Richard I. Hume

Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Highwire, a conserved axonal E3 ubiquitin ligase, regulates the initiation of axonal degeneration after injury in Drosophila by regulating the levels of the NAD+ biosynthetic enzyme, Nmnat, and the Wnd kinase.

The role of histidine residues in modulation of the rat P2X2 purinoceptor by zinc and pH

P2X2 receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. Wild‐type and mutant receptors were expressed in Xenopus oocytes and analysed with two‐electrode voltage clamp. All mutations caused less than a 2‐fold change in the EC50 of the ATP concentration‐response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 μm ATP of wild‐type P2X2 and eight mutant receptors more than 4‐fold, but the response of the mutant receptor H319A was potentiated only 1.4‐fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pKa (‐log of dissociation constant) of the potentiation to a more basic pH as compared with P2X2 and revealed a substantial pH‐dependent decrease in the maximum response with a pKa near 6.0. Substituting a lysine for H319 reduced the EC50 for ATP 40‐fold. Zinc (20 μm) potentiated the responses to 10 μm ATP of wild‐type P2X2 and seven histidine mutants by ∼8‐fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage‐independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

Excitatory action of ATP on embryonic chick muscle

It has been suggested that ATP might play a role in synaptic transmission at developing vertebrate neuromuscular junctions. To increase our understanding of the events underlying synapse formation, we have used intracellular recording and patch clamp recording to examine the response of chick myoblasts and myotubes to to ATP and other nucleotides, ATP, applied at micromolar concentrations, has a potent depolarizing action on chick myoblasts and myotubes. The ATP depolarization declines during prolonged application of ATP and shows no recovery for at least 20 min after the removal of ATP. The physiological event that underlies the ATP response has a reversal potential near O mV and is due to a conductance increase. However, contrary to our expectation, in a series of nearly 200 cell-attached and outside-out patch recordings, we did not detect single-channel currents that were related to ATP. The myotube ATP receptor is pharmacologically distinct from putative ATP receptors in other systems. It is not activated by ADP, AMP, or adenosine. Furthermore, the nonhydrolyzable ATP analogs, AMP-PNP, alpha,beta-meATP, and beta,gamma-meATP (respectively, 5-adenylylimido diphosphate; alpha,beta- methylene adenosine 5′-triphosphate; and beta,gamma-methylene adenosine 5′-triphosphate), which are potent ATP agonists in other systems, have no depolarizing action on myotubes. The ATP receptor is also distinct from the nicotinic ACh receptor since responses to ATP are unaffected by the nicotinic antagonists d-tubocurarine and alpha-bungarotoxin. We therefore applied alpha-bungarotoxin to nerve-muscle co-cultures in the hope of uncovering an additional component of the postsynaptic potential, which might represent a synaptic action of ATP. Under these experimental conditions no evidence indicative of a postsynaptic action of ATP released from nerve terminals was observed.

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular Junction In Vivo

In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

A receptor that is highly specific for extracellular ATP in developing chick skeletal muscle in vitro.

1 Extracellular adenosine 5′‐triphosphate (ATP) activated an early excitatory conductance followed by a late potassium conductance in developing chick skeletal muscle. A series of ATP analogues were tested for their ability to activate these two conductances. All compounds tested were either agonists for both responses or for neither. Furthermore, the potency of agonists was similar for the two responses. 2 The order of potency for agonists was ATP ≅ adenosine 5′‐O‐(3‐thio triphosphate) (ATP‐γ‐S) ≅ 2‐methylthio‐ATP (2‐CH3S‐ATP) > 2′‐deoxy‐ATP ≅ 3′‐deoxy‐ATP > adenosine 5′‐tetraphosphate (ATP‐OPO3) ≅ adenosine 5′‐diphosphate (ADP). Many other ATP analogues were not agonists. 3 Activation of the excitatory response did not require divalent cations. Furthermore, the concentration‐response relation of the excitatory response was similar when ATP was applied as the free anion of ATP (ATP4‐) or complexed with a divalent cation (M · ATP2‐). 4 Three antagonists of the ATP response were characterized. 8‐Br‐ATP was a weak antagonist, while 2′,3′‐dialdehyde‐ATP and DIDS (4,4′‐diisocyanatostilbene‐2,2′‐disulphonic acid) were potent irreversible inhibitors. The two conductances were equally affected by these antagonists. 5 These results suggest that both ATP responses are activated through the same receptor type, or two very similar receptors.

