N-type calcium current, Cav2.2, is enhanced in small-diameter sensory neurons isolated from Nf1+/- mice.

Major aspects of neuronal function are regulated by Ca(2+) including neurotransmitter release, excitability, developmental plasticity, and gene expression. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited both greater excitability and evoked release of neuropeptides compared to wildtype mice. Furthermore, augmented voltage-dependent sodium currents but not potassium currents contribute to the enhanced excitability. To determine the mechanisms giving rise to the enhanced release of substance P and calcitonin gene-related peptide in the Nf1+/- sensory neurons, the potential differences in the total voltage-dependent calcium current (ICa) as well as the contributions of individual Ca(2+) channel subtypes were assessed. Whole-cell patch-clamp recordings from small-diameter capsaicin-sensitive sensory neurons demonstrated that the average peak ICa densities were not different between the two genotypes. However, by using selective blockers of channel subtypes, the current density of N-type (Cav2.2) ICa was significantly larger in Nf1+/- neurons compared to wildtype neurons. In contrast, there were no significant differences in L-, P/Q- and R-type currents between the two genotypes. Quantitative real-time polymerase chain reaction measurements made from the isolated but intact dorsal root ganglia indicated that N-type (Cav2.2) and P/Q-type (Cav2.1) Ca(2+) channels exhibited the highest mRNA expression levels although there were no significant differences in the levels of mRNA expression between the genotypes. These results suggest that the augmented N-type (Cav2.2) ICa observed in the Nf1+/- sensory neurons does not result from genomic differences but may reflect post-translational or some other non-genomic modifications. Thus, our results demonstrate that sensory neurons from Nf1+/- mice, exhibit increased N-type ICa and likely account for the increased release of substance P and calcitonin gene-related peptide that occurs in Nf1+/- sensory neurons.

Dorsal root ganglia isolated from Nf1+/- mice exhibit increased levels of mRNA expression of voltage-dependent sodium channels.

We reported previously that sensory neurons isolated from mice with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited greater excitability and increased sodium current densities compared with wildtype mice. This raises the question as to whether the increased current density resulted from post-translational modifications or increased expression of sodium channels. Quantitative real-time polymerase chain reaction was used to measure expression levels of the nine different voltage-gated sodium channel α subunits and the four associated auxiliary ß subunits in the dorsal root ganglia (DRG) obtained from wildtype and Nf1+/- mice. The Relative Expression Software Tool indicated that Nav1.1, Nav1.3, Nav1.7, and Nav1.8 were significantly elevated in DRG isolated from Nf1+/- mice. Expression of Nav1.2, Nav1.5, Nav1.6, and Nav1.9 were not significantly altered. The gene transcript for Nav1.4 was not detected. There were no significant changes in the relative expression levels of ß subunits. The Nav1.9 subtype was the most abundant with Nav1.7 and Nav1.8 being the next most abundant subtypes, whereas Nav1.3 was relatively less abundant. For the ß subunits, ß1 was by far the most abundant subtype. These results demonstrate that the increased expression levels of Nav1.7, Nav1.8, and perhaps Nav1.1 in the Nf1+/- DRG make the largest contribution to the increased sodium current density and thus give rise to the enhanced excitability. Though the mechanisms by which many people with NF1 experience increased pain have not been elucidated, these abnormal painful states may involve elevated expression of specific sodium channel subtypes in small diameter nociceptive sensory neurons.

Ras signaling pathways mediate NGF-induced enhancement of excitability of small-diameter capsaicin-sensitive sensory neurons from wildtype but not Nf1+/- mice.

