Calcium signaling in intact dorsal root ganglia: new observations and the effect of injury.

BACKGROUND: Ca is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca recording techniques use high K or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. METHODS: Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. RESULTS: Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Abeta-type neurons. Unitary Ca transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca transients in C-type neurons, but increased transients in Abeta-type neurons. CONCLUSIONS: Refined observation of Ca signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state.

Depletion of calcium stores in injured sensory neurons: anatomic and functional correlates.

BACKGROUND: Painful nerve injury leads to disrupted Ca signaling in primary sensory neurons, including decreased endoplasmic reticulum (ER) Ca storage. This study examines potential causes and functional consequences of Ca store limitation after injury. METHODS: Neurons were dissociated from axotomized fifth lumbar (L5) and the adjacent L4 dorsal root ganglia after L5 spinal nerve ligation that produced hyperalgesia, and they were compared to neurons from control animals. Intracellular Ca levels were measured with Fura-2 microfluorometry, and ER was labeled with probes or antibodies. Ultrastructural morphology was analyzed by electron microscopy of nondissociated dorsal root ganglia, and intracellular electrophysiological recordings were obtained from intact ganglia. RESULTS: Live neuron staining with BODIPY FL-X thapsigargin (Invitrogen, Carlsbad, CA) revealed a 40% decrease in sarco-endoplasmic reticulum Ca-ATPase binding in axotomized L5 neurons and a 34% decrease in L4 neurons. Immunocytochemical labeling for the ER Ca-binding protein calreticulin was unaffected by injury. Total length of ER profiles in electron micrographs was reduced by 53% in small axotomized L5 neurons, but it was increased in L4 neurons. Cisternal stacks of ER and aggregation of ribosomes occurred less frequently in axotomized neurons. Ca-induced Ca release, examined by microfluorometry with dantrolene, was eliminated in axotomized neurons. Pharmacologic blockade of Ca-induced Ca release with dantrolene produced hyperexcitability in control neurons, confirming its functional importance. CONCLUSIONS: After axotomy, ER Ca stores are reduced by anatomic loss and possibly diminished sarco-endoplasmic reticulum Ca-ATPase. The resulting disruption of Ca-induced Ca release and protein synthesis may contribute to the generation of neuropathic pain.

Perioperative genomic profiles using structure-specific oligonucleotide probes.

OBJECTIVES: Many complications in the perioperative interval are associated with genetic susceptibilities that may be unknown in advance of surgery and anesthesia, including drug toxicity and inefficacy, thrombosis, prolonged neuromuscular blockade, organ failure and sepsis. The aims of this study were to design and validate the first genetic testing platform and panel designed for use in perioperative care, to establish allele frequencies in a target population, and to determine the number of mutant alleles per patient undergoing surgery. DESIGN/SETTING/PARTICIPANTS AND METHODS: One hundred fifty patients at Marshfield Clinic, Marshfield, Wisconsin, 100 patients at the Medical College of Wisconsin Zablocki Veteran's Administration Medical Center, Milwaukee, Wisconsin, and 200 patients at the University of Wisconsin Hospitals and Clinics, Madison, Wisconsin undergoing surgery and anesthesia were tested for 48 polymorphisms in 22 genes including ABC, BChE, ACE, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, beta2AR, TPMT, F2, F5, F7, MTHFR, TNFalpha, TNFbeta, CCR5, ApoE, HBB, MYH7, ABO and Gender (PRKY, PFKFB1). Using structure-specific cleavage of oligonucleotide probes (Invader, Third Wave Technologies, Inc., Madison, WI), 96-well plates were configured so that each well contained reagents for detection of both the wild type and mutant alleles at each locus. RESULTS: There were 21,600 genotypes confirmed in duplicate. After withdrawal of polymorphisms in non-pathogenic genes (i.e., the ABO blood group and gender-specific alleles), 376 of 450 patients were found to be homozygous for mutant alleles at one or more loci. Modes of two mutant homozygous loci and 10 mutant alleles in aggregate (i.e., the sum of homozygous and heterozygous mutant polymorphisms) were observed per patient. CONCLUSIONS: Significant genetic heterogeneity that may not be accounted for by taking a family medical history, or by obtaining routine laboratory test results, is present in most patients presenting for surgery and may be detected using a newly developed genotyping platform.

Hyperpolarization-activated current (I(h)) contributes to excitability of primary sensory neurons in rats.

