Stanislav Koulchitsky

The Nonpeptide Calcitonin Gene-Related Peptide Receptor Antagonist BIBN4096BS Lowers the Activity of Neurons with Meningeal Input in the Rat Spinal Trigeminal Nucleus

Calcitonin gene-related peptide (CGRP) has been suggested to play a major role in the pathogenesis of migraines and other primary headaches. CGRP may be involved in the control of neuronal activity in the spinal trigeminal nucleus (STN), which integrates nociceptive afferent inputs from trigeminal tissues, including intracranial afferents. The activity of STN neurons is thought to reflect the activity of central trigeminal nociceptive pathways causing facial pain and headaches in humans. In a rat model of meningeal nociception, single neuronal activity in the STN was recorded. All units had receptive fields located in the exposed parietal dura mater. Heat and cold stimuli were repetitively applied to the dura in a fixed pattern of ramps and steps. The nonpeptide CGRP receptor antagonist BIBN4096BS was topically applied onto the exposed dura or infused intravenously. BIBN4096BS (300 μg/kg, i.v.) reduced spontaneous activity by ∼30%, the additional dose of 900 μg/kg intravenously by ∼50% of the initial activity, whereas saline had no effect. The activity evoked by heat ramps was also reduced after BIBN4096BS (900 μg/kg, i.v.) by ∼50%. Topical administration of BIBN4096BS (1 mm) did not significantly change the spontaneous neuronal activity within 15 min. We conclude that the endogenous release of CGRP significantly contributes to the maintenance of spontaneous activity in STN neurons. Blockade of CGRP receptors, possibly at central and peripheral sites, may therefore be an effective way to decrease nociceptive transmission. This may offer a new therapeutic strategy for the treatment of facial pain and primary headaches.

Calcitonin gene-related peptide receptor inhibition reduces neuronal activity induced by prolonged increase in nitric oxide in the rat spinal trigeminal nucleus.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 μg/kg/h) or glycerol trinitrate (250 μg/ kg/h) for 2h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 μg/ kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

Differential effects of cocaine on dopamine neuron firing in awake and anesthetized rats.

Cocaine (benzoylmethylecgonine), a natural alkaloid, is a powerful psychostimulant and a highly addictive drug. Unfortunately, the relationships between its behavioral and electrophysiological effects are not clear. We investigated the effects of cocaine on the firing of midbrain dopaminergic (DA) neurons, both in anesthetized and awake rats, using pre-implanted multielectrode arrays and a recently developed telemetric recording system. In anesthetized animals, cocaine (10 mg/kg, intraperitoneally) produced a general decrease of the firing rate and bursting of DA neurons, sometimes preceded by a transient increase in both parameters, as previously reported by others. In awake rats, however, injection of cocaine led to a very different pattern of changes in firing. A decrease in firing rate and bursting was observed in only 14% of DA neurons. Most of the other DA neurons underwent increases in firing rate and bursting: these changes were correlated with locomotor activity in 52% of the neurons, but were uncorrelated in 29% of them. Drug concentration measurements indicated that the observed differences between the two conditions did not have a pharmacokinetic origin. Taken together, our results demonstrate that cocaine injection differentially affects the electrical activity of DA neurons in awake and anesthetized states. The observed increases in neuronal activity may in part reflect the cocaine-induced synaptic potentiation found ex vivo in these neurons. Our observations also show that electrophysiological recordings in awake animals can uncover drug effects, which are masked by general anesthesia.

Ongoing activity in trigeminal wide-dynamic range neurons is driven from the periphery.

