Perfusion Of Artificial Cerebrospinal Fluid Research Articles

Ischemic postconditioning prevents surge of presynaptic glutamate release by activating mitochondrial ATP-dependent potassium channels in the mouse hippocampus.

A mild ischemic load applied after a lethal ischemic insult reduces the subsequent ischemia–reperfusion injury, and is called ischemic postconditioning (PostC). We studied the effect of ischemic PostC on synaptic glutamate release using a whole-cell patch-clamp technique. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) from CA1 pyramidal cells in mouse hippocampal slices. The ischemic load was perfusion of artificial cerebrospinal fluid (ACSF) equilibrated with mixed gas (95% N2 and 5% CO2). The ischemic load was applied for 7.5 min, followed by ischemic PostC 30 s later, consisting of three cycles of 15 s of reperfusion and 15 s of re-ischemia. We found that a surging increase in sEPSCs frequency occurred during the immediate-early reperfusion period after the ischemic insult. We found a significant positive correlation between cumulative sEPSCs and the number of dead CA1 neurons (r = 0.70; p = 0.02). Ischemic PostC significantly suppressed this surge of sEPSCs. The mitochondrial KATP (mito-KATP) channel opener, diazoxide, also suppressed the surge of sEPSCs when applied for 15 min immediately after the ischemic load. The mito-KATP channel blocker, 5-hydroxydecanoate (5-HD), significantly attenuated the suppressive effect of both ischemic PostC and diazoxide application on the surge of sEPSCs. These results suggest that the opening of mito-KATP channels is involved in the suppressive effect of ischemic PostC on synaptic glutamate release and protection against neuronal death. We hypothesize that activation of mito-KATP channels prevents mitochondrial malfunction and breaks mutual facilitatory coupling between glutamate release and Ca2+ entry at presynaptic sites.

Mechanism of neuronal activity and synaptic transmission in rostral ventrolateral medulla

Rostral ventrolateral medulla (RVLM) plays an essential role in blood pressure homeostasis. This study was aimed to investigate the mechanism of neuronal activity and synaptic transmission in the RVLM. Medulla oblongata slices were carefully obtained from brainstem of rats. With continues perfusion of artificial cerebrospinal fluid (ACSF), the spontaneous firing of slices and amplitudes were assayed by conventional whole cell patch-clamp recording after addition of gamma-aminobutyric acid (GABA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl d-aspartate (NMDA). Furthermore, the effects of agonist or antagonist targeted Type-A GABA (GABAA) or glutamate receptors on spontaneous excitatory postsynaptic potential (sEPSP) and spontaneous inhibitory postsynaptic potential (sIPSP) of neurons in RVLM were determined. The spontaneous firing of neurons in RVLM were inhibited by GABA (P<0.001) while were promoted by NMDA or AMPA (P<0.01 or P<0.001). In terms of sEPSP and sIPSP, the numbers of firing neurons in RVLM were both improved by GABAA receptor antagonist (P<0.01 or P<0.001) while were both decreased by GABAA receptor agonist or glutamate receptor antagonist (P<0.05, P<0.01 or P<0.001). The corresponding effects of agonist and antagonist on amplitudes were the same as the effects on number of firing neurons in RVLM. The spontaneous firing, sEPSP and sIPSP of neurons in RVLM were all activated by GABAA receptor antagonist while were all suppressed by GABAA receptor agonist or glutamate receptor antagonist.

Ventriculolumbar Perfusion Chemotherapy for the Treatment of Leptomeningeal Carcinomatosis

