Continuous infusion of substance P inhibits acute, but not subacute, inflammatory pain induced by complete Freund's adjuvant.

Previous studies have reported that continuous infusion with substance P (SP) into rat dorsal striatum ameliorated both mechanical allodynia in both formalin-evoked transient inflammatory pain and neuropathic pain models. However, a role of striatal SP in persistent inflammatory pain has not been demonstrated. The current study examined the effect of continuous infusion of SP into the rat dorsal striatum by reverse microdialysis on persistent inflammatory pain induced by complete Freund's adjuvant (CFA). Intraplantar injection of CFA evoked both mechanical allodynia and paw edema 3 and 7 days post-injection. The continuous infusion of SP ameliorated the CFA-evoked mechanical allodynia, but not paw edema, 3 days after the CFA injection. This antinociceptive effect of SP was partially inhibited by co-infusion with the neurokinin-1 (NK1) receptor antagonist CP96345. Conversely, at 7 days both CFA-evoked mechanical allodynia and paw edema were not affected by SP treatment. To clarify why the effect of SP treatment on CFA-induced pain changed, we evaluated NK1 receptor protein levels at both time points. The NK1 receptor protein level was decreased at 7, but not 3, days post CFA injection. These data suggest that persistent inflammatory pain can downregulate the striatal NK1 receptor. The current study demonstrates that striatal SP-NK1 receptor pathway can exert antinociceptive effect only on the third days of inflammatory pain phase defined as an acute but not the 7 days defined as a subacute.

Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice.

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer's solution (control) or CO (60-125 µM) in Ringer's solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20 min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p = 0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.

GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum.

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease.

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal-regulated kinase 1/2.

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an 'acute' phase (0-15 min) and 'tonic' phase (16-120 min), which is accompanied by significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post-formalin injection. To uncover a possible relationship between the slow-onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 µg/mL in microdialysis fiber, 1 µL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of 'tonic' phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre-treatment with NK1 receptor antagonist CP96345 (10 µM) blocked the effect of SP infusion. However, formalin-induced 'tonic' phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the 'tonic' phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP-NK1 receptor-ERK1/2 system. Noxious stimulation induces a slow-onset substance P (SP) release as a volume transmitter, activating extra-synaptic NK1 receptors, and evokes phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The SP-NK1-ERK1/2 system in the striatum decreases tonic nociception.

In Vivo Study of Monoamine Oxidases Using Multisite Intracerebral Microdialysis.

The activity of monoamine oxidases (MAOs) in the brain is often associated with neurodegenerative diseases. The study of MAOs in vivo or ex vivo is generally performed using MAO inhibitors and rarely using substrates. We present a pharmacological approach using intracerebral microdialysis to study the activity of MAO in the striatum and the prefrontal cortex of rats. It consists of applying ascending concentrations of 3-methoxytyramine (3-MT) as a substrate via the probes and measuring the indirect product homovanillic acid generated by MAO activity. We present herein the methodologies comprising our in-house stereotaxic procedures in rats, the microdialysis perfusion system and the substrate application, and the neurochemical analysis of the samples.

Nitric oxide signalling underlies salicylate-induced increases in neuronal firing in the inferior colliculus: A central mechanism of tinnitus?

The anti-inflammatory drug salicylate induces tinnitus in animals and man. Salicylate reduces cochlear output but causes hyperactivity in higher auditory centres, including the inferior colliculus (the auditory midbrain). Using multi-electrode recording in anaesthetised guinea pigs (Cavia porcellus), we addressed the hypothesis that salicylate-induced hyperactivity in the inferior colliculus involves nitric oxide signalling secondary to increased ascending excitatory input. Systemic salicylate (200 mg/kg i.p., 0 h) markedly increased spontaneous and sound-driven neuronal firing in the inferior colliculus (3-6 h post drug), with both onset and sustained responses to pure tones being massively increased. Reverse microdialysis of increasing concentrations of salicylate directly into the inferior colliculus (100 µM-10 mM, from 0 h) failed to mimic systemic salicylate. In contrast, it caused a small, transient, increase in sound-driven firing (1 h), followed by a larger sustained decrease in both spontaneous and sound-driven firing (2-5 h). When salicylate was given systemically, reverse microdialysis of the neuronal nitric oxide synthase inhibitor L-methyl arginine into the inferior colliculus (500 mM, 2-6 h) completely blocked the salicylate-induced increase in spontaneous and sound-driven neuronal firing. Our data indicate that systemic salicylate induces neuronal hyperactivity in the auditory midbrain via a mechanism outside the inferior colliculus, presumably upstream in the auditory pathway; and that the mechanism is ultimately dependent on nitric oxide signalling within the inferior colliculus. Given that nitric oxide is known to mediate NMDA receptor signalling in the inferior colliculus, we propose that salicylate activates an ascending glutamatergic input to the inferior colliculus and that this is an important mechanism underlying salicylate-induced tinnitus.

