Maxim Dobretsov

Presynaptic Ca2+ buffers control the strength of a fast post-tetanic hyperpolarization mediated by the α3 Na+/K+-ATPase

The excitability of CNS presynaptic terminals after a tetanic burst of action potentials is important for synaptic plasticity. The mechanisms that regulate excitability, however, are not well understood. Using direct recordings from the rat calyx of Held terminal, we found that a fast Na+/K+-ATPase (NKA)-mediated post-tetanic hyperpolarization (PTH) controls the probability and precision of subsequent firing. Notably, increasing the concentration of internal Ca2+ buffers or decreasing Ca2+ influx led to larger PTH amplitudes, indicating that an increase in [Ca2+]i regulates PTH via inhibition of NKAs. The characterization for the first time of a presynaptic NKA pump current, combined with immunofluorescence staining, identified the α3-NKA isoform on calyx terminals. Accordingly, the increased ability of the calyx to faithfully fire during a high-frequency train as it matures is paralleled by a larger expression of α3-NKA during development. We propose that this newly discovered Ca2+ dependence of PTH is important in the post-burst excitability of nerve terminals.

Stretch receptor-associated expression of α3 isoform of the Na+, K+-ATPase in rat peripheral nervous system

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.

Mechanical hyperalgesia in rat models of systemic and local hyperglycemia

Mechanical hyperalgesia is an early symptom of diabetic neuropathy. To evaluate the mechanisms underlying this symptom, it was studied and compared in rat models of systemic and local hyperglycemia. Systemic hyperglycemia was induced by a single injection of streptozotocin (STZ, 50 mg/kg). Local hyperglycemia either in L(5) dorsal root ganglion (DRG) or a segment of the sciatic nerve at mid-thigh level was maintained by perfusion with 30-mM glucose solution delivered from a surgically implanted osmotic minipump. Mechanical hyperalgesia was assessed using modified von Frey filaments and hind limb withdrawal threshold measurements. During 2 weeks of STZ-induced diabetes rat systemic blood glucose level increased from 5.1+/-0.3 to 23+/-1.9 mM and limb withdrawal threshold decreased by approximately 30% bilaterally. During 2 weeks of local perfusion systemic blood glucose did not change; however, rats that underwent perfusion of the DRG or sciatic nerve with glucose exhibited a rapid (completed in approximately 1 week) 40-50% decrease in ipsilateral limb withdrawal threshold. Perfusion of the sciatic nerve with the normoglycemic buffer solution did not affect withdrawal thresholds. The aldose reductase inhibitor sorbinil (2.5 mg/ml) when added to 30-mM glucose perfusion solution prevented hyperalgesia. These data suggest that mechanical hyperalgesia in diabetic animals may, at least in part, result from focal injury caused by a direct toxic effect of glucose in the peripheral nervous system. These data also support the idea of activation of aldose reductase and polyol pathway as an important mechanism of hyperglycemia-induced impairment of nerve function.

Mechanical hyperalgesia correlates with insulin deficiency in normoglycemic streptozotocin-treated rats

The triggers and pathogenesis of peripheral diabetic neuropathy are poorly understood, and this study evaluated the role of insulinopenia in nociceptive abnormalities in the streptozotocin (STZ) rat model of diabetes to test the hypothesis that, in addition to hyperglycemia, impairment of insulin signaling may be involved in progression of neuropathy. We measured blood glucose, plasma insulin, and sciatic nerve glucose and sorbitol levels, and withdrawal thresholds for hind limb pressure pain and heat pain in STZ-injected rats that developed hyperglycemia or remained normoglycemic. The pressure pain threshold did not change in vehicle-injected controls, but during the 2 weeks after STZ, it decreased by 25-40% in STZ-hyperglycemic and STZ-normoglycemic animals (P<0.05). Mean heat pain threshold did not change in STZ-normoglycemic rats, but increased by about 1.5 degrees C in STZ-hyperglycemic rats (P<0.05). These pain thresholds did not correlate with blood or nerve glucose or sorbitol levels, but both correlated with plasma insulin level in STZ-normoglycemic rats, and low-dose insulin replacement normalized the pressure threshold without affecting blood glucose level. Thus, at least one of early signs of diabetic neuropathy in STZ-treated rats, mechanical hyperalgesia, can be triggered by moderate insulinopenia, irrespective of glycemic status of the animals.

Mechanical hyperalgesia in rats with chronic perfusion of lumbar dorsal root ganglion with hyperglycemic solution

In diabetes, chronic systemic hyperglycemia is associated with pain and other symptoms of peripheral neuropathy. Evaluation of mechanisms causing these symptoms is complicated because of the overlap between the systemic effects of hyperglycemia and its toxic effects within the peripheral nervous system. To address this problem we developed a technique for chronic local in vivo perfusion of rat lumbar dorsal root ganglion (DRG) with a hyperglycemic solution. Osmotic pumps were filled with 30 mM glucose in physiological buffer and implanted in normal adult rats. The output of the catheter attached to the pump was positioned in a hole drilled through the right transverse process of the L(5) vertebrae to perfuse the corresponding DRG. Repetitive tests of foot withdrawal to mechanical stimuli have shown that chronic hyperglycemia localized to the L(5) DRG causes hyperalgesia in the hind limb innervated by perfused ganglion but not in the contralateral limb. Control experiments (DRG perfusion with 5 mM glucose or 5 mM glucose+25 mM mannitol solution) have shown that hyperglycemia-induced hyperalgesia can not be attributed to surgery-related injury or hyperosmolality of the ganglion-perfusing solution. These data demonstrate direct functional toxicity of hyperglycemia in the peripheral nervous system. This technique provides a new approach for in vivo study of chronic effects of physiologically active factors on DRG neuron function.