To define the maximum tolerated dose, toxicities, and pharmacokinetics of the ventriculolumbar perfusion (VLP) chemotherapy for leptomeningeal carcinomatosis, according to different modes of administration, perfusion rate, and daily methotrexate (MTX) dose. For bolus injection modes, a designated MTX dose was given every 12 hours, for 3 consecutive days, with artificial cerebrospinal fluid perfusion. The starting daily MTX dose was 40 mg for 40 mL/h perfusion, and 24 mg for 20 mL/h, respectively. Although in continuous infusion modes, MTX premixed in artificial cerebrospinal fluid was infused for 3 consecutive days. The dose-limiting toxicity (DLT) was defined to be equal to or more than grade 3 side effects, as per the Common Terminology Criteria for Adverse Events. The perfusion rate of 40 mL/h provoked perfusion-related symptoms, such as nausea/vomiting and insomnia, and revealed DLTs including encephalopathy in more than 1/3 of patients in both administration modes when the daily MTX dose of 40 mg was given. The schedule of 20 mL/h perfusion with the daily MTX dose of 24 mg showed milder perfusion-related side effects and provoked no DLT in both administration modes. The relationship between the peak MTX concentration and encephalopathy was not statistically significant. Of 19 patients with increased intracranial pressure, the intracranial pressure was normalized in 14 patients after VLP therapy. This study disclosed the VLP chemotherapy with a perfusion rate of 20 mL/h and the daily MTX dose of 24 mg could be adopted for future study.

Testing neocortical slice viability in non-perfused no-magnesium artificial cerebrospinal fluid solutions

The acute in vitro brain slice model is a widely used neurophysiological research tool. When applying this method, most researchers continuously perfuse slices with carbogenated artificial cerebrospinal fluid (ACSF) to maintain pH balance and tissue oxygen delivery. Common wisdom suggests that static recordings are incompatible with submerged bath methodology because of deficiency in tissue oxygen supply. However, to our knowledge this has not been tested. In this study, we wanted to determine whether neocortical mouse slice viability could be maintained in the medium term (up to 2h) in a shallow, submerged recording bath under non-perfused, static conditions. Seizure-like events (SLEs) were generated in the slices utilizing no-magnesium ACSF and recorded for 2h under three conditions: (1) perfused ACSF condition (n=8), where slices were perfused continuously with carbogenated no-magnesium ACSF; (2) static ACSF condition (n=12), where slices were recorded in pre-carbogenated, but non-perfused (static) no-magnesium ACSF; and (3) static HEPES ACSF condition (n=12), where slices were recorded in non-perfused (static) no-magnesium ACSF with no pre-carbogenation but buffered with HEPES. SLE activity was stable for 2h across all three conditions. There was no statistically significant difference in SLE frequency, amplitude or length between static and perfused conditions. SLE frequency and amplitude were generally lower in the static HEPES buffer condition. The data indicate that robust and stable neocortical SLE activity can be generated for at least 2h in a submersion bath without ACSF perfusion if pH is adequately controlled.

Lactate Fuels the Neonatal Brain

Lactate Fuels the Neonatal Brain

Role of adenosine A 1 receptor in the perifornical–lateral hypothalamic area in sleep–wake regulation in rats

The perifornical–lateral hypothalamic area (PF-LHA) has been implicated in the regulation of arousal. The PF-LHA contains wake-active neurons that are quiescent during non-REM sleep and in the case of neurons expressing the peptide hypocretin (HCRT), quiescent during both non-REM and REM sleep. Adenosine is an endogenous sleep factor and recent evidence suggests that adenosine via A 1 receptors may act on PF-LHA neurons to promote sleep. We examined the effects of bilateral activation as well as blockade of A 1 receptors in the PF-LHA on sleep–wakefulness in freely behaving rats. The sleep–wake profiles of male Wistar rats were recorded during reverse microdialysis perfusion of artificial cerebrospinal fluid (aCSF) and two doses of adenosine A 1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX; 5 μM and 50 μM) or A 1 receptor agonist, N 6-cyclopentyladenosine (CPA; 5 μM and 50 μM) into the PF-LHA for 2 h followed by 4 h of aCSF perfusion. CPDX perfused into the PF-LHA during lights-on phase produced arousal ( F = 7.035, p < 0.001) and concomitantly decreased both non-REM ( F = 7.295, p < 0.001) and REM sleep ( F = 3.456, p < 0.004). In contrast, CPA perfused into the PF-LHA during lights-off phase significantly suppressed arousal ( F = 7.891, p < 0.001) and increased non-REM ( F = 8.18, p <0.001) and REM sleep ( F = 30.036, p < 0.001). These results suggest that PF-LHA is one of the sites where adenosine, acting via A 1 receptors, inhibits PF-LHA neurons to promote sleep.

