In Vivo Monitoring of Catecholaminergic Metabolism in the C1 Region of Rat Medulla Oblongata: A Comparative Study by Voltammetry and Intracerebral Microdialysis

In vivo voltammetry or microdialysis was used to monitor catecholaminergic metabolism in the C1 region of the ventrolateral medulla oblongata of anesthetized rats. In vivo voltammetry allowed the recording of a catechol oxidation current (CA.OC) peak in this region. This CA.OC was suppressed after inhibition of monoamine oxidase by pargyline or after inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine and was markedly increased after blockade of dopamine-beta-hydroxylase by FLA 63. Similar results were found when intracerebral microdialysis coupled with HPLC and electrochemical detection was used to measure the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) in the dialysates obtained from the C1 region: The changes in CA.OC and DOPAC concentration in the dialysates exhibited very similar kinetic characteristics in the three pharmacological experiments. These results support the involvement of DOPAC as a major component of the electrochemical signal recorded by voltammetry in the C1 group of adrenergic neurons.     

Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Somatodendritic α2-Adrenoceptors in the Locus Coeruleus Are Involved in the In Vivo Modulation of Cortical Noradrenaline Release by the Antidepressant Desipramine

Abstract: The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DMI) on noradrenergic transmission was evaluated in vivo by dual-probe microdialysis. DMI (1, 3, and 10 mg/kg, i.p.) dose-dependently increased extracellular levels of noradrenaline (NA) in the locus coeruleus (LC) area. In the cingulate cortex (Cg), DMI (3 and 10 mg/kg, i.p.) also increased NA dialysate, but at the lowest dose (1 mg/kg, i.p.) it decreased NA levels. When the α₂-adrenoceptor antagonist RX821002 (1 µ M ) was perfused in the LC, DMI (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the Cg. In electrophysiological experiments, DMI (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821002. Local DMI (0.01–100 µ M ) into the LC increased concentration-dependently NA levels in the LC and simultaneously decreased NA levels in the Cg. This decrease was abolished by local RX821002 administration into the LC. The results demonstrate in vivo that DMI inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LC inhibits noradrenergic activity, which in part counteracts the effects of DMI on the Cg. The modulation of cortical NA release by activity of DMI at the somatodendritic level is mediated through α₂-adrenoceptors located in the LC.     

Effects of Activation of NMDA and AMPA Glutamate Receptors on the Extracellular Concentrations of Dopamine, Acetylcholine, and GABA in Striatum of the Awake Rat: A Microdialysis Study

The effects of activation of the AMPA and NMDA ionotropic glutamate receptors on the extracellular concentration of dopamine, acetylcholine, (ACh) and GABA in striatum of the awake rat was investigated. Also the levels of DOPAC, HVA, and choline (Ch) were included in this study. Seven to eight days after stereotaxical implantation of a guide-cannulae assembly, microdialysis experiments were performed. The dopamine and ACh content of samples were measured by HPLC coupled to electrochemical detection. GABA was measured using fluorometric detection. Perfusion of AMPA (1, 20, 100 mM) produced a dose-related increase of dopamine and a dose-related decrease of DOPAC and HVA. AMPA 100 M decreased extracellular concentrations of ACh and increased the extracellular concentration of Ch and GABA. Perfusion of NMDA 500 M increased the concentration of dopamine and decreased DOPAC and HVA. Also, NMDA 100 M decreased DOPAC. NMDA 500 M decreased the extracellular concentrations of ACh and increased the concentrations of Ch and GABA. Perfusion of the AMPA/kainate-antagonist DNQX (100 M) blocked the effects of AMPA (100 M) on dopamine, DOPAC, HVA, ACh, and GABA concentrations. Perfusion of the NMDA-antagonist CPP (100 M) blocked the effects of NMDA 500 M on dopamine, DOPAC, HVA, ACh, Ch, and GABA concentrations. These results suggest an interaction between glutamate-dopamine-ACh-GABA in striatum of the awake rat.     