Relevance of hyperglycemia to early mechanical hyperalgesia in streptozotocin-induced diabetes.

Abstract  A modified von Frey filament test and an algesiometer paw pressure test were used to measure mechanical nociceptive withdrawal thresholds of the hind limb of control rats and rats injected with streptozotocin (STZ, 50 mg/kg). STZ treatment induced hyperglycemia (HG rats) in about 40% of treated animals. The rest of the STZ‐treated and control rats remained normoglycemic (NG rats) throughout the entire experiment. No indications of mechanical hyperalgesia were observed in control groups of animals injected with physiological buffer only. However, both the behavioral tests used detected a 15–30% decrease in the mechanical nociceptive threshold of rats treated with STZ. Furthermore, mechanical nociceptive threshold changes were statistically indistinguishable between NG and HG rats. Glucose tolerance test did not reveal abnormalities of glucose metabolism in NG rats (compared to control animals). However, 1 week after STZ injection, the serum insulin level of NG rats was significantly lower than that of age‐matched control rats (0.81 ± 0.16 vs. 3.5 ± 0.4 ng/mL; p < 0.01). These data strongly argue that systemic hyperglycemia is not the only factor triggering the development of mechanical hyperalgesia in the STZ rat model of diabetes. Other than hyperglycemia, consequences of insulinemia or insulinemia itself may play an important role in early impairment of mechanical nociception in this animal model.

The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test

ABSTRACT It is widely appreciated that there is significant inter‐individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β3 subunit of the Na+,K+‐ATPase pump (Atp1b3) contributes to inter‐strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β3 subunit produced strain‐specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β3 subunit of the Na+,K+‐ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.

Functional Na+/K+ pump in rat dorsal root ganglia neurons.

Steady-state Na+/K+ pump current (Ip) in isolated adult rat dorsal root ganglia neurons was studied to determine if the plasma membrane Na+/K+ pump activity is uniform across the population of dorsal root ganglia neurons. Cells were voltage-clamped at -40 mV and holding current (Ih) was recorded using whole-cell patch-clamp techniques under conditions that stimulate the Na+/K+ pump (60 mM intracellular Na+ and 5.4 mM extracellular K+). Ip was defined as the 1 mM ouabain-sensitive fraction of Ih. Data suggest the existence of three subpopulations of dorsal root ganglia neurons having mean steady-state Ip densities of 1.6+/-0.1, 3.8+/-0.2 and 7.5+/-0.4 pA/pF. Neurons with small Ip had an average soma perimeter of 95+/-3 microm, while neurons with medium and large Ip density had significantly larger soma sizes (131+/-8 and 141+/-7 microm, respectively). Neurons with a large Ip density had a significantly lower specific membrane resistance (Rm; mean 4.0+/-0.3 kohm x cm2) than neurons with medium or small Ip density (19+/-6 and 31+/-6 kohm x cm2, respectively). Regardless of these differences, in all groups of neurons Ip had a low sensitivity to ouabain (Ip half inhibition by ouabain was observed at 80-110 microM). These data suggest that the Na+/K+ pump site density and/or its activity is not uniform throughout the dorsal root ganglia neuron population; however, this non-uniformity does not appear to relate to the functional expression of the different alpha isoforms of the Na+/K+ pump. The major functional Na+/K+ pump in the dorsal root ganglia neuron plasma membrane appeared to be the low ouabain affinity (alpha1) isoform.

Non-uniform expression of α subunit isoforms of the Na+/K+ pump in rat dorsal root ganglia neurons

Abstract Tissue sections and antibodies selectively recognizing isoforms of the α subunit of the Na + /K + pump were used to determine the expression of α 1 , α 2 and α 3 pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the α 2 isoform of the Na + /K + pump. The α 1 isoform of the Na + /K + pump was found in most (77±4%) studied DRG neurons, regardless of cell size. Only 16±7% of the neurons expressed a detectable level of the α 3 Na + /K + pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the α 3 positive DRG neurons about 75% coexpressed the α 1 isoform of the Na + /K + pump. These data show that the expression of the protein of the α subunit isoforms of the Na + /K + pump is not uniform throughout the population of DRG neurons and that α 1 is the predominant isoform in the plasma membrane of these neurons.

COMPARISON OF METABOLIC AND NEUROPATHY PROFILES OF RATS WITH STREPTOZOTOCIN-INDUCED OVERT AND MODERATE INSULINOPENIA

To assess the relative roles of insulinopenia, hyperglycemia and dyslipidemia in pathogenesis of diabetic neuropathy, we compared plasma insulin, glucose and lipid metabolism and peripheral nerve function in rats with streptozotocin (STZ)-induced overt and moderate insulinopenia (hyperglycemic, STZ-HG; random glucose>11 mM and normoglycemic, STZ-NG rats). While being slightly insulinopenic, STZ-NG rats are metabolically not different from control, naive animals, by having normal glucose tolerance and normal levels of plasma glucose, glycated HbA1c, cholesterol and triglycerides. Two weeks following injection of STZ, STZ-HG but not STZ-NG rats had suppressed motor nerve conduction velocity, F-wave prevalence, withdrawal responses to heat and von Frey filament stimuli. In apparent correlation with plasma insulin level, both STZ-HG and -NG rats manifested exaggerated responses in paw pressure and colorectal distension tests. These data suggest that insulinopenia may play a leading role in the diabetic impairment of deep muscle and visceral afferent pathways while hyperglycemia/dyslipidemia may represent a key requirement for the onset and progression of electrophysiological nerve impairment and loss of superficial heat and tactile perception. STZ-NG rats offer a convenient model for the investigation of the short-term effects of insulinopenia on peripheral nerve function.