Acetylcholine and Glutamate PDF

**Acetylcholine and Glutamate.** **Myasthenia Gravis.** **Myasthenia Gravis** is a chronic **autoimmune condition** that disrupts **communication** between **nerves** and **muscles** at the neuromuscular junction (NMJ), leading to muscle weakness, especially in the eyes, eyelids, and face. **Sympt...

**Acetylcholine and Glutamate.** **Myasthenia Gravis.** **Myasthenia Gravis** is a chronic **autoimmune condition** that disrupts **communication** between **nerves** and **muscles** at the neuromuscular junction (NMJ), leading to muscle weakness, especially in the eyes, eyelids, and face. **Symptoms** include droopy eyelids (ptosis), double vision, and difficulty with facial expressions. The condition arises from **antibodies** that **attack acetylcholine (ACh) receptors**, preventing effective signalling for **muscle contraction**. Acetylcholine is synthesized in cholinergic neurons using **dietary choline** (primarily from fats) and **acetyl coenzyme A,** with the help of the enzyme **choline acetyltransferase**. ACh is essential for **muscle contractions** and functions as a neuromodulator in the brain, **regulating memory, attention, and sleep**. In Myasthenia Gravis, the loss of ACh receptor function at the NMJ impairs muscle activation, highlighting the importance of ACh in both peripheral and central nervous system processes. **Anatomy of cholinergic pathways: (the circuits and regions where acetylcholine (ACh) acts as the primary neurotransmitter)** **Rat brain.** **Major Brainstem Nuclei:** Key nuclei in the brainstem play a critical role in neural communication by: ACh is **synthesized** in the **presynaptic terminal**, where the enzyme choline acetyltransferase combines choline and acetyl coenzyme A. It is then packed into **vesicles** and released into the synaptic cleft upon **action potential**, binding to ionotropic receptors that allow **ion flow**. Once released, ACh is **broken down** by acetylcholinesterase into choline and acetic acid, with choline transported back into the presynaptic terminal through a transporter system for **reuse**. **Nicotinic and Muscarinic Acetylcholine Receptors (AChRs):** **Cholinergic receptors** are named after their selective agonists, which mimic the effects of acetylcholine (**ACh**). **Nicotine** is the agonist for nicotinic receptors, while **muscarine** activates muscarinic receptors. These receptors play distinct roles in the body. **Curare** acts as an antagonist for nicotinic receptors, and **atropine** inhibits muscarinic receptors, blocking their effects. **Nicotinic receptors** are ion channels composed of **five subunits** arranged in a ring. Subunits include **α, β, γ, δ, and ε**, with variations depending on the tissue. **Muscle nicotinic receptors** have a distinct composition compared to **neuronal nicotinic receptors**, which consist of combinations of **10 α-subunits** and **4 β-subunits**. Nicotinic receptors are permeable to **Na⁺** and **K⁺**, and some subtypes also allow **Ca²⁺** influx. Subunit-specific nicotinic receptors include the **α7-containing receptor**, which is highly permeable to **Ca²⁺** and requires all five ACh binding sites for activation. The **α4β2 receptor** primarily conducts **Na⁺** and has two binding sites for ACh. **Muscle nicotinic receptors**, which contain either a **δ** or **ε** subunit, are permeable to **Na⁺** and have two ACh binding sites. Muscarinic receptors differ as they are **G-protein-coupled receptors**, mediating slower responses such as smooth muscle contraction and glandular secretion. Together, nicotinic and muscarinic receptors adapt their structure and function to regulate critical physiological processes like muscle activation and neuronal signalling. **Myasthenia Gravis: An Autoimmune Disease:** To manage MG, increasing the availability of ACh at the synaptic cleft is essential. This is achieved using a **cholinesterase inhibitor**, which prevents the breakdown of ACh, allowing it to act longer on the remaining receptors. - **Neostigmine Test:** This test involves administering neostigmine (or edrophonium) to temporarily improve muscle strength, confirming the diagnosis of MG. - **Botox (Botulinum Toxin):** While not a treatment for MG directly, Botox is an injectable neurotoxin that can block ACh release at the synapse, used for other neuromuscular conditions or cosmetic purposes. **Muscarinic Acetylcholine Receptors (AchR):** Only a **specific agonist** activates the **M1 receptor**, while the **others** are activated by **non-specific agonists**. The most **well-known** agonists for muscarinic receptors are **carbachol and pilocarpine**. **Prototypical muscarinic antagonists** include **atropine** and **scopolamine**, but neither is subtype selective. **Atropine** is a derivative of **belladonna**, a plant whose name (\"beautiful woman\") reflects its historical use as a pupillary dilator. Belladonna is produced by the deadly nightshade plant, so named due to the dangerous effects of ingesting excessive amounts. Overuse of antimuscarinic drugs can lead to **cognitive impairment**, and at higher doses, delirium, along with tachycardia and other dangerous autonomic symptoms. **Ligands affecting cholinergic transmission:**  **Glutamate:** **Case Study: Early Onset Alzheimer's Disease.