Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application.

Ncm, 6-nitrocoumarin-7-ylmethyl, is a photolabile protective group useful for making "caged" molecules. Ncm marries the reliable photochemistry of 2-nitrobenzyl systems with the excellent stability and spectroscopic properties of the coumarin chromophore. From simple, commercially available starting materials, preparation of Ncm and its caged derivatives is both quick and easy. Photorelease of Ncm-caged molecules occurs on the microsecond time scale, with quantum efficiencies of 0.05-0.08. We report the synthesis and physical properties of Ncm and its caged derivatives. The utility of Ncm-caged glutamate for neuronal photostimulation is demonstrated in cultured hippocampal neurons and in brain slice preparations.

Excitation of primary afferent neurons by near-infrared light in vitro.

Near-infrared light therapy is an emerging neurostimulation technology, but its cellular mechanism of action remains unresolved. Using standard intracellular recording techniques, we observed that 5-10 ms pulses of 1889 nm light depolarized the membrane potential for hundreds of milliseconds in more than 85% of dorsal root ganglion and nodose ganglion neurons tested. The laser-evoked depolarizations (LEDs) exhibited complex, multiphasic kinetics comprising fast and slow components. There was no discernable difference in the LEDs in intact ganglion neurons and in acutely isolated neurons. Thus, the LED sensor seems to reside within the neuronal membrane. The near-uniform distribution of responsive neurons increased membrane conductance, and the negative reversal potential value (-41+/-2.9 mV) suggests that LED is unrelated to the activation of heat-sensitive transient receptor potential cation channel subfamily V member 1 channels. The long duration of LEDs favors an involvement of second messengers.

BKCa currents are enriched in a subpopulation of adult rat cutaneous nociceptive dorsal root ganglion neurons.

The biophysical properties and distribution of voltage-dependent, Ca(2+) -modulated K(+) (BK(Ca)) currents among subpopulations of acutely dissociated DiI-labeled cutaneous sensory neurons from the adult rat were characterized with whole-cell patch-clamp techniques. BK(Ca) currents were isolated from total K(+) current with iberiotoxin, charybdotoxin or paxilline. There was considerable variability in biophysical properties of BK(Ca) currents. There was also variability in the distribution of BK(Ca) current among subpopulations of cutaneous dorsal root ganglia (DRG) neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BK(Ca) current was present in the vast majority (> 90%) of small-diameter IB4+ neurons, but was present in only a minority of neurons in subpopulations defined by other criteria (i.e. small-diameter IB4-). Current-clamp analysis indicated that in IB4+ neurons, BK(Ca) currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. Reverse transcriptase-polymerase chain reaction analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BK(Ca) channel alpha-subunit, rslo and all four of the accessory beta-subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BK(Ca) current in cutaneous neurons reflects, at least in part, the differential distribution of splice variants and/or beta-subunits. Because even a small decrease in BK(Ca) current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury.

Inflammatory hyperalgesia: a role for the C-fiber sensory neuron cell body?

UNLABELLED: Peripheral nerve injury increases the chemosensitivity and excitability of injured afferents, resulting in ectopic activity arising from within dorsal root ganglia. Studies of dissociated sensory ganglion neurons in vitro suggest afferent somata might be sensitized by persistent inflammation. However, it is unknown whether this inflammation-induced sensitization is manifest in somata within the intact ganglia. To explore this possibility, intracellular electrophysiologic recording was used with a sciatic nerve-L4-dorsal root ganglia preparation to compare excitability and chemosensitivity of cutaneous C-fiber somata from control and inflamed rats. Cutaneous afferents were identified with the retrograde dye DiI. Excitability was assessed before and after the application of inflammatory soup (IS) containing bradykinin, serotonin, and prostaglandin E2 all at a pH of 7.0. Persistent inflammation decreased the excitability of cutaneous afferents in intact ganglia and had no significant influence on the magnitude of IS-induced increase in excitability. Opposite to the effects observed in intact ganglia, excitability was greater in dissociated cutaneous nociceptors obtained from inflamed rats, although the magnitude of the IS-induced increase in excitability was not significantly affected by inflammation. These results suggest that the cell bodies of putative cutaneous nociceptors in the intact ganglia contribute minimally to pain and hyperalgesia associated with persistent inflammation. PERSPECTIVE: Results of the present study suggest that inflammation-induced changes in afferent somata are minimal. However, they also suggest that inflammatory mediator-induced increase in the excitability of sensory neuron somata might contribute to global changes in nociception observed under high systemic inflammatory mediator loads.