-regulated, and Slit receptors are expressed, causing the axon to grow away from the high concentration of Slit.

• Non-diffusible factors: Extracellular matrix and cell adhesion molecules.

Synaptogenesis

1. Recognition of postsynaptic position and clustering of receptors.

2. Trans-synaptic protein complexes link the pre- and post-synaptic membranes.

3. Initial activation of voltage-gated $Ca^{2+}$ channels and the spontaneous release of the first vesicles (ensuring the contact is active).

4. Silent Synapses: Glutamatergic synapses are initially "silent" and become functional only after the insertion of $AMPA$ receptors.

Neurotrophins and Trophic Signaling

Discovery and Function

• Historical Context: Rita Levi-Montalcini and Stanley Cohen won the $1986 \text{ } Nobel \text{ } Prize$ in physiology or medicine for discovering Nerve Growth Factor ($NGF$).

• Role: Neurotrophins are signaling molecules (typically target-derived) essential for the survival, maintenance, and growth of neurons. Neurons compete for these factors; cells that do not receive sufficient trophic support undergo apoptosis.

The Neurotrophin Family

• Members: $NGF, BDNF$ (Brain-derived neurotrophic factor), $NT3$ (Neurotrophin-3), and $NT4/5$.

• Specificity: Different neurons require different factors. For example:

  • NGF: Sympathetic ganglion.

  • BDNF: Cortical neurons.

  • NT3: Parasympathetic ganglion.

  • Multiple factors: Dorsal root ganglion and basal forebrain cholinergic neurons.

Receptors and Signaling Pathways

1. $p75$ Receptor: A low-affinity receptor for mature neurotrophins but high-affinity for immature precursors (pro-neurotrophins: $pro-NGF, pro-BDNF$, etc.). It belongs to the $TNFR$ (Tumor Necrosis Factor receptor) family.

2. Trk Receptors (Tyrosine Kinase): High-affinity receptors for mature factors.

   • $TrkA$: Binds $NGF$.

   • $TrkB$: Binds $BDNF, NT-4/5, \text{ and } NT-3$.

   • $TrkC$: Binds $NT-3$.

Signaling Endosomes

When $NGF$ binds to $TrkA$ at axon terminals, it forms signaling endosomes. These are transported via retrograde transport to the cell body. There, $TrkA$ activates the $MEK/ERK$ signaling pathway to induce transcription factors for survival and extension.

Synapse Elimination

In early postnatal life, trophic interactions modulate innervation patterns. Initially, targets have polyneuronal innervation. Through competition for neurotrophic support, the number of axonal inputs is reduced to the mature pattern where each target is innervated by the correct number of inputs.

Questions & Discussion

Lecture 4 Review Questions

• Question: What will be one of the immediate consequences of the selective block of the fast axonal transport (in the absence of any stimulation) on neurotransmission machinery?

  • Answer: Block of the transport of neuropeptide precursors (e.g., endorphins) to the presynapse. (Note: Acetylcholine synthesis occurs locally in the terminal, thus is not immediately blocked).

• Question: What role does $Ca^{2+}$ influx play in the process of exocytosis?

  • Answer: $Ca^{2+}$ influx is essential for the synaptotagmin-mediated fusion of the vesicular and plasma membranes during the vesicular release of neurotransmitters to a synaptic cleft.

• Question: Which statement is true regarding neurotransmitters?

  • Answer: A neurotransmitter (interacting with ionotropic receptors) is considered excitatory if the reversal potential for the current through the cation-selective (permeable for $Na^+$ and $K^+$) receptor-channel is significantly above the threshold for generation of action potential. (Note: Glutamate removal involves reuptake, not largely enzymatic degradation; GABA in mature neurons is inhibitory due to $Cl^-$ influx, not efflux).

Lecture 5 Review Questions

• Question: Which region (structure) of the developing embryo sends inductive signals to the ectodermal cells resulting in neural plate formation?

  • Answer: Notochord.

• Question: What are two main mechanisms that regulate early development of the nervous system?

  • Answer: Neural induction via signals (molecules) released by neighboring cells and early Homeobox genes-dependent regionalization.

• Question: What would be sufficient to guide the growing axons across the midline of the spinal cord?

  • Answer: The interaction of diffusible tropic molecules (like netrin) with their corresponding receptors that, upon reaching the midline by axons, is replaced by interaction of chemorepellent molecules (like slit) with their corresponding receptors.