Michelle Mynlieff

Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons

&NA; Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger‐regulating neuronal function. We compared calcium currents (ICa) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch‐clamp techniques in current clamp mode, and in voltage‐clamp mode using solutions and conditions designed to isolate ICa. Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased ICa. Peak calcium channel current density was diminished by injury from 3.06±0.30 pS/pF to 2.22±0.26 pS/pF in medium neurons, and from 3.93±0.38 pS/pF to 2.99±0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium‐sized neuropathic cells lacked obvious T‐type calcium currents which were present in 25% of medium‐sized cells from control animals. Altered Ca2+ signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.

Loss of T-type calcium current in sensory neurons of rats with neuropathic pain.

Background Pathophysiology in the primary sensory neuron may contribute to chronic neuropathic pain. Ca channels play a central role in neuronal processes, and sensory neurons are rich in low-voltage–activated calcium channels (LVACCs). However, the physiologic function of these channels is unknown. Their possible role in rebound burst firing makes them a candidate for increased excitability after neuropathic injury. Methods This study uses pharmacological methods to isolate LVACC in cells from the dorsal root ganglia of neuropathic and sham-operated rats, including the blockade of high-voltage–activated Ca channels with fluoride and selective toxins. LVACCs were examined with conventional whole cell patch clamp electrophysiology techniques. Results After chronic constriction injury of the peripheral axon, LVACC was significantly reduced compared to sham rats as shown by a 60% reduction in peak current density and an 80% reduction in total calcium influx. A depolarizing shift in the voltage dependence of activation and an increase in the rate of deactivation and inactivation appear to cause this reduction of LVACC. Either Ni2+ or mibefradil, blockers of LVACC, applied in the bath to normal dorsal root ganglion cells during current clamp significantly and reversibly increased excitability. Conclusions These results suggest that loss of LVACC may contribute to decreased spike frequency adaptation and increased excitability after injury to sensory neurons. Through decreased Ca2+ influx, the cell becomes less stable and more likely to initiate or transmit bursts of action potentials. Consequently, modulation of Ca2+ currents at the dorsal root ganglion may be a potential method of therapeutic intervention.

γ-Aminobutyric acid type B receptors facilitate L-type and attenuate N-type Ca2+ currents in isolated hippocampal neurons

Activation of presynaptic γ‐aminobutyric acid type B (GABAB) receptors inhibits neurotransmitter release at many synapses (both excitatory and inhibitory), and activation of postsynaptic GABAB receptors leads to a general inhibition of the postsynaptic cell in mature neurons. Although the action of GABAB receptors at the soma of excitatory hippocampal pyramidal cells has been resolved to be regulation of a potassium or calcium conductance, it is not clear that all neurons in the hippocampus demonstrate similar effects of GABAB receptor activation. In the current study, GABAB receptor‐mediated effects on calcium currents in acute cultures composed of heterogeneous cells from the superior region of neonatal hippocampi were studied. In 54.5% of cells, the GABAB receptor agonist baclofen (10 μM) attenuated the whole‐cell calcium current by 21.0% ± 1.1%. In 29.9% of cells, baclofen facilitated the calcium current by 43.5% ± 8.1%. The component of current attenuated by baclofen was blocked by the N‐type calcium channel antagonist ω‐conotoxin GVIA (3 μM). The component of current facilitated by baclofen was blocked by the L‐type channel antagonist nimodipine (20 μM). For cells that showed calcium current facilitation, baclofen shifted the half‐maximal activation by approximately –14 mV. The data indicate that activation of GABAB receptors in neurons of the superior hippocampus attenuates current through N‐type channels and facilitates current through L‐type channels. The two opposing effects of GABAB receptor activation may reflect the heterogeneity of the cultured cells or may be a developmentally regulated phenomenon.

Involvement of protein kinase C and protein kinase A in the enhancement of L-type calcium current by GABAB receptor activation in neonatal hippocampus.

In the early neonatal period activation of GABAB receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of Gi/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6-8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of Gi/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABAB agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the Gi/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF-109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19-36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6-22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM-1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABAB receptor mediated effect.

