METABOLISM, NEUROTRANSMITTERS & NEUROMODULATORS

The brain weight is 2% of total body weight, but the brain attracts 15% of the cardiac output and consumes 20% of the total oxygen used by the body, and 75% of the glucose released rom the liver at rest. Its stores of glycogen, creatine phosphate and ATP are minute, and would last only about 2 min at normal consumption rates. If supplies were suddenly cut off, optimal functioning would be impaired long before this, within a few seconds. The brain therefore relies on a constant supply of glucose, oxygen and blood to remove waste products. Blood exits the brain at a PO₂ of 35 mmHg, but that leaving grey matter areas may be at 17 mm Hg, equivalent to that leaving the myocardium. In rats, total anoxemia produces an isoelectric EEG in 60 sec. Hypoxemia has less drastic effects, but ischemia is worse, because glucose as well as O₂ delivery is reduced, and wastes also can build up. The maximal period of total ischemia compatible with recovery is 30 min in the brainstem and about 5 min in the cerebral hemispheres.

An O₂ consumption rate of c. 3.5 ml/100 g/min is maintained down to plasma glucose levels of c. 20 mg/dl, below which O₂ consumption falls. At plasma glucose levels of c. 8 mg/dl, EEG changes and coma may occur. The brain almost exclusively burns glucose, aerobically, as a fuel (normal RQ c. 0.97). During fasting however, ketone bodies may enter the brain via a specific carrier, and be used. Deficient protein intake has little effect on brain protein metabolism in the adult, the brain protein being spared relative to muscle. Perhaps related to this, the capacity for gluconeogenesis in brain is very restricted.

Brain blood flow is rigorously regulated at c. 55 ml/100 g/min, between the perfusion pressures of 65-150 mm Hg. The vessels are innervated by sympathetic (NE and Neuropeptide Y) fibres from the superior cervical, and cholinergic parasympathetic innervation (with co-transmitters VIP & PHM-27) but the ANS effects seem most important towards the ends of the regulatory range. For the most part autoregulatory influences predominate. Brain blood flow remains remarkably constant under most conditions, but increases in REM sleep, in seizures, fever etc., and falls slightly in SWS. In chronic hypertension the regulatory range is shifted to higher pressures.

The outstanding feature of cerebral blood flow is the sensitive regional redistribution according to need, based on local concentrations of CO₂ and O₂.

Increased metabolism in an area will raise PCO₂ and reduce PO₂, leading to vasodilation and increased blood supply to that area in order to meet the increased demand. PO₂ only becomes critical at very low levels, but its effect is potentiated at high PCO₂.

Blockage or rupture of blood vessles leads rapidly to damage or death of cells in the affected region, producing ischemic or haemorrhagic STROKE. Global ischemia associated with heart failure etc., can also produce stroke. Cells in the affected area die due to nutritional (O₂, glucose) starvation and metabolite build-up. But dying cells also depolarise and release excitatory neurotransmitters (e.g. glutamate) which can damage intact cells via the phenomenon of EXCITOTOXICITY, mainly by the action of excess glutamate on NMDA receptors (see below) in depolarised cells. There is a gradient in vulnerability in response to global ischemia, with the cerebral cortex being most susceptible, leading to clouding of consciousness at an early stage. Mid-brain reflexes will persist longer, and if these become more than transiently impaired, cerebral cortical recovery is unlikely. Functions of the hindbrain are most robust, and can persist, sustaining respiratory and vasomotor processes even after "brain death"- loss of cerebral cortical function.

A vast part of the brain's metabolism goes into the regulation of ionic gradients in neurones and glial cells, and into the synthesis of neurotransmitter substances. Much of the glucose taken up is used in amino acid synthesis, and quickly appears in glutamic acid, aspartic acid, alanine and GABA. Essential amino acids except lysine compete for a Large Neutral Amino-Acid transporter in the Blood-Brain Barrier. Lysine is carried by a Basic Amino-Acid transporter. Macro-nutrients (e.g. glucose, amino acids) are transported into brain by selective carriers on endothelial cells of the brain capillaries. Glucose is carried by facilitated transport, and amino acids by active transport. Micro-nutrients (e.g. vitamins) are selectively transported into brain by carriers on cells of the choroid plexus.

