Dialysis Probe Research Articles

L-DOPA induced extracellular dopamine increases in the ventromedial hypothalamus affects food intake by chickens on a lysine-free diet

Previous work from our laboratory suggests that ventromedial hypothalamic (VMH) dopamine levels were associated with decreased in food intake by chicken on a lysine-free diet. Dopamine in the VMH started to decrease from its baseline after presenting a lysine-free diet and subsequently food intake decreased. In the present study, the dopamine levels were manipulated by perfusing L-3-4-dihydroxyphenylalanine (L-DOPA) into the VMH of chicken using the in vivo microdialysis technique and food intake was concomitantly measured when chickens received an experimental lysine-free diet. A microdialysis probe was implanted into the VMH. L-DOPA was then administered locally at 2 μg/ml through the dialysis probe into the VMH of free moving chicken for 15 min and the extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) were measured. Hourly food intake was also measured simultaneously both for control and experimental groups. Microdialysates collected from the VMH were analyzed using high performance liquid chromatography with electrochemical detection. Local administration of L-DOPA in chicken VMH increased extracellular levels of DA, which was observed at 1–2.5 h. There were no differences of NE and 5-HT levels from baseline in either group. Food intake was higher in L-DOPA treated chickens than controls at 2 h. Chickens received the lysine-free diet ate as much of their diet as the controls in the subsequent 2 h when the DA level was kept higher than the baseline. The findings suggest that L-DOPA induced extracellular DA increased in the VMH which was temporarily followed by the restoration of food intake in the lysine-free group.

Central nervous system sites of the sleep promoting effects of eszopiclone in rats

We examined the effects of eszopiclone (ESZ), a GABA-A receptor agonist in current clinical use as a hypnotic medication, on the activity of subcortical wake- and sleep-active neuronal populations in the rat brain. Sleep-wake states were quantified after i.p. injections of ESZ (3 and 10 mg/kg) or vehicle administered early in the dark phase, when rats are spontaneously awake. Rats were euthanized 2 h post-injection and brain tissue was processed for c-Fos protein immunoreactivity (IR) and for neurotransmitter markers. ESZ at 3 and 10 mg/kg increased time spent in non-rapid-eye-movement (nonREM) sleep, but had no significant effect on Fos-IR in GABAergic neurons in the preoptic hypothalamus that normally express c-Fos during sleep. Among wake-active cell types examined, Fos-IR in hypocretin (HCRT) neurons in the perifornical lateral hypothalamus (LH) was reduced following 3 and 10 mg/kg ESZ. At 10 mg/kg, ESZ suppressed Fos-IR in cholinergic and noncholinergic neurons in the basal forebrain and in serotonergic and nonserotonegic neurons in the dorsal raphe. Having determined that HCRT neurons were responsive to the low dose of systemic ESZ, we unilaterally perfused ESZ directly into the LH of awake rats, using reverse microdialysis. Perfusion of ESZ at 50 μM into the LH for 2 h suppressed waking-related Fos-IR in HCRT neurons, but not in nonHCRT neurons ipsilateral to the dialysis probe. Bilateral LH perfusion of ESZ at 50 μM for 2 h early in the dark phase significantly increased sleep. These findings demonstrate that sleep induction by ESZ does not require activation of GABAergic sleep-regulatory neurons in the preoptic hypothalamus, and identify suppression of HCRT neurons in the LH and suppression of basal forebrain and dorsal raphe neurons as potential mechanisms underlying the sleep-promoting effects of ESZ.