** A 58-year-old woman was referred to a memory clinic with a **2-year history** of **memory loss**, **repetitiveness**, and **executive dysfunction**. Imaging showed **mild cortical atrophy**. She retired at 48 for financial reasons, not due to cognitive issues, but over the next 9 months, her cognitive decline became evident with difficulties in daily activities. Neurocognitive testing revealed an **MMSE score of 14/30** and poor **verbal fluency**. The diagnosis was likely **early-onset Alzheimer\'s disease (EOAD)**, though other forms were considered. After treatment with a **cholinesterase inhibitor**, her cognition stabilized temporarily, but over the following years, she became **totally dependent** and required **care facility admission**. She experienced **muscle rigidity**, **motor apraxia**, and **possible hallucinations**. She passed away at 63 from **pneumonia**, and the **autopsy** confirmed **Alzheimer's disease** with **Lewy body pathology**. **Glutamate synthesis and inactivation:** **Glutamine** is converted into **glutamate** by the enzyme **glutaminase**. The **glutamate** is then packed into **vesicles** for **synaptic release**. Upon release, glutamate binds to **neurotransmitter receptors** to activate downstream signalling. **Deactivation of glutamate** occurs through **cholinesterase**, which breaks down glutamate and transports it back into the presynaptic terminal. The **SN1 transporter** pumps glutamate from **glial cells** back into the synapse for recycling. **Glutamatergic Transmission:** **Two receptor types** , **iGluR** which conduct ions, and **mGluR**. **iGluR subunits:** **Activation by NMDAR, AMPAR (Quis) and Kainate R:** In **panel a**, the addition of **NMDA alone** produces minimal current, but when **glycine** is applied simultaneously (**NMDA + Gly**), a substantial inward current is observed, confirming glycine\'s role in NMDAR activation. **Panel b** shows that **glutamate (Glu)** alone produces a current, but when combined with glycine (**Glu + Gly**), the current is significantly enhanced. **Panels c** and **d** involve **quisqualate (Quis)** and **Kainate (Kai)**, agonists for **AMPA** and **Kainate receptors**, respectively. These panels reveal no significant change in current when glycine is added (**Quis + Gly** or **Kai + Gly**), indicating glycine specifically modulates NMDARs. The recordings underscore the unique co-agonist requirement of glycine for NMDAR activation. **Ketamine; an antagonist of NMDA receptors:** This graph shows neuronal firing rates (**spikes per second**) in response to the application of specific agonists: **Quisqualate (Q)** for **AMPA receptors**, **Kainate (K)** for **Kainate receptors**, and **NMDA (N)** for **NMDA receptors**. The consistent firing indicates receptor activation by these agonists. Upon the addition of **ketamine** (a known **NMDA receptor antagonist**), firing associated with **NMDA receptor activation** is significantly suppressed, while responses to **AMPA** and **Kainate receptor activation** remain unaffected. This demonstrates **ketamine\'s selective inhibition** of **NMDA receptor-mediated signalling**. **Types of NMDA receptors:** **Conventional NMDA Receptors**: These contain **2 GluN1 subunits** and **2 GluN2 subunits**. They are the most well-studied and understood type of NMDA receptor. **Unconventional NMDA Receptors**: These incorporate **GluN3 subunits** in addition to either **GluN1** or both **GluN1 and GluN2 subunits**. Less is known about their functional and pharmacological roles. **Subunit Composition**: **Di-heteromeric NMDARs**: Incorporate **up to 2 types of different subunits** (e.g., GluN1 + GluN2). **Tri-heteromeric NMDARs**: Contain **3 different types of subunits** (e.g., GluN1 + GluN2 + GluN3). **Conventional NMDARs:** NMDA receptors are composed of two GluN1 subunits and two GluN2 subunits, forming a tetrameric complex. The receptor is depicted as an ion channel permeable to sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺) ions. **Ligands:** **Agonists:** Glutamate is the primary neurotransmitter that activates NMDARs. **Co-agonists:** Glycine or D-serine must bind to a separate site to enable receptor activation. Once activated by glutamate and glycine/D-serine, the receptor allows the flow of ions. **Glu and gly sensitivity of GluN1/N2 receptors:** **Panels A & B:** Show receptor currents in response to **10 µM glutamate** when glycine is present. The receptor sensitivity and current amplitude differ between subunit combinations (e.g., GluN1/2A vs. GluN1/2C). **Panels C & D:** Show receptor currents in response to **10 µM glycine** when glutamate is present. Again, receptor behaviour varies based on the GluN2 subunit type. Different GluN2 subunits (e.g., GluN2A vs. GluN2C) affect the receptor\'s