Extracellular Somatostatin Measured by Microdialysis in the Hippocampus of Freely Moving Rats: Evidence for Neuronal Release

Abstract: Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect <1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 μl/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 ± 6.4% ( p < 0.05) when Ca²⁺ was omitted and 1 m M EGTA added to the Krebs-Ringer solution and by 65.5 ± 3.2% ( p < 0.05) in the presence of 1 μ M tetrodotoxin. Depolarizing concentrations of the Na⁺ channel opener veratridine (6.25, 25, 100 μ M ) induced 11 ± 2 ( p < 0.05), 17 ± 2 ( p < 0.05), and 21 ± 5 ( p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 μ M veratridine was blocked by coperfusion with 5 μ M tetrodotoxin ( p < 0.01) and reduced by 79% ( p < 0.01) in the virtual absence of Ca²⁺. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 m M KCl and proportionally lowered Na⁺ increased the dialysate SLI 4.4 ± 1 ( p < 0.05) and 17 ± 3 ( p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na⁺,K⁺-ATPase, increased the dialysate SLI 15-fold baseline, on average ( p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.     

Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats

Noradrenergic and corticotropin-releasing factor (CRF) neuronal systems within the brain have been implicated in stress and anxiety. Synaptic release of cerebral norepinephrine (NE) is increased during stress, and following intracerebral CRF administration. Benzodiazepines are commonly used anxiolytic drugs but information on their effects on the stress- and CRF-related release of NE is limited. We have used in vivo microdialysis to test the effects of the benzodiazepine, chlordiazepoxide (CDP) on the noradrenergic responses to footshock and intracerebroventricular CRF in the medial hypothalamus and the medial prefrontal cortex (PFM) of freely moving rats. Footshock (60 x 0.1-0.2 mA shocks in 20 min) significantly increased microdialysate concentrations of NE in the first sample collected after initiating the footshock. In the hypothalamus, microdialysate NE was augmented 64% above baseline. A second footshock session (100 min after the first footshock) increased microdialysate NE to 313% of the baseline. Thus the noradrenergic responses to footshock were enhanced by preceding footshocks. CRF (100 ng) administered into the locus coeruleus (LC) almost tripled microdialysate concentrations of NE in the PFM. CDP (5mg/kg, i.p.) had no statistically significant effects on the basal dialysate concentrations of NE, but it significantly attenuated both footshock- and CRF-induced increases in dialysate NE. CDP may exert a direct inhibitory effect on the noradrenergic neurons, alter the input to LC noradrenergic neurons, or alter the ability of CRF to activate the LC noradrenergic system.     

S(-)-Nornicotine Increases Dopamine Release in a Calcium-Dependent Manner from Superfused Rat Striatal Slices

Abstract: The present study demonstrates that S (-)-nornicotine evoked a concentration-dependent increase in dopamine (DA) release from superfused rat striatal slices. The increase in DA release was indicated by an S (-)-nornicotine-induced overflow of endogenous 3,4-dihydroxyphenyl-acetic acid (DOPAC) in the striatal superfusate and by an S (-)-nornicotine-induced increase in tritium overflow from striatal slices preloaded with [³H]DA. Low concentrations (0.01–1.0 μ M ) of S (-)-nornicotine, which did not evoke endogenous DOPAC overflow, also were unable to modulate electrically evoked DOPAC overflow. The increase in DOPAC overflow induced by S (-)-nornicotine was compared with that produced by S (-)-nicotine. Comparing equimolar concentrations (0.1-100 μ M ) of S (-)-nornicotine and S (-)-nicotine, superfusion with S (-)-nornicotine resulted in a significantly greater DOPAC overflow. In contrast to the effect of S (-)-nicotine, S (-)-nornicotine evoked a sustained increase in DOPAC over-flow for the entire period of S (-)-nornicotine exposure. Furthermore, DOPAC overflow evoked by S (-)-nornicotine in control Krebs buffer was inhibited by superfusion with a low-calcium buffer. Moreover, in the low-calcium buffer, DOPAC overflow induced by 30 and 100 μ M S (-)-nornicotine was not different from that with no S (-)-nornicotine. The results indicate that S (-)-nornicotine, a constituent of tobacco products and a known metabolite of S (-)-nicotine, increases DA release in a calcium-dependent manner in superfused rat striatal slices. It is interesting that unlike S (-)-nicotine, there does not appear to be desensitization to this effect of S (-)-nornicotine.     