Continuous infusion of substance P into rat striatum relieves mechanical hypersensitivity caused by a partial sciatic nerve ligation via activation of striatal muscarinic receptors.

Previous studies have demonstrated that continuous substance P (SP) infusion into the rat striatum attenuated hind paw formalin-induced nociceptive behaviors and mechanical hypersensitivity via a neurokinin-1 (NK1) receptor dependent mechanism. However, whether there is a role of striatal infusion of SP on chronic, neuropathic pain has yet to be demonstrated. The present study investigated the effect of continuous SP infusion into the rat striatum using a reverse microdialysis method is antinociceptive in a rat model of chronic, mononeuropathic pain. Two weeks after partial sciatic nerve injury, the ipsilateral hind paw demonstrated mechanical hypersensitivity. Infusion of SP (0.2, 0.4, or 0.8 µg/mL, 1 µL/min) for 120 min into the contralateral striatum dose-dependently relieved mechanical hypersensitivity. The antinociceptive effect of SP infusion was inhibited by co-infusion with the NK1 receptor antagonist CP96345 (10 µM). Neither ipsilateral continuous infusion nor acute microinjection of SP (10 ng) into the contralateral striatum was antinociceptive. A role of striatal muscarinic cholinergic neurons is suggested since co-infusion of SP with atropine (10 µM), but not the nicotinic receptor mecamylamine (10 µM), blocked antinociception. The current study suggests that activation of striatal muscarinic receptors through NK1 receptors could be a novel approach to managing chronic pain.

Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study.

Dopamine (DA) and serotonin (5-HT) seem to be involved in several of the effects of ethanol (EtOH). Acetaldehyde (AcH), especially in the brain, induces effects that mimic those of EtOH. The purpose of this study was to investigate the effects of local perfusion of EtOH and AcH on extracellular DA and 5-HT in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Aldh2-KO mice were used as a model of aldehyde dehydrogenase 2 deficiency in humans to examine the effects of AcH. Mice were perfused with Ringer's solution (control), EtOH (100, 200, or 500mM) and AcH (100, 200, or 500µM) into the dorsal striatum. Dialysate samples were collected every 5min, and then analyzed with HPLC coupled to an ECD. We found that local perfusion with 500mM EtOH increased extracellular levels of DA (p<0.05) in both Aldh2-KO and WT mice, while 5-HT levels remain unchanged. EtOH at a dose of 200mM also increased DA in WT mice, but this was limited to a 30-40-min time-point. In contrast, perfusion with 200 and 500µM AcH decreased both DA and 5-HT (p<0.05) in Aldh2-KO mice, but this decrease was not found in WT mice at any AcH dose, indicating an effect of AcH on DA and 5-HT levels. There were no genotype effects on the basal levels of DA and 5-HT. These results indicate that high EtOH can stimulate DA, whereas high AcH can depress both DA and 5-HT in the dorsal striatum of mice.

In vivo dialysis setup with a loop injection valve facilitates retrodialysis studies.

INTRODUCTION: Retrodialysis, as used in neuropharmacological research, is a technique for in vivo delivery of neuroactive agents with concurrent monitoring of their effects on cellular activity with a separation between certain degree of spatial and temporal resolution. Typically, this is accomplished either by the use of a liquid-switch requiring multiple pumps, or by exchange of flow tubing requiring stopping and restarting dialysis. In the present study, we describe the use of a medium pressure injection valve for retrodialysis that overcomes these problems. METHODS: The valve was configured with a loop to deliver 20µL of solution, and artificial CSF flow from the pump to the probe was established via this device. The application of this setup was evaluated in urethane anesthetized adult male C57BL/6J mice prepared with a CMA 11 probe implanted in the ventral hippocampus. By switching between the load and inject positions, the loop was filled with escitalopram solution (0.3µM) and delivered at a rate of 1µL/min at the probe for retrodialysis. Escitalopram (2mg/kg BW) was administered subcutaneously for microdialysis studies. During these treatments, dialysate fractions were collected for the determination of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). RESULTS: Irrespective of route of escitalopram administration, the pattern of dialysate 5-HT, and 5-HIAA response was comparable to that reported by other investigators. Accordingly, the in-line valve assembly did not compromise retrodialysis or microdialysis sampling. The manipulations to carry out retrodialysis using the valve setup are easy and simple. DISCUSSION: An in-line injection valve is a promising adaptation for retrodialysis studies and can be incorporated as a standard part of in vivo dialysis instrumentation.