A linear relationship between lamotrigine and GABA in cerebrospinal fluid

The J-amino butyric acid (GABA)-mediated neurotransmission is useful in treat- ing conditions like anxiety, sleep disturbances, depression and bipolar disorders. The aim of the present study is to supply evidence about neurochemical effects of acute lamotrigine treatment on GABA and L-glutamic acid levels in the cerebrospinal fluid of Wistar Albino rats and the involvement of cholinergic system. The day after the placement of concentric micro- dialysis probes into the lateral ventricles of rats, microdialysis experiments were performed in conscious rat model. The rats either received intraperitoneal injections of physiological saline or 20 mg/kg lamotrigine. For assessing the cholinergic involvement 0.5 mg/kg phys- ostigmine or 2 mg/kg atropine sulfate pre-treatments were given prior to lamotrigine injec- tion. GABA, L-glutamic acid and lamotrigine concentrations in the dialysates were analyzed using High Performance Liquid Chromatography. Saline produced no change in GABA or L-glutamic acid levels, but lamotrigine treatment significantly elevated GABA concentrations (p<0.05). Pre-treatment with physostigmine or atropine sulfate did not affect either GABA or L-glutamic acid levels siginificantly. Physostigmine or atropine sulfate pre-treatments did not affect the lamotrigine-induced GABA levels. The results may imply that lamotrigine-in- duced GABA plays a role in the pharmacological effects of lamotrigine where a linear rela- tionship exists between lamotrigine and GABA. However, central cholinergic system fails to affect lamotrigine-induced GABA release.

Collection of nanoliter microdialysate fractions in plugs for off-line in vivo chemical monitoring with up to 2 s temporal resolution

An off-line in vivo neurochemical monitoring approach was developed based on collecting nanoliter microdialysate fractions as an array of “plugs” segmented by immiscible oil in a piece of Teflon tubing. The dialysis probe was integrated with the plug generator in a polydimethlysiloxane microfluidic device that could be mounted on the subject. The microfluidic device also allowed derivatization reagents to be added to the plugs for fluorescence detection of analytes. Using the device, 2nL fractions corresponding to 1–20ms sampling times depending upon dialysis flow rate, were collected. Because axial dispersion was prevented between them, each plug acted as a discrete sample collection vial and temporal resolution was not lost by mixing or diffusion during transport. In vitro tests of the system revealed that the temporal resolution of the system was as good as 2s and was limited by mass transport effects within the dialysis probe. After collection of dialysate fractions, they were pumped into a glass microfluidic chip that automatically analyzed the plugs by capillary electrophoresis with laser-induced fluorescence at 50s intervals. By using a relatively low flow rate during transfer to the chip, the temporal resolution of the samples could be preserved despite the relatively slow analysis time. The system was used to detect rapid dynamics in neuroactive amino acids evoked by microinjecting the glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) or K+ into the striatum of anesthetized rats. The resulted showed increases in neurotransmitter efflux that reached a peak in 20s for PDC and 13s for K+.

Role of Ca 2+-activated K + channels in catecholamine release from in vivo rat adrenal medulla

To elucidate the role of Ca 2+-activated K + (K Ca) channels in the presynaptic acetylcholine (ACh) release from splanchnic nerve endings and the postsynaptic catecholamine release from chromaffin cells, we applied microdialysis technique to the left adrenal medulla of anesthetized rats and investigated the effects of local administration of K Ca channel antagonists through dialysis probes on the release of ACh and/or catecholamine, induced by electrical stimulation of splanchnic nerves or local administration of ACh through the dialysis probes. Nerve stimulation-induced release: in the presence of a cholinesterase inhibitor, neostigmine, large-conductance K Ca (BK) channel antagonists, iberiotoxin and paxilline enhanced the presynaptic ACh release and postsynaptic norepinephrine (NE) and epinephrine (Epi) release. Small-conductance K Ca (SK) channel antagonists, apamin and scyllatoxin enhanced the Epi release without any changes in ACh or NE release. In the absence of neostigmine, ACh release was not detected. Iberiotoxin and paxilline enhanced NE and Epi release. Apamin and scyllatoxin had no effect on NE or Epi release. Exogenous ACh-induced release: iberiotoxin and paxilline enhanced the Epi release, but had no effect on the NE release. Apamin and scyllatoxin enhanced both NE and Epi release. In conclusion, BK channels on splanchnic nerve endings play an inhibitory role in the physiological catecholamine release from adrenal medulla by limiting presynaptic ACh release while SK channels do not. BK channels on Epi-storing cells may play an inhibitory role in nerve stimulation-induced Epi release. SK channels on NE- and Epi-storing cells play a minor role in nerve stimulation-induced catecholamine release.

Microfluidic chip for high efficiency electrophoretic analysis of segmented flow from a microdialysis probe and in vivo chemical monitoring.