Ongoing activity of spinal trigeminal neurons is observed under various conditions and suggested to be responsible for ongoing headache. It can be spontaneous, i.e. arising intrinsically from the neuron, or the product of descending influences from other central neurons, or maintained by ongoing afferent input. The aim of the present study was to examine if ongoing activity of neurons in different subnuclei of the spinal trigeminal nucleus is driven from peripheral afferent input. Experiments were performed in Wistar rats anesthetized with isoflurane or Nembutal/urethane. Ongoing activity of single wide-dynamic range (WDR) neurons was recorded with carbon fiber glass microelectrodes in two subnuclei of the spinal trigeminal nucleus: oral (Sp5O) and caudal (Sp5C). Peripheral receptive fields were evaluated using von Frey filaments. Sp5O neurons received peripheral input from facial areas innervated by the mandibular branch of the trigeminal nerve. Units in Sp5C had receptive fields in the surgically exposed dura mater and in facial areas innervated by the ophthalmic and maxillary branch of the trigeminal nerve. Saline or the local anesthetic lidocaine was locally applied onto the exposed dura mater or microinjected into V3 (for Sp5O units) or V1/V2 (for Sp5C units) divisions of the trigeminal ganglion via the infraorbital channel. Local application of lidocaine onto the exposed dura caused mechanical insensitivity of dural receptive fields but not significant decrease in ongoing activity. Microinjection of lidocaine but not saline into the trigeminal ganglion was followed by a substantial decrease in both the receptive field size and the activity of the recorded WDR units. Mechanical insensitivity of receptive fields after trigeminal ganglion blockade was accompanied by the disappearance of ongoing activity. We conclude that the ongoing activity of WDR neurons in the spinal trigeminal nucleus, which may be indicative for processes of sensitization, is driven remotely by ongoing afferent input.

Behavior in the open field predicts the number of KCl-induced cortical spreading depressions in rats

Anxiety disorders are known to be comorbid with migraine, and cortical spreading depression (CSD) is the most likely cause of the migraine aura. To search for possible correlations between susceptibility to CSD and anxiety we used the open field test in male Sprague-Dawley rats chronically treated with the preventive anti-migraine drugs valproate or riboflavin. Animals avoiding the central area of the open field chamber and those with less exploratory activity (i.e. rearing) were considered more anxious. After 4 weeks of treatment CSDs were elicited by application of 1M KCl over the occipital cortex and the number of CSDs occurring over a 2h period was compared to the previously assessed open field behavior. Higher anxiety-like behavior was significantly correlated with a higher frequency of KCl-induced CSDs. In saline-treated animals, fewer rearings were found in animals with more frequent CSDs (R=-1.00). The duration of ambulatory episodes in the open field center correlated negatively with number of CSDs in the valproate group (R=-0.83; p<0.005) and in riboflavin treated group (R=-0.69; p<0.05) as well as total time spent in the open field center in both groups (R=-0.75; p<0.05 and R=-0.58; p<0.1 respectively). These results suggest that anxiety symptoms are associated with susceptibility to CSD and might explain why it can be an aggravating factor in migraine with aura.

Role of the solitary tract nucleus and caudal ventrolateral medulla in temperature responses in endotoxemic rats

In experiments on conscious rats it was found that preliminary microinjection of 100 nl 100 microM glutamic acid to the rostral commissural part of the solitary tract nucleus or to the caudal ventrolateral medulla increased a rise in colonic temperature induced by systemically applied endotoxin (3 microg/kg Escherichia coli lipopolysaccharide, i.p.) as compared to animals with intrabulbar injection of vehicle (control group). Preliminary microinjection of glutamate to the caudal commissural part of the solitary tract nucleus levelled the endotoxin-induced temperature response. After glutamate treatment of the caudal ventrolateral medulla there was a significant decrease in the noradrenaline content and decrease in the adrenaline level in the caudal (not significant) and rostral ventrolateral medulla (significant), as well as a small rise in noradrenergic activity at the solitary tract nucleus as compared to control animals. The post-mortem measurement of the optical density of brainstem tissues revealed its significant attenuation at the solitary tract nucleus and caudal ventrolateral medulla after glutamate as compared with these structures after vehicle. The involvement of monoaminergic systems of both structures under study in the initiation and control of temperature responses during endotoxemia is suggested.

Release of calcitonin gene-related peptide from the jugular--nodose ganglion complex in rats--A new model to examine the role of cardiac peptidergic and nitrergic innervation