Nerve growth factor (NGF) activates multiple downstream effectors, including Ras, phosphoinositide-3 kinase, and sphingomyelins. However, pathway mediating the NGF-induced augmentation of sensory neuronal excitability remains largely unknown. We previously reported that small-diameter sensory neurons with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited increased excitability. The protein product of the Nf1 gene is neurofibromin, a guanosine triphosphatase-activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. Thus, Nf1+/- cells have augmented basal and stimulated Ras activity. To investigate whether NGF-induced increases in excitability of small-diameter sensory neurons are dependent on Ras signaling, an antibody that blocks the activation of Ras, Y13-259, was perfused into the cell. Under these conditions, the enhanced excitability produced by NGF was suppressed in wildtype neurons but the excitability of Nf1+/- neurons was unaltered. In addition, expression of a dominant-negative form of Ras abolished the ability of NGF to increase the excitability of small-diameter sensory neurons. These results demonstrate that NGF enhances excitability of small-diameter sensory neurons in a Ras-dependent manner while the consequences of decreased expression of neurofibromin cannot be restored by blocking Ras signaling; suggesting that Ras-initiated signaling pathways can regulate both transcriptional and posttranslational control of ion channels important in neuronal excitability.

Signaling pathways that mediate nerve growth factor-induced increase in expression and release of calcitonin gene-related peptide from sensory neurons.

Nerve growth factor (NGF) can augment transmitter release in sensory neurons by acutely sensitizing sensory neurons and by increasing the expression of calcitonin gene-related peptide (CGRP) over time. The current study examined the intracellular signaling pathways that mediate these two temporally distinct effects of NGF to augment CGRP release from sensory neurons. Growing sensory neurons in 30 or 100 ng/mL of NGF for 7 days increases CGRP content and this increase augments the amount of CGRP that is released by high extracellular potassium. Overexpressing a dominant negative Ras, Ras(17N) or treatment with a farnesyltransferase inhibitor attenuates the NGF-induced increase in CGRP content. Conversely, overexpressing a constitutively active Ras augments the NGF-induced increase in content of CGRP. Inhibiting mitogen activated protein kinase (MEK) activity also blocks the ability of NGF to increase CGRP expression. In contrast to the ability of chronic NGF to increase peptide content, acute exposure of sensory neurons to 100 ng/mL NGF augments capsaicin-evoked release of CGRP without affecting the content of CGRP. This sensitizing action of NGF is not affected by inhibiting Ras, MEK, or PI3 kinases. In contrast, the NGF-induced increase in capsaicin-evoked release of CGRP is blocked by the protein kinase C (PKC) inhibitor, BIM and the Src family kinases inhibitor, PP2. These data demonstrate that different signaling pathways mediate the alterations in expression of CGRP by chronic NGF and the acute actions of the neurotrophin to augment capsaicin-evoked release of CGRP in the absence of a change in the content of the peptide.

Augmented sodium currents contribute to the enhanced excitability of small diameter capsaicin-sensitive sensory neurons isolated from Nf1+/⁻ mice.

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Glial cell line-derived neurotrophic factor family ligands enhance capsaicin-stimulated release of calcitonin gene-related peptide from sensory neurons.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are a group of peptides that have been implicated as important factors in inflammation, since they are released in increased amounts during inflammation and induce thermal hyperalgesia upon injection. Mouse isolated sensory neurons in culture and freshly dissociated spinal cord slices were used to examine the enhancement in stimulated-release of the neuropeptide, calcitonin gene-related peptide (CGRP), as a measure of sensitization. Exposure of isolated sensory neurons in culture to GDNF, neurturin, and artemin enhanced the capsaicin-stimulated release of immunoreactive calcitonin gene-related peptide (iCGRP) two- to threefold, but did not increase potassium-stimulated release of iCGRP. A similar profile of sensitization was observed in freshly dissociated spinal cord slices. Persephin, another member of the GFL family thought to be important in development, was unable to induce an enhancement in the release of iCGRP. These results demonstrate that specific GFLs are important mediators affecting sensory neuronal sensitivity, likely through modulation of the capsaicin receptor. The sensitization of sensory neurons during inflammation, and the pain and neurogenic inflammation resulting from this sensitization, may be due in part to the effects of these selected GFLs.

Stimulus-evoked release of neuropeptides is enhanced in sensory neurons from mice with a heterozygous mutation of the Nf1 gene.

