Increased noradrenergic neurotransmission to a pain facilitatory area of the brain is implicated in facilitation of chronic pain.

BACKGROUND: Noradrenaline reuptake inhibitors are known to produce analgesia through a spinal action but they also act in the brain. However, the action of noradrenaline on supraspinal pain control regions is understudied. The authors addressed the noradrenergic modulation of the dorsal reticular nucleus (DRt), a medullary pronociceptive area, in the spared nerve injury (SNI) model of neuropathic pain. METHODS: The expression of the phosphorylated cAMP response element-binding protein (pCREB), a marker of neuronal activation, was evaluated in the locus coeruleus and A5 noradrenergic neurons (n = 6 rats/group). pCREB was studied in noradrenergic DRt-projecting neurons retrogradely labeled in SNI animals (n = 3). In vivo microdialysis was used to measure noradrenaline release in the DRt on nociceptive stimulation or after DRt infusion of clonidine (n = 5 to 6 per group). Pharmacology, immunohistochemistry, and western blot were used to study α-adrenoreceptors in the DRt (n = 4 to 6 per group). RESULTS: pCREB expression significantly increased in the locus coeruleus and A5 of SNI animals, and most noradrenergic DRt-projecting neurons expressed pCREB. In SNI animals, noradrenaline levels significantly increased on pinprick (mean ± SD, 126 ± 14%; P = 0.025 vs. baseline) and acetone stimulation (mean ± SD, 151 ± 12%; P < 0.001 vs. baseline), and clonidine infusion showed decreased α2-mediated inhibitory function. α1-adrenoreceptor blockade decreased nociceptive behavioral responses in SNI animals. α2-adrenoreceptor expression was not altered. CONCLUSIONS: Chronic pain induces brainstem noradrenergic activation that enhances descending facilitation from the DRt. This suggests that antidepressants inhibiting noradrenaline reuptake may enhance pain facilitation from the brain, counteracting their analgesic effects at the spinal cord.

GABA acting on GABAB receptors located in a medullary pain facilitatory area enhances nociceptive behaviors evoked by intraplantar formalin injection.

The dorsal reticular nucleus (DRt) plays a key role in facilitation of nociceptive transmission at the spinal cord. In this study, we evaluated the mechanisms involved in GABA-mediated control of the DRt focusing on the role of local GABAB receptors. First, we used in vivo microdialysis to study the release of GABA in the DRt during the course of the formalin test. An increase of GABA levels in comparison with baseline values was detected in the second phase of the test. Because we previously showed that GABAB receptors are expressed by opioidergic DRt neurons, which respond to nociceptive stimuli and inhibit spinally projecting DRt neurons involved in descending pronociception, we then interfered with local GABAB receptors using gene transfer and pharmacological approaches. Lentiviral-mediated knockdown of GABAB1a expression decreased nociceptive responses during the second phase of the test. Local administration of the GABAB receptor antagonist CGP 35348 also decreased nociceptive responses in the second phase of the test, whereas the opposite was detected after injection of the GABAB agonist baclofen. Finally, we determined the GABAergic afferents of the DRt, namely those arising from its main brain afferents, which are located at the telencephalon and diencephalon. For that purpose, we combined retrograde tract-tracing from the DRt with immunodetection of glutamate decarboxylase, the GABA-synthesizing enzyme. The higher numbers of retrogradely labelled glutamate decarboxylase-immunoreactive neurons were located at insular, somatosensory, and motor cortices. Collectively, the results suggest that GABA acting on GABAB receptors may enhance pain facilitation from the DRt during inflammatory pain.

Microdialysis-coupled enzymatic microreactor for in vivo glucose monitoring in rats.

Continuous glucose monitoring (CGM) is an important aid for diabetic patients to optimize glycemic control and to prevent long-term complications. However, current CGM devices need further miniaturization and improved functional performance. We have coupled a previously described microfluidic chip with enzymatic microreactor (EMR) to a microdialysis probe and evaluated the performance of this system for monitoring subcutaneous glucose concentration in rats. Nanoliter volumes of microdialysis sample are efficiently reacted with continuously supplied glucose oxidase (GOx) solution in the EMR. The hydrogen peroxide produced is amperometrically detected at a (polypyrrole (PPy)-protected) thin-film Pt electrode. Subcutaneous glucose concentration was continuously monitored in anesthetized rats in response to intravenous injections of 20% glucose (w/v), 5 U/kg insulin, or saline as a control. In vitro evaluation showed a linear range of 2.1-20.6 mM and a sensitivity of 7.8 ± 1.0 nA/mM (n = 6). The physical lag time between microdialysis and the analytical signal was approximately 18 min. The baseline concentration of blood glucose was 10.2 ± 2.3 mM. After administering glucose to the rats, glucose levels increased by about 2 mM to 12.1 ± 2.3 mM in blood and 11.9 ± 1.5 mM in subcutaneous interstitial fluid (ISF). After insulin administration, glucose levels decreased by about 8 mM relative to baseline to 2.1 ± 0.6 mM in blood and 2.1 ± 0.9 mM in ISF. A microfluidic device with integrated chaotic mixer and EMR has been successfully combined with subcutaneous microdialysis to continuously monitor glucose in rats. This proof-of-principle demonstrates the feasibility of improved miniaturization in CGM based on microfluidics.

Quantitative microdialysis using modified ultraslow microdialysis: direct rapid and reliable determination of free brain concentrations with the MetaQuant technique.