Two mechanisms for inward rectification of current flow through the purinoceptor P2X2 class of ATP‐gated channels

1 The ATP receptor subunit P2X2 was expressed in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. ATP‐activated currents were studied with two‐electrode voltage clamp recordings from oocytes, whole‐cell recordings from HEK 293 cells, and outside‐out patch clamp recordings from both cell types. The steady‐state current‐voltage (I‐V) relation showed profound inward rectification in all recording configurations. 2 Recordings from outside‐out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch. 3 Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast voltage jumps, instantaneous current jumps were followed by substantial relaxations to the steady state. The time course of the current relaxations could be fitted by single exponential functions. The instantaneous I‐V relation was less inwardly rectifying than the steady‐state I‐V relation; however, it was not linear. 4 Single channel recordings indicated that the single channel conductance became smaller when the membrane potential became more positive. This decrease could quantitatively account for inward rectification of the instantaneous I‐V relation. 5 We conclude that inward rectification of P2X2 is due to two mechanisms: voltage‐dependent gating and voltage dependence of the single channel conductance.

TRPM7 Is Required within Zebrafish Sensory Neurons for the Activation of Touch-Evoked Escape Behaviors

Mutations in the gene encoding TRPM7 (trpm7), a member of the Transient Receptor Potential (TRP) superfamily of cation channels that possesses an enzymatically active kinase at its C terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations that abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons, we found that TRPM7s kinase activity and selectivity for divalent cations over monovalent cations were dispensable for touch-evoked activation of escape behaviors in zebrafish. Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively, these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses.

Apportionment of the terminals from single preganglionic axons to target neurones in the rabbit ciliary ganglion.

We have studied the apportionment of terminals from single preganglionic axons to target neurones in the ciliary ganglion of adult rabbits. Both electrical recording and intra‐axonal injection of horseradish peroxidase (HRP) showed that each preganglionic axon innervates only a small fraction of the ganglion cell population (about 10‐20 of the approximately 400 ganglion cells). Examination of ganglia in whole mounts showed that neurones whose cell bodies were enveloped by HRP‐labelled boutons from a single axon were often surrounded by other neurones which received no contacts from the labelled fibre. Electron microscopical examination of labelled presynaptic terminals on individual ganglion cells confirmed that the boutons of single axons were sharply confined to particular target cells. This suggests that individual target neurones (or portions of them) are the unit of innervation during the development of these synaptic connexions. Comparison of the amplitudes of synaptic responses in singly and multiply innervated ganglion cells indicated that, on average, an individual axon made a weaker synaptic connexion with a multiply innervated neurone than with neurone that received only one input. Moreover, neurones innervated by several different axons tended to have fewer synapses on their somata than neurones innervated by only one or two preganglionic axons. Individual post‐synaptic profiles were often contacted exclusively by labelled terminals when examined in the electron microscope. Since many of these neurones are multiply innervated, this observation suggests some regional separation of the several inputs contacting the same cell. For several reasons, however, this inference must be regarded as tentative. Taken together, these findings provide a possible explanation of the correlation between the dendritic geometry of ganglion cells and the number of different axons that innervate them (Purves & Hume, 1981). The several axons that initially innervate ganglion cells without dendrites evidently compete during early life until only a single input remains. On ganglion cells with dendrites, however, the number of inputs that persists is proportional to dendritic complexity. The present results suggest that the diminished competition between axons innervating neurones with dendrites may result from some degree of terminal segregation on dendritic arborizations.

Dihydropyridines and verapamil inhibit voltage-dependent K+ current in isolated outer hair cells of the guinea pig

Dihydropyridines and verapamil are widely used as blockers of voltage-dependent Ca++ channels. In this work we show that these compounds can have a direct blocking action on a class of voltage-activated potassium channels. Voltage-dependent whole-cell currents were recorded from isolated guinea-pig outer hair cells (OHCs) under conditions such that the free Ca++ concentration in both the internal and external solutions was minimized. A substantial Ca(++)-independent K+ current was revealed by this procedure. Both conventional K+ and Ca++ channel ligands inhibited this current. The order of potency (in terms of the half inhibitory concentrations (IC50) of channel inhibitors) was: nimodipine (6 microM) > Bay K 8644 (8 microM) > verapamil (11 microM) > 4-aminopyridine (22 microM) > nifedipine (32 microM) > quinine (49 microM) > TEA (10236 microM). Except for verapamil, these channel ligands reduced the size of the K+ currents without much alteration of the time course of the currents. In contrast, verapamil caused a more than 10-fold increase in the apparent inactivation rate of the K+ currents without significantly altering the activation of the currents. The observation that relatively low concentrations of calcium channel ligands can directly inhibit potassium currents in isolated OHCs indicates that caution should be taken when these pharmacological agents are used as tools for studying cochlear hair cell physiology.