Effect of microdialysis perfusion of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol in the perifornical hypothalamus on sleep–wakefulness: Role of δ-subunit containing extrasynaptic GABA A receptors

Gaboxadol or 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP) is a selective agonist for the δ-subunit containing extrasynaptic GABA A receptors that will soon enter the U.S. market as a sleep aid [Winsky-Sommerer R, Vyazovskiy VV, Homanics GE, Tobler I (2007) The EEG effects of THIP (gaboxadol) on sleep and waking are mediated by the GABA(A)delta-subunit-containing receptors. Eur J Neurosci 25:1893–1899]. Numerous studies have shown that systemic administration of THIP reduces wakefulness and increases sleep both in humans and rats [ Lancel M, Langebartels A (2000) Gamma-aminobutyric acid(A) (GABA(A)) agonist 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol persistently increases sleep maintenance and intensity during chronic administration to rats. J Pharmacol Exp Ther 293:1084–1090; Walsh JK, Deacon S, Dijk DJ, Lundahl J (2007) The selective extrasynaptic GABAA agonist, gaboxadol, improves traditional hypnotic efficacy measures and enhances slow wave activity in a model of transient insomnia. Sleep 30:593–602]. However, it is yet unclear where in the brain THIP acts to promote sleep. Since the perifornical lateral hypothalamus (PFH) contains orexin neurons and orexin neurons are critical for maintenance of arousal [McCarley RW (2007) Neurobiology of rapid eye movement (REM) and NREM sleep. Sleep Med 8:302–330], we hypothesized that THIP may act on PFH neurons to promote sleep. To test our hypothesis, we used reverse microdialysis to perfuse THIP unilaterally into the PFH and studied its effects on sleep–wakefulness during the light period in freely behaving rats. Microdialysis perfusion of THIP (100 μM) into the PFH produced a significant reduction in wakefulness with a concomitant increase in non-rapid eye movement or slow wave sleep as compared with artificial cerebrospinal fluid perfusion. REM sleep was unaffected. This is the first study implicating the δ-subunit containing extrasynaptic GABA A receptors in PFH in control of sleep–wakefulness in freely behaving rats.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Genioglossus muscle activity and serotonergic modulation of hypoglossal motor output in obese Zucker rats

Obese Zucker rats have a narrower and more collapsible upper airway compared with lean controls, similar to obstructive sleep apnea (OSA) patients. Genioglossus (GG) muscle activity is augmented in awake OSA patients to compensate for airway narrowing, but the neural control of GG activity in obese Zucker rats has not been investigated to determine whether such neuromuscular compensation also occurs. This study tests the hypotheses that GG activity is augmented in obese Zucker rats compared with lean controls and that endogenous 5-hydroxytryptamine (5-HT) contributes to GG activation. Seven obese and seven lean Zucker rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and they were implanted with GG and diaphragm wires for respiratory muscle recordings. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid and the 5-HT receptor antagonist mianserin (100 microM). Compared with lean controls, respiratory rates were increased in obese rats across sleep-wake states (P=0.048) because of reduced expiratory durations (P=0.007); diaphragm activation was similar between lean and obese animals (P=0.632). Respiratory-related, tonic, and peak GG activities were also similar between obese and lean rats (P>0.139). There was no reduction in GG activity with mianserin at the hypoglossal motor nucleus, consistent with recent observations of a minimal contribution of endogenous 5-HT to GG activity. These results suggest that despite the upper airway narrowing in obese Zucker rats, these animals have a sufficiently stable airway such that pharyngeal muscle activity is normal across sleep-wake states.