The only method to quantify free extracellular levels of drugs in the brain of living animals is microdialysis. However, quantitative microdialysis has been hampered by methodological issues for decades. The problems arise from the need to establish the in vivo recovery for appropriate quantitation. In dealing with these issues the "dynamic no-net-flux" (DNNF) method seemed to be the experimental method of choice. Major disadvantages were, however, the need for a very high degree of bioanalytical precision and accuracy and the need for a large number of animals. Moreover, today we know that the experimental data are not always straightforward. To improve robustness and practicality of quantitative microdialysis sampling we modified the ultraslow microdialysis approach. Ultraslow microdialysis uses very low microdialysis flow rates (<200 nl/min) which increase recovery (both in vivo and in vitro) to over 90%. However, new practical issues arise when attempting to work with these flow rates. The resulting very low volumes and long lag times make this method very impractical for general application. In the modified version, addition of a carrier flow after the dialysis process has been completed, which negates the problems of long lag times and low volumes. The resulting dilution of the dialysis sample concentration can simply be mathematically corrected. In the current study we measured the free brain levels of two CNS compounds using the classic DNNF and the new modified ultraslow dialysis method. Modified ultraslow microdialysis was shown to generate robust data with the use of only small numbers of rats. The method is a promising tool for common straightforward screening of blood-brain barrier penetration of compounds into the brain.

Hypoglycemia-induced noradrenergic activation in the VMH is a result of decreased ambient glucose.

During insulin-induced hypoglycemia, there is an increase in extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH). This brain area is known to play an important role in integrated hormonal and behavioral responses to systemic hypoglycemia. Selective glucoprivation restricted to the VMH is both necessary and sufficient to initiate secretion of counterregulatory hormones. The present study was designed to investigate whether increased release of NE in the VMH depends on detection of glucoprivation localized in this area. In awake, chronically catheterized male Sprague-Dawley rats, extracellular NE in the VMH was monitored using 1-mm microdialysis probes perfused with Krebs Ringer buffer (KRB) or KRB + 100 mM d-glucose (d-Glc). During insulin-induced hypoglycemia (glycemic nadir approximately 2.4 mM) extracellular NE was increased to >160% of baseline (P < 0.01) only in the KRB + insulin group. There was no increase in NE from baseline when glucose was added to the perfusate to maintain euglycemia at the periprobe environment. The sympathoadrenal response to hypoglycemia, present in the KRB + insulin group, was attenuated in the d-Glc + insulin group. The present results confirm that noradrenergic activation in the VMH during systemic hypoglycemia depends on detection of glucoprivation locally in this area. These data provide additional support for the importance of increased noradrenergic activity in the VMH in the counterregulatory hormonal responses to hypoglycemia.

Dissociation of hypothalamic noradrenergic activity and sympathoadrenal responses to recurrent hypoglycemia.

This study evaluated whether attenuation of sympathoadrenal responses to recurrent hypoglycemia is mediated by diminished noradrenergic activity in the hypothalamus. Male Sprague-Dawley rats received either once daily insulin (1.0 units/kg) injections or an equal administration of saline for 3 days. Both groups received an administration of insulin on the fourth day, during which blood glucose and plasma catecholamines were determined, and extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH) or paraventricular hypothalamic nucleus (PVN) was monitored with microdialysis. The peak response of plasma epinephrine to insulin-induced hypoglycemia (nadir approximately 3.2 mmol/l) was significantly reduced during the fourth hypoglycemic episode (774 +/- 134 pg/ml) compared with the first episode (2,561 +/- 410 pg/ml, P < 0.001). Baseline levels of extracellular NE were elevated approximately 25% (P = 0.07) in the VMH and approximately 46% (P = 0.03) in the PVN after multiple hypoglycemic episodes. There was no difference in noradrenergic activity during the first or fourth hypoglycemic episode in either brain area. The reduced sympathoadrenal output after recurrent hypoglycemia is likely postsynaptic from hypothalamic NE release or is mediated via a collateral pathway.

Extracellular glucose in rat ventromedial hypothalamus during acute and recurrent hypoglycemia.

The activity of neurons in the ventromedial hypothalamus (VMH) important for initiating compensatory responses to hypoglycemia is influenced by ambient glucose concentration. In the present study, we used in vivo microdialysis to evaluate interstitial glucose concentrations in rat VMH under various glycemic conditions. Using the zero-net-flux method, steady-state glucose concentration in the VMH was approximately 20% of blood glucose (approximately 1.4 mmol/l) in fed rats but approximately 14% of blood glucose (approximately 0.7 mmol/l) in overnight-fasted rats. During moderate hypoglycemia VMH glucose declined in parallel with blood glucose; however, VMH glucose decreased to a greater degree than blood glucose during a more severe hypoglycemic episode, falling to 10 +/- 1.2% of blood levels (P < 0.01). To determine whether VMH glucose concentrations were influenced by recurrent episodes of hypoglycemia a second zero-net-flux study was conducted. Steady-state glucose concentrations in the VMH were approximately 20% lower after three episodes of recurrent hypoglycemia, a value 17.8 +/- 0.8% of blood glucose, although the relative change in VMH glucose levels during the first and fourth hypoglycemic episodes were similar. From these results, we conclude that interstitial glucose concentrations in the VMH are not maintained at a constant level and are more dynamic than previously proposed.