pharmacological properties, such as sensitivity to glutamate and glycine, and the resulting ion currents. **Mg2+ dependence of NMDA receptor-opening:** **Panel (a): Current-voltage relationship** The graph illustrates NMDA receptor current as a function of membrane voltage. At negative potentials (below 0 mV), Mg²⁺ blocks the receptor channel, resulting in minimal ion flow. As the membrane potential becomes more positive, the Mg²⁺ block is relieved, allowing ion flow through the receptor. This behaviour demonstrates the voltage-dependent block of NMDA receptors by Mg²⁺. **Panel (b): Traces of receptor activity**. The receptor activity (current traces) is shown under different conditions (e.g., with or without Mg²⁺). In the presence of Mg²⁺, receptor currents are strongly reduced at negative potentials. Removing Mg²⁺ or applying positive voltages enables robust ion flow through the channel. **Differential Mg2+ sensitivity:** The four graphs depict current-voltage relationships for different receptor subtypes (e.g., GluN1/2A, GluN1/2B, GluN1/2C, and GluN1/2D). In the presence of 1 mM Mg²⁺, the curves show suppression of current at negative potentials due to Mg²⁺ block. Mg²⁺-free conditions (control) demonstrate unblocked receptor currents across all potentials. **GluN2A and GluN2B:** Show strong Mg²⁺ block at negative potentials, indicating high sensitivity to Mg²⁺.**GluN2C and GluN2D:** Show weaker Mg²⁺ block, meaning these subtypes are less sensitive to Mg²⁺ **Developmental regulation of GluN2 subunits:** GluN2A, GluN2B, GluN2C, and GluN2D are expressed in distinct brain regions, indicating functional specialization. **GluN2B**: Expressed strongly at **P0 (birth)** and remains prominent throughout development (P7, P12) and into adulthood. **GluN2A**: Absent at birth (**P0**) but increases during development, becoming strongly expressed by **P12** and into adulthood. This shows a **developmental shift** where GluN2A expression increases as the brain matures, while GluN2B remains consistently expressed. **Conventional vs nonconventional NMDARs:** **Assembly of NMDARs:** GluN1 (light grey) and GluN2 (dark grey) subunits combine to form functional receptor complexes. The process involves individual subunits (A), dimerization (B), and full receptor assembly into a tetramer (C). **GluN1/N3 excitatory glycine receptors (NMDARs):** Electrophysiological recordings of receptor responses. Indicates that glycine alone activates the GluN1/N3 receptors, while glutamate (Glu) or Kainate (KA) do not. Demonstrates selective glycine-induced currents in GluN1/N3 assemblies. How GluN1/N3A receptors respond to 100 µM glycine. Each trace corresponds to a different subunit combination, highlighting that glycine elicits inward currents only when specific subunit pairs (GluN1/N3) are present. The uniform response across conditions emphasizes the functional importance of glycine in activating these receptors, with minimal variation among subunit pairings. **Electrophysiological recordings**: The left panel shows glycine-induced currents in GluN1/N3A receptors. The first trace demonstrates the response to glycine (100 µM), while the second shows a significantly reduced current when CGP-78608, a glycine-site antagonist, is applied. **Dose-response curve (right)**: This curve quantifies the inhibitory effect of CGP-78608, showing how increasing concentrations progressively reduce receptor activity. The sigmoidal shape reflects competitive antagonism, and the calculated IC50 value represents the concentration required to inhibit 50% of receptor activity. **Chemical structure of CGP-78608**: Depicted above the graph, highlighting its role as a glycine antagonist. Shows a schematic of electrophysiological recordings from the juvenile hippocampus, targeting CA1 pyramidal cells. - Glycine is applied (via puff) to activate GluN1/N3 receptors. **Electrophysiological Traces**: - WT (wild-type) neurons exhibit glycine-induced inward currents, blocked by CGP (glycine antagonist) or CGP-DCKA (a dual antagonist for glycine binding sites). - GluN3A KO (knockout) neurons show no glycine responses, confirming the role of GluN3A in these currents. **Quantitative Analysis (Bar Graph)**: - Shows significant glycine-induced responses in WT neurons compared to negligible activity in GluN3A KO neurons. - The effect of CGP and CGP-DCKA further confirms receptor specificity. **NMDARs in Diseases**: - Implicated in epilepsy, stroke, pain, schizophrenia, psychosis, depression, autism, and Alzheimer\'s disease. **Representative Drugs**: - **Ketamine**: A notable NMDAR antagonist with anaesthetic and antidepressant effects. - **Memantine**: Used in Alzheimer's disease to prevent excitotoxicity. - **MK-801** and **Phencyclidine (PCP)**: Research compounds acting as NMDAR antagonists. **Mechanisms**: - Diagrams illustrate how drugs target different sites on the receptor to modulate activity. **Research Evidence**: - Studies on neurodegeneration and NMDAR blockade (e.g., MK-801) demonstrate its influence on signalling and potential therapeutic uses.