An effective method for in vivo chemical monitoring is to couple sampling probes, such as microdialysis, to online analytical methods. A limitation of this approach is that in vivo chemical dynamics may be distorted by flow and diffusion broadening during transfer from sampling probe to analytical system. Converting a homogeneous sample stream to segmented flow can prevent such broadening. We have developed a system for coupling segmented microdialysis flow with chip-based electrophoresis. In this system, the dialysis probe is integrated with a PDMS chip that merges dialysate with fluorogenic reagent and segments the flow into 8-10 nL plugs at 0.3-0.5 Hz separated by perfluorodecalin. The plugs flow to a glass chip where they are extracted to an aqueous stream and analyzed by electrophoresis with fluorescence detection. The novel extraction system connects the segmented flow to an electrophoresis sampling channel by a shallow and hydrophilic extraction bridge that removes the entire aqueous droplet from the oil stream. With this approach, temporal resolution was 35 s and independent of distance between sampling and analysis. Electrophoretic analysis produced separation with 223,000 +/- 21,000 theoretical plates, 4.4% RSD in peak height, and detection limits of 90-180 nM for six amino acids. This performance was made possible by three key elements: (1) reliable transfer of plug flow to a glass chip; (2) efficient extraction of aqueous plugs from segmented flow; (3) electrophoretic injection suitable for high efficiency separation with minimal dilution of sample. The system was used to detect rapid concentration changes evoked by infusing glutamate uptake inhibitor into the striatum of anesthetized rats. These results demonstrate the potential of incorporating segmented flow into separations-based sensing schemes for studying chemical dynamics in vivo with improved temporal resolution.

In vivo direct monitoring of interstitial norepinephrine levels at the sinoatrial node

We assessed in vivo interstitial norepinephrine (NE) levels at the sinoatrial node in rabbits, using microdialysis technique. A dialysis probe was implanted adjacent to the sinoatrial node of an anesthetized rabbit and dialysate was sampled during sympathetic nerve stimulation. Atrial dialysate NE concentration correlated well with heart rate. Desipramine significantly increased dialysate NE concentrations both before and during sympathetic nerve stimulation compared with the absence of desipramine. However, desipramine did not affect the relation between heart rate and dialysate NE concentration. These results suggest that atrial dialysate NE level reflects the relative change of NE concentration in the synaptic cleft. Microdialysis is a powerful tool to assess in vivo interstitial NE levels at the sinoatrial node.

Current separation and detection methods in microdialysis the drive towards sensitivity and speed

This review outlines some of the analytical challenges associated with the analysis of microdialysis (MD) samples, in particular, the minute complex sample volumes that are often encountered. In MD sampling many different low-molecular-weight molecules can be collected, but the research findings are often limited by the sensitivity, specificity, and reliability of the analytical technique that is coupled to the dialysis probe. Therefore it is critical that a lot of consideration is given in selecting the most suitable analytical method including the most appropriate detector. This review aims to highlight the strengths and weaknesses of a range of commonly used analytical methods employed in MD. In Section 1, a brief overview of the MD technique is described, followed by a discussion on some of the advantages and drawbacks of this sampling technique. Sections 2 and 3 examine analytical and other technical considerations regarding analysis, with special emphasis on the factors that specifically influence analytical detection. Section 4 outlines the most commonly employed analytical techniques used in MD, including HPLC coupled with various detectors. Detail is given regarding the LOD and LOQ for many applications using each detector. As MS is of such high importance in MD, a special sub-section has been devoted to it. The importance of CE is also highlighted, with specific applications described. In addition, analytical techniques that do not appear to have found routine use in MD are discussed. Section 5 is concerned with recent innovations in chemical separation techniques, in particular MCE and ultra-performance liquid chromatography. Specific applications of the coupling of these techniques with MD are highlighted, along with technical challenges associated with miniaturization. In the Section 6, the future outlook of MD is discussed. Techniques other than electrophoretic- and chromatographic based separation methods are outside the scope of this review.

