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BSci Neuroscience: exam practice – guideline answers Q1a. What is meant by an electrochemical gradient for Na+ ions, and how is it established and utilized by excitable cells? The Na+ ion concentrations inside and outside a neuronal membrane are different – about 10 mM inside, 110-140 mM. Non-equilibrium scenario. Distribution of ions is generated by the Na-pump (on Na/K ATPase) utilizing metabolic energy in the form of ATP. Na+ ions from inside the membrane are exchanged for K+ ions from outside, with a stoichiometry of 3/2. Combination of Na pumping and low resting membrane permeability to Na+ ions means Na+ largely excluded from the intracellular compartment – helps balance the osmotic effects of non-permeant intracellular protein (double Donnan equilibrium) The neuronal membrane carries a resting potential that is largely explained by the resting K+ ion permeability of the membrane. Because of the non-equilibrium distribution of K+ ions, the membrane potential is negative inside with respect to outside, ie it is much closer to the K equilibrium potential than to the Na equilibrium potential. The equilibrium potential for Na+ or for K+ ions is given by the Nernst equation: • At 20 ºC, 58.2 log [out]/[in] • At 37 ºC, 61.5 log [out]/[in] • Thus a normal value of EK is negative and ENa is positive. For Na+, given the internal and external concentrations of 10 mM and 110 mM respectively, and at 37 ºC, ENa = +64 mV. Can give alternative concentrations, so long as sensible At the equilibrium potential for a given ion, the propensity for ions to move down the electrical potential gradient across the membrane is exactly balanced by their propensity to move in the opposite direction down their concentration gradient. Where the membrane potential is different from the equilibrium potential, the driving force on the ion species is given by the potential difference between the membrane potential and the reversal potential. This is the ‘electrochemical’ gradient, and for Na+ ions, under normal circumstances, this will drive an inward current. Therefore, under normal circumstances there is a chemical gradient driving Na+ inwards, and also an electrical potential gradient driving Na+ inwards, and the electrochemical gradient is the sum. The electrochemical gradient for Na+ represents a form of stored energy that can be utilized by excitable cells for the generation of impulses and long distance signalling through propagated action potentials. Na+ channels incorporated in neuronal membranes selectively allow Na+ ions to cross the membrane, and while they conduct briefly, the membrane potential approaches closer to the Na+ equilibrium potential, giving the upswing and peak of the action potential. Q 1b. Referring to the Na+ channel subtypes normally expressed in peripheral sensory neurones, in what ways are these channels functionally similar to each other, and in what ways do they differ? (this is a non-exhaustive list) Na channel subtypes for which there is evidence of normal primary sensory neuronal expression: NaV1.1, NaV1.2, NaV1.6, NaV1.7, NaV1.8, NaV1.9. SIMILAR: Peripheral sensory neurones express a variety of Na+ channel sub-types. They are similar in that they all comprise alpha and beta subunits. Alpha subunits in all tissues show over 70 % homology in pore and gating regions. SIMILAR: All voltage-gated Na channel sub-types, including all those in sensory neurones, have alpha subunits based on the 4 domain repeat structure, with 4 S4 activation gates that incorporate static +ve charge, and an intracellular inactivation gate with an IFM motif. The 4 domains coalesce to form a central aqueous pore. SIMILAR: All Na channels expressed in sensory neurones can be found in several states, including closed, open and inactivated, and they all normally generate inward currents when activated (or journeying through the open state), that can contribute to neuronal excitation. DIFFER: Na+ channels in peripheral sensory neurones can be pharmacologically discriminated on the basis of their TTX sensitivity, the toxin pharmacology being critically dependent on the amino acid sequence of each channel sub-type. TTX tetrodotoxin, from the puffer fish, blocks within the channel vestibule close to the ion selectivity filter. TTX-sensitive Na+ channels are blocked by single nM concentrations, whereas TTX-resitant channels are resistant to 3 orders of magnitude higher concentrations. DIFFER: the TTX-resistant Na+ channels in sensory neurones have slow activation and inactivation kinetics, in comparison with the TTX-sensitive channel expressed in the same cells. The TTX-resistant channel expressed in primary sensory neurones are NaV1.8 and NaV1.9 DIFFER: NaV1.7, NaV1.8 and NaV1.9 have special roles to play in pain signalling. NaV1.8 and NaV1.9 expression are markers for nociceptors DIFFER: NaV1.6 is the channel that confers excitability at nodes of Ranvier. In A-fibres NaV1.2 is expressed in the juvenile and is later replaced by NaV1.6 DIFFER: NaV1.8 is essential for pain signalling in the cold Q 2. Discuss the statement “Glial function after injury – a double-edged sword” Glia have good and bad roles; good roles largely restricted to normal functions and bad roles most often occur after injury/inflammation Should describe normal roles of astrocytes, microglia, oligodendrocytes and Schwann cells. After injury/infection; General bad role is in production &maintenance of pain – exemplified by lack of pain in neo injury where glial immune response is weak. Acute roles – mostly good; destruction of pathogens etc. Clearance of debris; Limiting size of injury/infection e.g. glial scar Schwann cells good roles – description of Wallerian degeneration & preparation for PN regeneration; production of proinflam mediators (cytokines) – pain (bad) or protection of injury site (good?) Oligodendrocytes – bad roles production of inhibitory substances MOG, MAG, OMPG – that prevent axonal regeneration; good role remyelination after CNS injury Astrocytes – activated – good role provide structural support for degenerating structures. Bad role production of proinflammatory substances, alterations in neurotransmitter transporters. Glial scar formation becomes a barrier to regeneration. Roles in neuropathic pain maintenance. Microglia – good roles - scavenging “Pac-man” role clearing debrs away.Signalling to T cells to help in immune response. Bad roles, contribution to neuropathic pain and with opioid hyperalgesia… early roles in development of neuropathic pain Extra points for details about signalling pathways & mechanisms.