In various excitable tissues, the hyperpolarization-activated, cyclic nucleotide-gated current (I(h)) contributes to burst firing by depolarizing the membrane after a period of hyperpolarization. Alternatively, conductance through open channels I(h) channels of the resting membrane may impede excitability. Since primary sensory neurons of the dorsal root ganglion show both loss of I(h) and elevated excitability after peripheral axonal injury, we examined the contribution of I(h) to excitability of these neurons. We used a sharp electrode intracellular technique to record from neurons in nondissociated ganglia to avoid potential artefacts due to tissue dissociation and cytosolic dialysis. Neurons were categorized by conduction velocity. I(h) induced by hyperpolarizing voltage steps was completely blocked by ZD7288 (approximately 10 microM), which concurrently eliminated the depolarizing sag of transmembrane potential during hyperpolarizing current injection. I(h) was most prominent in rapidly conducting Aalpha/beta neurons, in which ZD7288 produced resting membrane hyperpolarization, slowed conduction velocity, prolonged action potential (AP) duration, and elevated input resistance. The rheobase current necessary to trigger an AP was elevated and repetitive firing was inhibited by ZD7288, indicating an excitatory influence of I(h). Less I(h) was evident in more slowly conducting Adelta neurons, resulting in diminished effects of ZD7288 on AP parameters. Repetitive firing in these neurons was also inhibited by ZD7288, and the peak frequency of AP transmission during tetanic bursts was diminished by ZD7288. Slowly conducting C-type neurons showed minimal I(h), and no effect of ZD7288 on excitability was seen. After spinal nerve ligation, axotomized neurons had less I(h) compared to control neurons and showed minimal effects of ZD7288 application. We conclude that I(h) supports sensory neuron excitability, and loss of I(h) is not a factor contributing to increased neuronal excitability after peripheral axonal injury.

Restoration of calcium influx corrects membrane hyperexcitability in injured rat dorsal root ganglion neurons.

BACKGROUND: We have previously shown that a decrease of inward Ca(2+) flux (I(Ca)) across the sensory neuron plasmalemma, such as happens after axotomy, increases neuronal excitability. From this, we predicted that increasing I(Ca) in injured neurons should correct their hyperexcitability. METHODS: The influence of increased or decreased I(Ca) upon membrane biophysical variables and excitability was determined during recording from A-type neurons in nondissociated dorsal root ganglia after spinal nerve ligation using an intracellular recording technique. RESULTS: When the bath Ca(2+) level was increased to promote I(Ca), the after-hyperpolarization was decreased and repetitive firing was suppressed, which also followed amplification of Ca(2+)-activated K(+) current with selective agents NS1619 and NS309. A decreased external bath Ca(2+) concentration had the opposite effects, similar to previous observations in uninjured neurons. CONCLUSIONS: These findings indicate that at least a part of the hyperexcitability of somatic sensory neurons after axotomy is attributable to diminished inward Ca(2+) flux, and that measures to restore I(Ca) may potentially be therapeutic for painful peripheral neuropathy.

Modulators of calcium influx regulate membrane excitability in rat dorsal root ganglion neurons.

BACKGROUND: Chronic neuropathic pain resulting from neuronal damage remains difficult to treat, in part, because of incomplete understanding of underlying cellular mechanisms. We have previously shown that inward Ca2+ flux (I(Ca)) across the sensory neuron plasmalemma is decreased in a rodent model of chronic neuropathic pain, but the direct consequence of this loss of I(Ca) on function of the sensory neuron has not been defined. We therefore examined the extent to which altered membrane properties after nerve injury, especially increased excitability that may contribute to chronic pain, are attributable to diminished Ca2+ entry. METHODS: Intracellular microelectrode measurements were obtained from A-type neurons of dorsal root ganglia excised from uninjured rats. Recording conditions were varied to suppress or promote I(Ca) while biophysical variables and excitability were determined. RESULTS: Both lowered external bath Ca2+ concentration and blockade of I(Ca) with bath cadmium diminished the duration and area of the after-hyperpolarization (AHP), accompanied by decreased current threshold for action potential (AP) initiation and increased repetitive firing during sustained depolarization. Reciprocally, elevated bath Ca2+ increased the AHP and suppressed repetitive firing. Voltage sag during neuronal hyperpolarization, indicative of the cation-nonselective H-current, diminished with decreased bath Ca2+, cadmium application, or chelation of intracellular Ca2+. Additional recordings with selective blockers of I(Ca) subtypes showed that N-, P/Q, L-, and R-type currents each contribute to generation of the AHP and that blockade of any of these, and the T-type current, slows the AP upstroke, prolongs the AP duration, and (except for L-type current) decreases the current threshold for AP initiation. CONCLUSIONS: Taken together, our findings show that suppression of I(Ca) decreases the AHP, reduces the hyperpolarization-induced voltage sag, and increases excitability in sensory neurons, replicating changes that follow peripheral nerve trauma. This suggests that the loss of I(Ca) previously demonstrated in injured sensory neurons contributes to their dysfunction and hyperexcitability, and may lead to neuropathic pain.