Early postnatal stress affects 5-HT 1A receptor function in the medial prefrontal cortex in adult rats

Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT) 1A receptors are known to play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. The goal of the present study was to investigate whether early postnatal stress influences 5-HT 1A receptor function in the medial prefrontal cortex in adult rats. Rats were subjected to aversive foot shock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10–14 weeks postnatal), immunohistochemical experiments were carried out to investigate c-Fos expression following the administration of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT 1A receptor agonist. In the 3wFS group, the 8-OH-DPAT-induced c-Fos expression in the medial prefrontal cortex was significantly attenuated compared to that in the non-FS control group. A dual immunofluorescence study revealed that a small proportion of c-Fos positive cells co-express parvalbumin, and a relatively large proportion of c-Fos positive cells co-express glutaminase, suggesting that most c-Fos positive cells are glutamatergic neurons. We found that local perfusion of 8-OH-DPAT via a dialysis probe decreased extracellular 5-HT levels in the medial prefrontal cortex of the non-FS group, but not in the 3wFS group. However, the levels of 8-OH-DPAT-induced 5-HT syndrome were not significantly different between the non-FS and 3wFS groups. Therefore, aversive stress in the third week of the postnatal period attenuates 5-HT 1A receptor function in the medial prefrontal cortex in adulthood and produces feedback inhibition of the raphe nuclei via postsynaptic 5-HT 1A receptors.

A cancer research UK pharmacokinetic study of BPA-mannitol in patients with high grade glioma to optimise uptake parameters for clinical trials of BNCT

This paper describes results to-date from a human pharmacokinetic study which began recruitment in December 2007. Results are presented for a single patient recruited in December 2007. A second patient was recruited in July 2008 but detailed data are not available at the time of writing. The trial is an open-label, non-comparative, non-therapeutic study of BPA-mannitol in patients with high-grade glioma, who will be undergoing stereotactic brain biopsy as part of the diagnostic process before definitive treatment. The study investigates the route of infusion (intra-venous (IV) or intra-carotid artery) and in each case will assess the effect of administration of mannitol as a blood–brain barrier disrupter. All cohorts will receive a 2 h infusion of BPA-mannitol, and for some cohorts an additional mannitol bolus will be administered at the beginning of this infusion. Measurements are made by inductively coupled plasma mass spectrometry (ICP-MS) of 10B concentration in samples of blood, urine, extra-cellular fluid in normal brain (via a dialysis probe), brain tissue around tumour and tumour tissue. Additional analysis of the tumour tissue is performed using secondary ion mass spectrometry (SIMS). The first patient was part of the cohort having intra-venous infusion without mannitol bolus. No serious clinical problems were experienced and the assay results can be compared with available patient data from other BNCT centres. In particular we note that the peak 10B concentration in blood was 28.1 mg/ml for a total BPA administration of 350 mg/kg which is very consistent with the previous experience with BPA-fructose reported by the Helsinki group.

Single administration of 1-benzyl-1,2,3,4-tetrahydroisoquinoline increases the extracellular concentration of dopamine in rat striatum

We performed a combined neurochemical and behavioral study to determine the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) on the extracellular dopamine concentrations in the striatum. Single dose administration of 1-BnTIQ (20, 40, and 80 mg/kg i.p.) increased striatal dopamine extracellular levels in a dose-dependent manner when an in vivo microdialysis technique was used to assess dopamine levels in the striatum of rats. Enhancement of striatal dopamine levels by systemic administration of a single dose of 1-BnTIQ was suppressed by perfusion of tetrodotoxin and a calcium ion-free solution into the striatum. This 1-BnTIQ-induced increase in extracellular dopamine concentration was also inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (1-(2-[bis(4-Fluorophenyl)-4-(3-phenylpropyl)piperazine dihydrochloride). Local application of 1-BnTIQ into the striatum via a dialysis probe failed to enhance the extracellular concentration of dopamine. However, microinjection of 1-BnTIQ into the substantia nigra pars compacta increased the extracellular dopamine levels in the striatum. Locomotor activity was increased by systemic administration of a single dose of 1-BnTIQ in a dose-dependent manner. This 1-BnTIQ-induced locomotor activity was attenuated by pre-treatment with SCH23390 (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlodride) and raclopride, D 1 and D 2 dopaminergic receptor antagonists, respectively. Moreover, 1-BnTIQ induced ipsilateral rotational behavior in 6-hydroxydopamine–lesioned rats. These results suggest that systemic administration of a single dose of 1-BnTIQ increases striatal extracellular dopamine concentration through activation of dopaminergic nigra striatal neurons via the dopamine transporter.

Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats

We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 μl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at < 0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 μM, 1.5 μl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 μM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 μM paraoxon but was transiently elevated (0.5–1.5 h) with 10 μM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme–inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.

Local salsolinol modulates dopamine extracellular levels from rat nucleus accumbens: Shell/core differences

Salsolinol (SAL), a condensation product of dopamine and acetaldehyde that appears in the rat and human brain after ethanol ingestion, has been largely implicated in the aetiology of alcoholism. Although the behavioural consequences of systemic or intracerebral SAL administrations have been described, the neurochemical effects of pharmacologically relevant doses of SAL and other tetrahydroisoquinolines (THIQs) in the brain areas involved in alcohol addiction are practically unknown. To gain an insight into this topic, male Wistar rats were stereotaxically implanted with one concentric microdialysis probe in either the shell or the core of the nucleus accumbens (NAc). Treatments involved local administration of 0.1, 5 and 25 μM SAL for 20 min through the dialysis probe. Dopamine (DA) concentrations in the shell or core of the NAc were on-line analyzed every 20 min by HPLC with electrochemical detection. Implantation of the probe was histologically evaluated at the end of the experiments. Our results indicate that dialysis application of 5 and 25 μM SAL into the core increased the dialysate levels of DA. Conversely, the administration of the same doses of this drug into the shell significantly reduced the DA levels in this subregion. In conclusion, these data reveal that local application of SAL affects the DA levels in the NAc subterritories in a region-specific manner. These findings may prove useful in probing CNS sites and mechanisms involved in alcohol consumption.

Reliability of results and interpretation of measures of 3-methylhistidine in muscle interstitium as marker of muscle proteolysis

TO THE EDITOR: We were interested in the contribution by Tesch and colleagues to the evidence base concerning the events subsequent to acute immobilization in human muscle. Tesch and colleagues (14) used a microdialysis technique applied to human muscle in an attempt to measure an index of myofibrillar proteolysis 72 h after immobilization using the unilateral leg suspension technique. The authors discuss their results as though they had good evidence that the technique they used produces results that are sufficiently firm to be the basis of conjecture about mechanisms of protein balance in muscle after immobilization. We demur and propose that they are on very shaky ground. First, we wonder about the size of the effect reported. The authors claim it to be 44%, but calculation of the change shows it to be actually 29% with the SEs sufficiently large that they almost span the difference; SDs must overlap. Significance at the 5% level can only barely have been reached. Are the authors convinced that they have not ignored a type I error? The authors (14) begin their explanation of the advantages of the dialysis technique using 3-methylhistidine (3MeHis) by describing the gold standard method, i.e., the stable isotopelabeled amino acid tracer dilution method, as cumbersome and possibly unjustified in study of otherwise healthy subjects. We find this notion strange since Ferrando and colleagues (6, 12) have used the technique in many studies of how immobilization affects muscle protein turnover subsequent to disuse. We have also used the techniques in a wide variety of studies of proteolysis in healthy and infirm subjects (1, 2, 10, 11, 15) with no problems and, we would propose, producing results with a greater degree of certainty and biological appropriateness than those obtained by the 3MeHis dialysis technique.

Involvement of dopaminergic mechanisms in the nucleus accumbens core and shell subregions in the expression of fear conditioning

The involvement of dopamine (DA) mechanisms in the nucleus accumbens (NAC) in fear conditioning has been proposed by many studies that have challenged the view that the NAC is solely involved in the modulation of appetitive processes. However, the role of the core and shell subregions of the NAC in aversive conditioning remains unclear. The present study examined DA release in these NAC subregions using microdialysis during the expression of fear memory. Guide cannulae were implanted in rats in the NAC core and shell. Five days later, the animals received 10 footshocks (0.6 mA, 1 s duration) in a distinctive cage A (same context). On the next day, dialysis probes were inserted through the guide cannulae into the NAC core and shell subregions, and the animals were behaviorally tested for fear behavior either in the same context (cage A) or in a novel context (cage B). Dialysates were collected every 5 min for 90 min and analyzed by high-performance liquid chromatography. The rats exhibited a significant fear response in cage A but not in cage B. Moreover, increased DA levels in both NAC subregions were observed 5–25 min after the beginning of the test when the animals were tested in the same context compared with accumbal DA levels from rats tested in the different context. These findings suggest that DA mechanisms in both the NAC core and shell may play an important role in the expression of contextual fear memory.