Neurofibromatosis type I is a common autosomal dominant disease characterized by formation of multiple benign and malignant tumors. People with this disorder also experience chronic pain, which can be disabling. Neurofibrinomin, the protein product of the NF1 gene (neurofibromin gene (human)), is a guanosine triphosphate activating protein for p21(ras). Loss of NF1 results in an increase in activity of the p21(ras) transduction cascade. Because of the growing evidence suggesting involvement of downstream components of the p21(ras) transduction cascade in the sensitization of nociceptive sensory neurons, we examined the stimulus-evoked release of the neuropeptides, substance P and calcitonin gene-related peptide, from primary sensory neurons of mice with a mutation of the Nf1 gene (neurofibromin gene (mouse)) (Nf1+/-). Measuring immunoreactive substance P and immunoreactive calcitonin gene-related peptide by radioimmunoassay, we demonstrated that capsaicin-stimulated release of neuropeptides is three to five-fold higher in spinal cord slices from Nf1+/- mice than from wildtype mouse tissue. In addition, the potassium and capsaicin-stimulated release of immunoreactive calcitonin gene-related peptide from cultures of sensory neurons isolated from Nf1+/- mice was more than double that from cultures of wildtype neurons. Treatment of wildtype sensory neurons with nerve growth factor for 5-7 days mimicked the enhanced stimulus-evoked release observed from the Nf1+/- neurons. When nerve growth factor was removed 48 h before conducting release experiments, nerve growth factor-induced augmentation of immunoreactive calcitonin gene-related peptide release from Nf1+/- neurons was more pronounced than in Nf1+/- sensory neurons that were treated with nerve growth factor continuously for 5-7 days. Thus, sensory neurons from mice with a heterozygous mutation of the Nf1 gene that is analogous to the human disease neurofibromatosis type I, exhibit increased sensitivity to chemical stimulation. This augmented responsiveness may explain the abnormal pain sensations experienced by people with neurofibromatosis type I and suggests an important role for guanosine triphosphate activating proteins, in the regulation of nociceptive sensory neuron sensitization.

Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.

Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.

The painful perils of a pair of pianists: the chronic pain of Clara Schumann and Sergei Rachmaninov.

Clara Wieck Schumann (1819-1896) and Sergei Vassilievich Rachmaninov (1873-1943) were two of the greatest pianists that ever lived. They had full lives composing and performing music. Each also had more than his or her fair share of hardships. In addition to all the pressures that are part of a performer's life, both Clara Schumann and Sergei Rachmaninov also suffered from chronic pain. This article discusses the pain syndromes that plagued these great musicians and the effect of chronic illness on their music.

The tragedy of Sergei Prokofiev.

Sergei Sergeevich Prokofiev (1891-1953) was one of the great Russian composers. He died, at age 61, of a supposed intracerebral brain hemorrhage. During the 8 years before his death, he suffered from episodic headaches, nausea and dizziness. This article reviews the historical information about Prokofiev's illness leading to a plausible underlying etiology for his death.

The treatment of Parkinson's disease: current concepts and rationale.

The recent advances in the treatment of Parkinson's disease have made for significant improvements in the quality of life and mortality rate of those who suffer from this neurodegenerative disease. At the same time, the number of options and the complexity of multi-drug regimens have posed a great challenge for the clinician caring for the patient with Parkinson's disease. Though there are still many questions to be answered in regard to the potential neuroprotective effects of several medications, a few general rational treatment plans can be outlined. In patients requiring treatment in the early stages of the disease, especially with a predominance of tremor, anticholinergics or amantadine should be considered initially. At this point, it would be reasonable to add selegiline for both therapeutic and possible neuroprotective effects. As a patient becomes more affected by the disease and additional therapy is necessary, starting either a dopamine agonist or levodopa would be a rational choice. Continuation of selegiline and, possibly, amantadine for neuroprotective reasons should be contemplated. Titration in levodopa therapy (with controlled-release or standard levodopa) to higher levels should prompt addition of a dopamine receptor agonist if one has not been started previously. Conversely, if a patient is receiving only a dopamine receptor agonist and is becoming progressively disabled, levodopa should be added to the regimen. Fluctuations in motor abilities may be improved further by the use of a COMT inhibitor. Patients with uncontrollable motor fluctuations should be considered for surgery. Undoubtedly, the coming years will bring more treatment options and more evidence on which sequences and combinations of therapies are the most beneficial. Differences in efficacy and adverse effects for each patient must be taken into consideration when outlining and carrying out a treatment plan. By using a rational approach to the treatment of Parkinson's disease, with the above guidelines in mind, the patient should be able to enjoy a good quality of life and level of function for many years.