OBJECTIVE Afferent information from the heart and the lung is conveyed to the brainstem by primary afferent fibers originating from vagal sensory neurons (jugular-nodose ganglion complex, JNC). The present study was made to evaluate if release of the sensory neuropeptide calcitonin gene-related peptide (CGRP) from the JNC can be used as a model for future studies on changes in neuropeptide release under pathological conditions of the heart. METHODS Freshly isolated rat JNCs were passed through a series of solutions based on oxygenated synthetic interstitial fluid (SIF). Substances such as the TRPV1 receptor agonist capsaicin and the nitric oxide (NO) donor sodium nitroprusside (SNP) were added as excitatory test stimuli. The eluates were processed using an enzyme immuno-assay (EIA) for measurement of CGRP concentrations. Immunohistochemistry was used to visualize CGRP containing and NO producing neurons in the JNC. RESULTS Both SNP and capsaicin caused significant increases in CGRP release. CGRP-immunoreactive neurons (somata) were preferentially found in the jugular ganglion, whereas neurons immunoreactive for neuronal NO synthase were mostly localized in the nodose ganglion. CONCLUSION The present study demonstrates an easily reproducible model for measuring stimulated CGRP release from vagal afferents arising from the JNC. Nitric oxide produced by vagal afferents may stimulate CGRP release upon afferent activation.

Long-term potentiation of the AMPA and NMDA components of minimal postsynaptic currents in rat hippocampal field Ca1.

The aim of the present work was to study the potentiation of the AMPA and NMDA components of minimal excitatory postsynaptic currents (EPSC) evoked by activation of restricted numbers of synapses. EPSC of neurons in field CA1 in hippocampal slices were recorded in whole-call patch-clamp conditions selected such that both (AMPA and NMDA) components were present, and these were measured in parallel using computational methods in combination with pharmacological receptor blockade. There was a quite strong correlation between the amplitudes of the AMPA and NMDA components and this was regarded as evidence that they were generated by the same synapses. In cases producing this correlation, both components showed essentially equal long-term potentiation lasting from 5 min to 2 h after afferent tetanization. The data did not support the postsynaptic hypothesis and were in better agreement with the concept that the major mechanism for the persistence of the initial phase of long-term potentiation (up to 1–2 h) is based on increases in the quantity of transmitter released presynaptically.

NO-ergic mechanisms are implicated in a disturbed cardiac rhythm after systemic application of lipopolysaccharide E. coli to rats

In acute experiments on nembutal-urethan-anaesthetized rats, a slow infusion of subseptic dose of lipopolysaccharide (LPS) Escherichia coli (1 mg/ml) via the right jugular vein immediately led to bradycardia and extrasystoles. Preliminary administration of 20 mg/kg N(G)-nitro-L-arginine methyl ester (L-NAME) or 30 mg/kg aminoguanidine hydrochloride prevented the LPS-induced extrasystoles but did not affect the pattern of bradycardia. We conclude that nitric oxide (NO)-ergic mechanisms are involved in provoking electrical instability of the heart in conditions of endotoxemia.

Effect of a calcitonin gene-related peptide-binding L-RNA aptamer on neuronal activity in the rat spinal trigeminal nucleus

BackgroundCalcitonin gene-related peptide (CGRP) plays a major role in the pathogenesis of migraine and other primary headaches. Spinal trigeminal neurons integrate nociceptive afferent input from trigeminal tissues including intracranial afferents, and their activity is thought to reflect facial pain and headache in man. CGRP receptor inhibitors and anti-CGRP antibodies have been demonstrated to be therapeutically effective in migraine. In parallel, CGRP receptor inhibition has been shown to lower spinal trigeminal neuron activity in animal models of meningeal nociception.MethodsIn a rat model of meningeal nociception, single cell activity of neurons in the spinal trigeminal nucleus with meningeal afferent input was recorded to test a further pharmacological approach, scavenging CGRP with a CGRP-binding l-RNA oligonucleotide, the l-aptamer NOX-C89. Cumulative ascending doses of NOX-C89 were intravenously infused.ResultsSpontaneous activity of spinal trigeminal neurons did not change after 0.05 mg/kg NOX-C89, however, after additional infusion of 0.5 mg/kg and 5 mg/kg NOX-C89, spontaneous activity was dose-dependently reduced. Identical doses of a control l-aptamer had no effect. This pharmacological effect of NOX-C89 was observed 10–25 min after infusion, but no difference was detected in the period 0–5 min. For comparison, the previously investigated CGRP receptor antagonist olcegepant had reduced activity within 5 min after infusion. Alongside the reduced spontaneous activity, after infusion of NOX-C89 the heat-induced neuronal activity was abolished.ConclusionsScavenging CGRP by mirror-image RNA aptamers provides further evidence that this approach can be used to control spinal trigeminal activity.