Stimulation by neurotensin of dopamine and 5-hydroxytryptamine (5-HT) release from rat prefrontal cortex: Possible role of NTR1 receptors in neuropsychiatric disorders

The modulation of cortical dopaminergic and serotonergic neurotransmissions by neurotensin (NT) was studied by measuring the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) from the prefrontal cortex (PFC) of freely moving rats. The samples were collected via transversal microdialysis. Dopamine and 5-HT levels in the dialysate were measured using high-performance liquid chromatography (HPLC) with an electrochemical detector. Local administration of neurotensin (1 μM or 0.1 μM) in the PFC via the dialysis probe produced significant, long-lasting, and concentration-dependent increase in the extracellular release of DA and 5-HT. The increase produced by 1 μM neurotensin reached a maximum of about 210% for DA and 340% for 5-HT. A high-affinity selective neurotensin receptor (NTR1) antagonist {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3yl)carbonylamino tricyclo (3.3.1.1. 3.7) decan-2-carboxylic acid} (SR 48692), perfused locally at a concentration of 0.1 μM and 0.5 μM in the PFC antagonized the effects of 1 μM neurotensin. Our in vivo neurochemical results indicate, for the first time, that neurotensin is able to regulate cortical dopaminergic and serotonergic neuronal activity in freely moving rats. These effects are possibly mediated by interactions of neurotensin with neurons releasing DA or 5-HT, projecting to the PFC from the ventrotegmental area (VTA) and from the dorsal raphe nuclei (DRN), respectively. The potentiating effects of neurotensin on DA and 5-HT release in the PFC are regulated by NTR1 receptors, probably located on dopaminergic and serotonergic nerve terminals or axons.

Intranasal administration of progesterone increases dopaminergic activity in amygdala and neostriatum of male rats

We evaluated the effects of intranasal administration of progesterone (PROG) on the activity of dopaminergic neurons in the brain of anesthetized rats by means of microdialysis. Male Wistar rats were implanted with guide cannulae in the basolateral amygdala and neostriatum. Three to 5 days later, they were anesthetized with urethane, and dialysis probes were inserted. After a stabilization period of 2 h, four 30-min samples were collected. Thereafter, the treatment (0.5, 1.0 or 2.0 mg/kg of PROG dissolved in a viscous castor oil mixture, or vehicle) was applied into the nose in a volume of 10 μl (5 μl in each nostril). In other animals, an s.c. injection of PROG (1.0, 2.0 or 4.0 mg/kg) or vehicle was given. Samples of both application ways were collected at 30-min interval for 4 h after the treatment and immediately analyzed with high performance liquid chromatography and electrochemical detection. Intranasal administration of 2 mg/kg of PROG led to an immediate (within 30 min after the treatment) significant increase in the basolateral amygdala dopamine levels. In the neostriatum, the 2 mg/kg dose led to a delayed significant increase in dopamine. S.c. administration of 4 mg/kg of PROG was followed by a delayed significant increase in dopamine, both, in the basolateral amygdala and neostriatum, but smaller in magnitude in comparison to the intranasal treatment. This is the first study to demonstrate dopamine-enhancing effects of PROG, not only in the neostriatum, but also in the basolateral amygdala. Our results indicate that the intranasal route of administration of PROG is a more efficacious way for targeting the brain than the s.c. route.