Prostaglandins facilitate peptide release from rat sensory neurons by activating the adenosine 3',5'-cyclic monophosphate transduction cascade.

Prostaglandins sensitize sensory neurons to activation by mechanical, thermal and chemical stimuli. This sensitization also results in an increase in the stimulus-evoked release of the neuroactive peptides, substance P and calcitonin gene-related peptide from sensory neurons. The cellular transduction cascade underlying the prostaglandin-induced augmentation of peptide release is not known. Therefore, we examined whether the sensitizing action of prostaglandins on peptide release from sensory neurons grown in culture is mediated by the second messenger, adenosine 3', 5' cyclic monophosphate (cAMP). Prostaglandin E2 and carba prostacyclin (a stable analog of prostaglandin I2) significantly increase the content of cAMP-like immunoreactive substance (icAMP) in the sensory neuron cultures at concentrations that also augment the bradykinin- or capsaicin-evoked release of peptides. Furthermore, pretreating sensory neurons with agents that increase intracellular cAMP mimics the sensitizing action of prostaglandins. Exposing cultures to either forskolin (0.1-10 microM), cholera toxin (1.5 micrograms), or 8-bromo-cAMP (100 microM) results in a significant enhancement of the bradykinin- or capsaicin-stimulated release of both substance P-like and calcitonin gene-related peptide-like immunoreactive substances. Pretreating sensory neurons with the adenylyl cyclase inhibitor, 9-tetrahydro-2-furyl adenine (5 mM), abolishes the prostaglandin-induced increases in icAMP content and attenuates the prostaglandin E2 or carba prostacyclin enhancement of the evoked release of calcitonin gene-related peptide-like immunoreactive substance. These results demonstrate that the cAMP transduction cascade mediates the sensitizing actions of prostaglandins on peptide release from sensory neurons.

Prostacyclin enhances the evoked-release of substance P and calcitonin gene-related peptide from rat sensory neurons.

Prostacyclin (PGI2) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI2-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI2 and carba prostacyclin (CPGI2), a stable analog of PGI2, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI2 (10-1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI2, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI2 did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2-3 fold. In contrast, prostaglandin F2 alpha was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI2 sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI2-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.

The phosphatase inhibitor, okadaic acid, increases peptide release from rat sensory neurons in culture.

We examined the effects of the phosphatase inhibitor, okadaic acid, on substance P and calcitonin gene-related peptide release from embryonic rat sensory neurons grown in culture. Exposing isolated sensory neurons to 500 or 1000 nM okadaic acid for 30 min resulted in a 2- to 5-fold increase in the release of either peptide above resting levels and this evoked release was dependent on extracellular calcium. Treating sensory neurons with 250 nM okadaic acid did not alter resting peptide release, but significantly enhanced peptide release evoked by either 50 nM capsaicin, 100 nM bradykinin, or 30 mM KCl. These results suggest that enhancing phosphorylation in sensory neurons is an important component in augmenting transmitter release.

Carba prostacyclin enhances the capsaicin-induced cobalt-loading of rat sensory neurons grown in culture.

Sensory neurons isolated from the dorsal root ganglia of embryonic rats and grown in culture are distributed into two populations based on cross-sectional areas and sensitivity to capsaicin. Of the total neuronal population, about 48% of the neurons are labelled by cobalt in the presence of 1 microM capsaicin. The distribution of the small-sized neurons coincides with that of the capsaicin-cobalt-labelled neurons. When a lower concentration of capsaicin (100 nM) is used, pretreatment of sensory neurons with carba prostacyclin, a stable analog of prostaglandin I2, produces a three-fold enhancement in the number of neurons that are cobalt-labelled by capsaicin compared with that in the absence of carba prostacyclin.