Role of Endogenous Serotonin in Modulating Genioglossus Muscle Activity in Awake and Sleeping Rats

Exogenous serotonin at the hypoglossal motor nucleus (HMN) stimulates genioglossus (GG) muscle activity. However, whether endogenous serotonin contributes to GG activation across natural sleep-wake states has not been determined, but is relevant given that serotonergic neurons have decreased activity in sleep and project to pharyngeal motoneurons. To determine the role of endogenous serotonin at the HMN in modulating GG activity across natural sleep-wake states. Ten rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm wires for respiratory muscle recordings. Microdialysis probes were implanted into the HMN for perfusion of artificial cerebrospinal fluid and the serotonin receptor antagonist mianserin (100 microM). In room air, there was no effect of mianserin on respiratory-related or tonic GG activities across sleep-wake states (p > 0.300). In hypercapnia, however, the normal declines in GG activity from non-REM to REM sleep, and wakefulness to REM sleep, were reduced with mianserin (p < 0.005). These data demonstrate a normally low endogenous serotonergic drive modulating GG activity unless augmented by reflex inputs. We also demonstrated a significant serotonergic drive modulating GG activity in vagotomized rats, but not in vagi-intact rats, under anesthesia, suggesting that previous results in reduced preparations may have been influenced by vagotomy. The results show a minimal endogenous serotonergic drive at the HMN modulating GG activity across sleep-wake states, unless augmented by reflex inputs. This result has implications for pharmacologic strategies aiming to increase GG activity by manipulating endogenous serotonin in patients with obstructive sleep apnea.

Wakefulness-inducing effects of histamine in the basal forebrain of freely moving rats

Histamine-containing neurons of the tuberomammillary nucleus (TMN) are implicated in facilitating wakefulness. They project to many brain areas, including the cholinergic basal forebrain (BF). The cholinergic magnocellular regions of the BF are important in the regulation of cortical arousal and wakefulness, and a role for histamine in this activity is suggested by in vitro data indicating histamine excites BF cholinergic and non-cholinergic neurons. To test the hypothesis that histamine induces wakefulness via actions in the BF, we performed microdialysis perfusion of different concentrations of histamine (100, 500 and 1000 μM) in the BF of Sprague-Dawley rats. A MANOVA analysis showed that histamine produced a highly statistically significant and dose-dependent increase in wakefulness and decrease in non-rapid eye movement (NREM) sleep compared with artificial cerebrospinal fluid perfusion. From a wakefulness baseline percentage time of about 12% with artificial cerebrospinal fluid, histamine perfusion increased this value to 26% (100 μM), 36% (500 μM), or 47% (1000 μM). There was no statistically significant change in rapid eye movement (REM) sleep. Histamine perfusion (500 μM) in a control site, the centromedian thalamic nucleus, did not produce any change in behavioral state. The results indicate a prominent role of histamine in wakefulness regulation via the BF and further support the hypothesis that the BF has an important role in EEG activation and wakefulness.

Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats.

The hypoglossal motor nucleus innervates the genioglossus (GG) muscle of the tongue, a muscle that helps maintain an open airway for effective breathing. Rapid-eye-movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity even during strong reflex stimulation such as by hypercapnia, effects that can predispose to sleep-related breathing problems in humans. We have developed an animal model to chronically manipulate neurotransmission at the hypoglossal motor nucleus using in vivo microdialysis in freely behaving rats. This study tests the hypothesis that glycine receptor antagonism at the hypoglossal motor nucleus, either alone or in combination with GABAA receptor antagonism, will prevent suppression of GG activity in natural REM sleep during room air and CO2-stimulated breathing. Rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) and strychnine (glycine receptor antagonist, 0.1 mM) either alone or combined with bicuculline (GABAA antagonist, 0.1 mM) during room air and CO2-stimulated breathing. Compared to ACSF controls, glycine receptor antagonism at the hypoglossal motor nucleus increased respiratory-related GG activity in room air (P = 0.010) but not hypercapnia (P = 0.221). This stimulating effect of strychnine in room air did not depend on the prevailing sleep-wake state (P = 0.625) indicating removal of a non-specific background inhibitory glycinergic tone. Nevertheless, GG activity remained minimal in those REM sleep periods without phasic twitches in GG muscle, with GG suppression from non-REM (NREM) sleep being > 85 % whether ACSF or strychnine was at the hypoglossal motor nucleus or the inspired gas was room air or 7 % CO2. While GG activity was minimal in these REM sleep periods, there was a small but measurable increase in GG activity after strychnine (P < 0.05). GG activity was also minimal, and effectively abolished, in the REM sleep periods without GG twitches with combined glycine and GABAA receptor antagonism at the hypoglossal motor nucleus. We conclude that these data in freely behaving rats confirm that inhibitory glycine and GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but that such inhibitory mechanisms make only a small contribution to the marked suppression of GG activity and reflex responses observed in periods of natural REM sleep.