Ammonia produced as a by-product of protein metabolism in brain, is combined with glutamate in glial cells, to form glutamine, which is at a higher concentration in venous blood leaving the brain, than in arterial blood. The glutamine may be transferred to synaptic terminals, and converted to glutamate by mitochondrial glutaminase. -ketoglutarate from the Krebs Cycle also contributes to glutamate formation. Glutamate in turn may be conveted to GABA by glutamic acid decarboxylase, or to glycine (below) by transamination.

Tyrosine is an essential amino-acid in brain, since phenylalanine cannot be converted to tyrosine there. Although the synthesis of many transmitters is regulated by feedback inhibition, dietary levels of precursors, notably tryptophan (serotonin precursor), and oerhaps l-dopa (dopamine precursor) can influence brain levels of the respective neurotransmitters. Choline in the diet can also promote ACh synthesis.

Neurotransmitter substances are generally classified as low molecular weight (LMW) substances and peptides. To qualify as a true neurotransmitter, it must be shown that a substance is synthesized, stored presynaptically, released by appropriate stimuli, and produces the expected post-synaptic effect. A system of termination of action by removal from the synapse and/or degradation should exist. Several substances meet some, but not all criteria, and are regarded as "putative" transmitters (e.g. aspartate; histamine; many peptides). The majority of synapses in the brain are amino-acidergic: glutamate (the most prevalent excitatory transmitter); glycine, g -aminobutyric acid, aspartate and maybe taurine (inhibitory). Next come the aminergic and cholinergic groups: dopamine, NE, E (catecholamines); 5-HT (indolealkylamine); histamine; and acetylcholine. Enzymes catalysing the synthesis of these transmitters are synthesized in the perikarya and carried by slow transport to the axon terminals. The transmitters are synthesized at the synaptic endings and actively transported into the vesicles there. The vesicles for the LMW transmitters are released exocytotically, on normal depolarization of the terminal, by fusion with active zones on the presynaptic membrane.

The peptide neurotransmitters are synthesized on the rough ER, usually by a process involving splicing and rearrangements, transferred to the Golgi apparatus, and packaged into vesicles, which are carried to the terminals by fast axoplasmic transport. Peptide transmitters frequently are members of "families" with related amino acid sequences. They are usually released by intense activation of the presynaptic terminal, and like exocytotic vesicles for hormones, may fuse with the membrane at any point, and not just at active zones. About 50 peptide transmitters have been characterised, but have not always satisfied all the necessary criteria. Many, if not most, coexist with LMW transmitters at terminals (e.g. CGRP with ACh in motoneurones; Substance P with glutamate in nociceptors). The LMW transmitter often mediates rapid immediate responses, while the peptide mediates slower, longer lasting, modulatory effects. This dichotomy however, is more properly applied not to the transmitter groups, but to the differing modes of action at the post synaptic membrane: the fast, short acting ionotrotic responses via directly gated ion channels vs the slower, more persistent metabotropic responses via second messenger systems.

The ionotropic receptors include the nicotinic ACh (N) receptor, the GABA and glycine receptors, and the glutamate receptors (AMPA, Kainate and NMDA). All are aggregates of sub-units which are integral (transmembrane) proteins, with 4 membrane spanning segments (M1 to M4). The N, AMPA and NMDA receptors all have negative side groups on membrane spanning segments, which, when the subunits aggregate, form an cation selective channel, allowing passage of both Na⁺ and K⁺ ions (plus Ca²⁺ for the NMDA type). The glutamate AMPA receptor, gates a low conductance Na⁺/K⁺ channel.