Influx of Calcium through L-type Calcium Channels in Early Postnatal Regulation of Chloride Transporters in the Rat Hippocampus

During the early postnatal period, GABAB receptor activation facilitates L‐type calcium current in rat hippocampus. One developmental process that L‐type current may regulate is the change in expression of the K+Cl− co‐transporter (KCC2) and N+K+2Cl− co‐transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L‐type current, GABAB receptors, and expression of chloride transporters during development. The facilitation of L‐type current by GABAB receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L‐type channel antagonist and a GABAB receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L‐type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L‐type channels in the early postnatal period in hippocampal neurons.

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Dissociation of postnatal hippocampal neurons for short term culture

A method is described to isolate hippocampal neurons from postnatal day 7 to postnatal day 21 rats for use in short term cultures using a combination of enzymatic and mechanical dissociation. A significant fraction of the cells (37.5 +/- 2.3%, n = 10 cultures) were labelled with anti-GABA antibodies and the neurons survived a minimum of 8 days in culture. Patch-clamp recording in the current clamp mode revealed an average membrane potential and input resistance of - 47.6 +/- 2.5 mV and 737 +/- 147 M omega respectively, after 24 h in culture. The cells exhibited normal excitability and fired multiple action potentials in response to a depolarizing pulse. The technique described here provides a good alternative to either the use of embryonic brain tissue for cultures used in electrophysiological studies which may not exhibit the mature phenotype or use of acutely isolated cells which may be unstable or have their channels and receptors modified by enzymatic treatment.

Amyotrophic lateral sclerosis patient IgG alters voltage dependence of Ca2+ channels in dissociated rat motoneurons

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) have detected IgG within motoneurons of patients and these IgGs have been shown to alter voltage dependent calcium channel activity in various cell types. The current study investigates whether IgG from categorized SALS patients alter voltage dependent calcium currents in rat motoneurons in culture. Patients were categorized based on onset and progression pattern. IgG yields were 38% higher in SALS patients compared to control subjects. Incubation with 1 mg/ml IgG from SALS patients did not cause visible toxicity, alter input resistance, capacitance or the maximal calcium conductance in rat motoneurons when compared to motoneurons incubated with control IgG. However, the activation curve of calcium current was shifted to the left in motoneurons treated with SALS IgG compared to control IgG.

PAINFUL NEUROPATHY DECREASES MEMBRANE CALCIUM CURRENT IN MAMMALIAN PRIMARY AFFERENT NEURONS

Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger-regulating neuronal function. We compared calcium currents (I-Ca) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch-clamp techniques in current clamp mode, and in voltage-clamp mode using solutions and conditions designed to isolate I-Ca. Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased I-Ca. Peak calcium channel current density was diminished by injury from 3.06 +/− 0.30 pS/pF to 2.22 +/− 0.26 pS/pF in medium neurons, and from 3.93 +/− 0.38 pS/ pF to 2.99 +/− 0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium-sized neuropathic cells lacked obvious T-type calcium currents which were present in 25% of medium-sized cells from control animals. Altered Ca2+ signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.

GABAB receptors couple to Gαq to mediate increases in voltage-dependent calcium current during development

Metabotropic GABAB receptors are known to modulate the activity of voltage‐dependent calcium channels. Previously, we have shown that GABAB receptors couple to a non‐Gi/o G‐protein to enhance calcium influx through L‐type calcium channels by activating protein kinase C in neonatal rat hippocampal neurons. In this study, the components of this signaling pathway were investigated further. Gαq was knocked down using morpholino oligonucleotides prior to examining GABAB‐mediated enhancement of calcium influx. When Gαq G‐proteins were eliminated using morpholino‐mediated knockdown, the enhancing effects of the GABAB receptor agonist baclofen (10 μM) on calcium current or entry were eliminated. These data suggest that GABAB receptors couple to Gαq to regulate calcium influx. Confocal imaging analysis illustrating colocalization of GABAB receptors with Gαq supports this hypothesis. Furthermore, baclofen treatment caused translocation of PKCα (protein kinase C α) but not PKCβ or PKCε, suggesting that it is the α isoform of PKC that mediates calcium current enhancement. Inhibition of calcium/calmodulin‐dependent kinase II did not affect the baclofen‐mediated enhancement of calcium levels. In summary, activation of GABAB receptors during development leads to increased calcium in a subset of neurons through Gαq signaling and PKCα activation without the involvement of calcium/calmodulin‐dependent kinase II.