Acamprosate blocks the increase in dopamine extracellular levels in nucleus accumbens evoked by chemical stimulation of the ventral hippocampus.

Recently, we have shown that acamprosate is able to modulate extracellular dopamine (DA) levels in the nucleus accumbens (NAc) and may act as an antagonist of N-methyl-D-aspartate (NMDA) receptors. Neurochemical studies show that chemical stimulation (using NMDA) of the ventral subiculum (vSub) of the hippocampus produces robust and sustained increases in extracellular DA levels in the NAc, an effect mediated through ionotropic glutamate (iGlu) receptors. The present study examines whether acamprosate locally infused in the NAc of rats could block or attenuate the increase in NAc extracellular DA elicited by chemical stimulation (with 5 mM NMDA) of the ventral subiculum of the hippocampus. The stimulation of the vSub during perfusion of artificial cerebrospinal fluid in NAc induced a significant and persistent increase in NAc DA levels. Reverse dialysis of 0.05 mM acamprosate in NAc blocked the increase in DA evoked by the chemical stimulation of the vSub. These data support the possibility that the antagonism at the NMDA receptors in NAc can explain, at least in part, the mechanism of action of this drug.

GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep.

The pharyngeal muscles, such as the genioglossus (GG) muscle of the tongue, are important for effective lung ventilation since they maintain an open airspace. Rapid-eye-movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity, even during strong reflex respiratory stimulation by elevated CO2. In vitro studies have demonstrated the presence of GABAA receptors on hypoglossal motoneurons, and these and other data have led to the speculation that GABAA mechanisms may contribute to the suppression of hypoglossal motor outflow to the GG muscle in REM sleep. We have developed an animal model that allows us to chronically manipulate neurotransmission at the hypoglossal motor nucleus using microdialysis across natural sleep-wake states in rats. The present study tests the hypothesis that microdialysis perfusion of the GABAA receptor antagonist bicuculline into the hypoglossal motor nucleus will prevent the suppression of GG muscle activity in REM sleep during both room-air and CO2-stimulated breathing. Ten rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) or 100 microM bicuculline during room-air and CO2-stimulated breathing (7 % inspired CO2). GABAA receptor antagonism at the hypoglossal motor nucleus increased respiratory-related GG activity during both room-air (P = 0.01) and CO2-stimulated breathing (P = 0.007), indicating a background inhibitory GABA tone. However, the effects of bicuculline on GG activity depended on the prevailing sleep-wake state (P < 0.005), with bicuculline increasing GG activity in non-REM (NREM) sleep and wakefulness both in room air and hypercapnia (P < 0.01), but GG activity was effectively abolished in those REM periods without phasic twitches in the GG muscle. This abolition of GG activity in REM sleep occurred regardless of ACSF or bicuculline at the hypoglossal motor nucleus, or room-air or CO2-stimulated breathing (P > 0.63). We conclude that these data in freely behaving rats confirm previous in vitro studies that GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but the data also show that GABAA receptor antagonism at the hypoglossal motor nucleus does not increase GG muscle activity in natural REM sleep.