The NMDA receptor gates a Na⁺/K⁺/Ca²⁺ channel with a much greater conductance, and with binding sites for glycine (agonist) as well as glutamate, and for Zn²⁺, PCP (phencyclidine or angel dust) and Mg²⁺, all of which can block the channel. The Mg²⁺ block normally prevents the NMDA receptor from contributing greatly to the EPSP produced in response to glutamate release. However, when depolarisation of the membrane via convergent pathways, removes the Mg²⁺ block preceding glutamate release, a large, late Ca²⁺ current flows through the NMDA channels in response to glutamate stimulation. This conditional responsiveness in the dually gated (voltage gated & chemically gated) NMDA channels, is important in producing long term potentiation (LTP) and associative learning in some cells. It is believed that strong stimulation of these channels when cells have been depolarised for some reason, can lead to excessive Ca²⁺ entry, activation of Ca²⁺-dependent proteases, and subsequent destruction of cells by autolysis. This is termed excitoxicity, and may be involved in promoting damage to cells after stroke, or in status epilepticus.

The GABA and glycine receptor sub-units have basic, positively charged residues on membrane spanning segments, and so, on aggregation, make anion selective Cl^- channels. The ACh nicotinic receptor has 5 subunits: two subunits carry the ACh binding sites. The GABA_A receptor has 4 different subunits, all of which bind GABA with differing affinities. Two subunits (a and b ) bind barbiturates, while one (g ) binds benzodiazepines. Combination with these agents potentiates binding mutualistically, and enhances inhibitory activity, by increasing Cl^- current.

Metabotropic receptors which activate second messenger systems include the muscarinic ACh (M) receptor, some glutamate receptors (e.g. kainate/quisqualate type), dopaminergic, a - and b -adrenergic receptors, serotonergic, peptidergic, nitric oxidergic (NO or EDRF) receptors, and rhodopsin. The receptors in this family have seven membrane spanning segments, with an extracellular, N-terminal, receptor portion, and a G-protein binding site. The G-protein, a peripheral protein bound to the cytoplasmic face of the membrane, and attached at rest to GDP, is activated by the transmitter-receptor complex. This allows the -subunit (GTP bearing) to dissociate and link with an effector protein - usually and enzyme whose action produces a soluble second messenger of some sort (cAMP, cGMP, DAG, IP₃, arachidonic acid). If the G-protein is a G_s it stimulates the effector protein; if it is G_i its action is inhibitory. The G-protein then splits the GTP to form GDP + P_i, and the G-protein/effector protein bond is broken. Second messengers produced in this process, can alter existing proteins, or can induce synthesis of new proteins by altering gene expression.

In certain hippocampal cells in which a Ca²⁺ activated K⁺ current causes accommodation, b -adrenergic stimulation prolongs excitability by activating a G_s protein which stimulates adenylate cyclase promoting formation of cAMP. A protein kinase activated by cAMP, phosphorylates the channel, inhibiting its opening. Acetylcholine acting on its M1 receptor in brain, activates a G protein which stimulates phospholipase C, a peripheral (cytoplasmic face) enzyme, which cleaves phosphoinositol (PI) in the membrane, to give diacyl glycerol (DAG) and inositol triphosphate (IP₃). IP₃ diffuses into the cytosol and stimulates ryanodine-related Ca²⁺ channels in the endoplasmic reticulum and mitochondria, to promote the release Ca²⁺ ion. IP₃ is degraded by phosphatases to inositol which is resynthesized to PI. Lithium blocks this degradation, an action which may explain its usefulness in treating manic depressive illness. Histamine acting at H1 receptors, stimulates a G protein which activates phospholipase A2 in the cell membrane, which catalyses the breakdown of PI to give arachidonic acid. Arachidonic acid can then be metabolised by cyclo-oxygenase to prostaglandins or thromboxanes, or by lipoxygenase to form leukotrienes. These products normally mediate inflammatory responses, but may play a neuromodulatory role in the brain.

Direct chemical gating of ion channels can produce rapid, short lived changes in neuronal activity, which may also lead to longer-term changes (c.f. the NMDA receptor). Activation of second messenger systems allows great flexibility and diversity of responses, ranging from opening or closing of ion channels, through activation of enzyme systems, to alteration of gene expression.

Read: Ganong, Review of Medical Physiology, Chapter 4 (selected portions) and Chapter 32 - Cerebral Circulation.

Berne & Levy, Principles of Physiology, Ch. 4 & Ch 24, pp306-307.

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