In vivo effects of phosphodiesterase inhibition on basal cyclic guanosine monophosphate levels in the prefrontal cortex, hippocampus and cerebellum of freely moving rats

We have characterized the various phosphodiesterases (PDE) that degrade cyclic GMP in the prefrontal cortex, hippocampus, and cerebellum using the microdialysis technique to measure in vivo extracellular cyclic GMP in awake rats. The following PDE blockers were used (100 and 1,000 microM): 8-methoxymethyl-IBMX (8-MM-IBMX), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), milrinone, rolipram, and zaprinast. For solubility reasons, sildenafil was tested only at 100 microM. All drugs were administered locally in the brain regions through the dialysis probe. At 100 microM, 8-MM-IBMX enhanced the cyclic nucleotide extracellular levels in the prefrontal cortex and hippocampus but not in the cerebellum; EHNA and milrinone were active only in the hippocampus; rolipram was devoid of any effect; zaprinast and sildenafil were effective in all three brain areas. At 1 mM, 8-MM-IBMX, milrinone, and zaprinast increased extracellular cyclic GMP in all the brain regions examined, EHNA became active also in the prefrontal cortex and rolipram showed a significant effect only in the cerebellum. This is the first in vivo functional study showing that, in cortex, PDE1, -2, and -5/9 degrade cGMP, with PDE9 probably playing a major role; in hippocampus, PDE5/9 and PDE1 are mainly involved and seem almost equally active, but PDE2 and -3 also contribute; in cerebellum, PDE5/9 are the main cGMP hydrolyzing enzymes, but also PDE1 and -4 significantly operate.

ESTIMATION OF TRABECULAR BONE PERMEABILITY USING FLUORESCENT IMAGE OF LACUNO-CANALICULAR SYSTEM

To examine the effects of altered nitric oxide (NO) levels in the paraventricular nucleus (PVN) on copulatory behavior and reflexive erections in male rats.Extracellular nitrite (NO2−) and nitrate (NO3−) levels were measured in the PVN following administration of the NO precursor L-arginine (L-arg, 10 mM), the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA, 10 mM), or Ringer’s solution via a dialysis probe to the PVN. The effects of alterations in extracellular NO on reflexive erections and copulatory behavior were assessed.L-arg administration was associated with significant elevations of extracellular NO2− and NO3− in the PVN, while L-NMMA significantly reduced NO2− and NO3− levels. A corresponding increase in reflexive erections was noted during infusion of L-arg in the PVN, with a corresponding decrease in reflexive erections observed during administration of L-NMMA into the PVN (Student’s t test for paired samples, p <0.05). Mount rate was unaffected by infusion of the either L-arg or L-NMMA.Altered NO levels in the PVN affected the frequency of reflexive erections, but not the mount rate. These studies contrast with previous observations of the effects of altered NO levels in the MPOA, and support the hypothesis that physiological specificity in the actions of NO on discrete brain nuclei may have important implications to erectile physiology and dysfunction.

The CRF-like peptide urocortin produces a long-lasting recovery in rats made hemiparkinsonian by 6-hydroxydopamine or lipopolysaccharide

We have recently observed that the corticotropin releasing factor related peptide urocortin (UCN) reverses key features of nigrostriatal neurodegeneration following intracerebral injection of either 6-hydroxydopamine (6-OHDA) or lipopolysaccharide (LPS). To determine the potential therapeutic utility of UCN here we have studied whether these effects are sustained for several weeks following peptide injection. In addition we have studied whether UCN still shows efficacy in rats with more pronounced nigrostriatal lesions. Rats were lesioned using 6-OHDA or LPS and injected with UCN either 7 or 14 days later. At different time points animals were tested for rotational behaviour (apomorphine, 0.5 mg/kg) and subsequently implanted with bilateral dialysis probes into the striata. The following day rats were dialysed to estimate extracellular striatal dopamine (DA) and then sacrificed for estimation of striatal tissue DA and subsequent immunohistochemistry of TH(+) cells in the substantia nigra (SN). Toxin treated rats given UCN 7 days later showed clear evidence of reduced nigrostriatal damage both 28 and 84 days following UCN compared with saline injection. In rats given UCN 14 days after toxin injection, by which time deficits were maximal, a restoration of nigrostriatal damage was observed. This suggests that UCN is able to elicit a sustained restoration of functional nigrostriatal integrity and has the ability to produce a recovery in severely lesioned rats. These findings suggest that stimulation of CRF (probably CRF(1)) receptors could have therapeutic utility in PD.