Fos expression is induced by increased nitric oxide release in rat spinal cord dorsal horn

The relationship between exogenous or endogenous nitric oxide and c-fos, an immediate-early gene which can further activate the production of other substances in the central nervous system, was investigated in this study. We found that Fos expression is increased after intradermal capsaicin injection, which also leads to endogenous nitric oxide release in the spinal cord. The increased Fos expression is distributed in neurons of the superficial layers and lamina V of the dorsal horn on the side ipsilateral to the injection. The increased Fos expression is blocked by N G-nitro- l-arginine methyl ester, a nitric oxide synthase inhibitor, but not by its inactive isomer N G-nitro- d-arginine methyl ester. Fos expression was also increased following the perfusion of 3-morpholino-sydnonimine, a nitric oxide donor, into the dorsal horn through a microdialysis fiber. The increased Fos was distributed within 400 μm from the edge of the microdialysis fiber. Although Fos expression was increased with 3-morpholino-sydnonimine perfusion compared to that seen with artificial cerebrospinal fluid perfusion, there was still some Fos immunostaining in the control sections. Following perfusion of artificial cerebrospinal fluid in the spinal cord of rats pretreated with N G-nitro- l-arginine methyl ester, it was found that Fos staining was reduced significantly compared to the control sections from animals without N G-nitro- l-arginine methyl ester pretreatment. These results suggest that nitric oxide helps mediate Fos expression induced by an intradermal capsaicin injection. We conclude that both endogenous and exogenous nitric oxide induce Fos expression. Involvement of nitric oxide in the development of central sensitization may affect nociceptive processing by increasing Fos expression. Since many other substances which are related to pain mechanisms can be induced by Fos, it is suggested that nitric oxide may regulate production of these substances through activation of Fos. Nitric oxide is not only involved in the development of central sensitization, but is also involved in the activation of control mechanisms affecting nociception.

Nicotine stimulation of extracellular glutamate levels in the nucleus accumbens: neuropharmacological characterization.

In the present study, we have characterized the neuropharmacological regulation of nicotine-induced increases in extracellular nucleus accumbens glutamate levels. Sprague-Dawley rats were stereotaxically implanted with 2 mm microdialysis probes in the nucleus accumbens and on the following day in vivo microdialysis experiments were performed in awake, freely moving animals. An acute dose of nicotine (0.3-0.6 mg/kg, s.c.) produced an increase in nucleus accumbens glutamate levels with a maximal increase of approximately 50% following the higher dose. No changes in nucleus accumbens aspartate levels were found. The increase in glutamate levels following nicotine (0.3 mg/kg, s.c.) was blocked by mecamylamine (1 mg/kg, i.p. ) but not by haloperidol (0.2 mg/kg, i.p.) pretreatment. Local perfusion of artificial cerebrospinal fluid (CSF) without calcium did not alter nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. Local perfusion with a selective blocker for the GLT-1 glutamate transporter, dihydrokainic acid (DHKA) (10(-4) M), had no effect, while local perfusion with a nonselective glutamate transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (10(-4) M), blocked nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. In animals previously dopamine denervated by local injections of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens, nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels was enhanced vs. sham-lesioned animals. These findings demonstrate a novel form of nucleus accumbens glutamate release that is dopamine- and calcium-independent. The ability of PDC to block the effects of nicotine suggest that a glutamate transporter may be involved in mediating the stimulation of glutamate release.

Adenosinergic modulation of rat basal forebrain neurons during sleep and waking: neuronal recording with microdialysis.

1. The cholinergic system of the basal forebrain (BF) is hypothesized to play an important role in behavioural and electrocortical arousal. Adenosine has been proposed as a sleep-promoting substance that induces sleep by inhibiting cholinergic neurons of the BF and brainstem. However, adenosinergic influences on the activity of BF neurons in naturally awake and sleeping animals have not been demonstrated. 2. We recorded the sleep-wake discharge profile of BF neurons and simultaneously assessed adenosinergic influences on wake- and sleep-related activity of these neurons by delivering adenosinergic agents adjacent to the recorded neurons with a microdialysis probe. Discharge rates of BF neurons were recorded through two to three sleep-wake episodes during baseline (artificial cerebrospinal fluid perfusion), and after delivering an adenosine transport inhibitor (s-(p-nitrobenzyl)-6-thioinosine; NBTI), or exogenous adenosine, or a selective adenosine A1 receptor antagonist (8-cyclopentyl-1, 3-dimethylxanthine; CPDX). 3. NBTI and adenosine decreased the discharge rate of BF neurons during both waking and non-rapid eye movement (NREM) sleep. In contrast, CPDX increased the discharge rate of BF neurons during both waking and NREM sleep. These results suggest that in naturally awake and sleeping animals, adenosine exerts tonic inhibitory influences on BF neurons, supporting the hypothesized role of adenosine in sleep regulation. 4. However, in the presence of exogenous adenosine, NBTI or CPDX, BF neurons retained their wake- and sleep-related discharge patterns, i.e. still exhibited changes in discharge rate during transitions between waking and NREM sleep. This suggests that other neurotransmitters/neuromodulators also contribute to the sleep-wake discharge modulation of BF neurons.

Effects of anion channel blockers on hyposmotically induced amino acid release from the in vivo rat cerebral cortex.

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM); 4-acetamido-4'-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 microM); niflumic acid (500 microM); tamoxifen (20 microM) and arachidonic acid (0.5 microM) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.

Sleep/waking effects of a selective 5-HT 1A receptor agonist given systemically as well as perfused in the dorsal raphe nucleus in rats

Sleep/waking stages and behavior were studied following the selective 5-HT 1A agonist 8-OH-DPAT given subcutaneously (s.c.) (0.010–0.375 mg/kg) as well as perfused continuously (10 μM) for 6 h into the dorsal raphe nucleus (DRN) using microdialysis. Given systemically, 8-OH-DPAT at 0.375 mg/kg s.c. induced 5-HT behavioral syndrome, increased waking to 149% and reduced slow wave sleep (SWS) to 86%, transition to 76% and rapid eye movement (REM) sleep to 73%. The effect on deep SWS (SWS-2) was biphasic, with an increase after 2 h. 8-OH-DPAT at 0.010 mg/kg did not have any vigilance effects. 8-OH-DPAT perfusion in DRN produced a fourfold increase in REM sleep compared to perfusion of artificial cerebrospinal fluid. This is consistent with the hypothesis that reduced 5-HT neurotransmission following 5-HT 1A autoreceptor stimulation will disinhibit cholinergic REM-promoting mesopontine neurons and thereby lead to a REM sleep increase. The other sleep/waking stages were not significantly affected by 8-OH-DPAT perfusion in DRN.

Effects of probenecid on the elicitation of spreading depression in the rat striatum

Spreading depression (SD) is a wave of cellular depolarization which contributes to neuronal damage in experimental focal ischaemia, and may also underlie the migraine aura. The purpose of this study was to examine the effects of probenecid, an inhibitor of organic anion transport, on K +-evoked SD in vivo. Microdialysis electrodes were implanted in the rat striatum, and recurrent SD elicited by perfusion of artificial cerebrospinal fluid containing 160 mM K + for 20 min. Probenecid was administered either directly through the microdialysis probe, starting 50 min before application of high K +, or intravenously. SD was markedly reduced by perfusion of 5 mM probenecid through the microdialysis probe. In contrast, a high intravenous dose of probenecid (250 mg/kg) only slightly inhibited SD elicitation 90 min after treatment, despite clear changes in the amplitude and spectrum of the electroencephalogram, as early as 10 min after drug administration, confirming that probenecid readily penetrated the central nervous system. As SD is inhibited by hypercapnia, we have examined the possibility that probenecid may inhibit SD through extracellular acidification subsequent to blockade of lactate transport. Perfusion of 1–20 mM probenecid increased dose-dependently the dialysate levels of lactate, but without extracellular acidosis since the dialysate pH was not significantly reduced. How probenecid inhibits SD deserves further investigation because it may help identify novel strategies to suppress this phenomenon, now recognized deleterious to neuronal function and survival.