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin-related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K⁺ concentrations. Increasing the K⁺ concentration in the perfusate from 4 to 15 or 17.5 m M did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 m M K⁺, dose-dependent reductions of both basal and MFB-stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB-stimulated DA release with 4 or 15 m M K⁺ in the perfusate. However, after perfusion with 17.5 m M K⁺, ceruletide significantly attenuated the basal and MFB-stimulated DA release. Carbachol (10 μ M ) locally applied via the dialysis probe also attenuated MFB-stimulated DA release after perfusion with 17.5 m M K⁺. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.     

Extracellular 5-Hydroxytryptamine in Median Raphe Nucleus of the Conscious Rat Is Decreased by Nanomolar Concentrations of 8-Hydroxy-2-(Di-n-Propylamino)tetralin and Is Sensitive to Tetrodotoxin

Abstract: Extracellular 5-hydroxytryptamine (5-HT) in the median raphe and dorsal hippocampus was measured using in vivo microdialysis. Administration of 60 m M K⁺ through the probe into the median raphe region significantly increased 5-HT output from the median raphe and the right dorsal hippocampus. Local infusion of 10 µ M tetrodotoxin into the median raphe region substantially decreased 5-HT in the median raphe and left and right dorsal hippocampus. Systemic administration (0.3 mg/kg s.c.) of 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) decreased the 5-HT levels in the dialysates from both the median raphe region and dorsal hippocampus. Administration of 30 n M 8-OH-DPAT through the dialysis probe into the median raphe region decreased 5-HT output from the median raphe and dorsal hippocampus significantly, whereas at concentrations from 60 n M to 10 µ M , no significant effects were found in either region. With 100 µ M 8-OH-DPAT, a significant increase was seen in the median raphe region, but not in dorsal hippocampus. Similar findings were obtained following microinjections of different doses of the compound into the median raphe region. The results of this study indicate that the somatodendritic release of 5-HT is impulse flow-dependent. Moreover, the decrease of 5-HT in the median raphe region by low nanomolar concentrations of 8-OH-DPAT supports the notion that somatodendritic 5-HT release is subject to a local negative feedback mechanism through 5-HT_1A autoreceptors.     

The effects of diclofop-methyl on root growth of wild oat

Diclofop-methyl severely reduced the growth of seminal roots of wild oat ( Avena fatua L.) when applied in hydroponics at 0.01 and 0.05 μ M . Lateral roots emerged closer to the seminal root apex than in the controls, but coronal root number and length were unaffected at 0.01 μ M . However, doses of 0.05 to 0.1 μ M induced more but shorter coronal roots to emerge than for controls. At 1 μ M the number and length of coronal roots were less than for controls. Root-applied diclofop-methyl at 1 μ M inhibited emerging second leaf growth to the same extent as a foliar dip in 1 μ M diclofop-methyl without causing chlorosis as foliar treatment does. Because of limited basipetal transport of foliarly-applied diclofop-methyl, shoot treatment was ineffective in inducing abnormal root morphogenesis of the seminal and lateral root systems, although it caused abnormalities of the coronal root system. Time course studies were initiated to examine the effect of root-applied diclofop-methyl at 0.05 μ M . Seminal root growth was inhibited (by diclofop-methyl) soon after treatment, while controls continued elongating. The distance between the seminal root apex and the first lateral primordia increased in the controls within one day after treatment, but decreased in the herbicide-treated roots. The distance between the seminal root apex and the first emerged lateral root was reduced by three days after treatment. The number of lateral primordia and emerged roots was unaffected three days after treatment. These dose-response and kinetic results suggested that diclofop-methyl caused a loss of apical dominance in the seminal root.     

Dopamine Release in Rat Striatum: Physiological Coupling to Tyrosine Supply

Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.     

Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat

By means of intracerebral microdialysis effects of cholecystokinin peptides and neurotensin administered via the microdialysis probe have been studied on dopamine release and metabolism in the nucleus accumbens and neostriatum of the halothane anaesthetized male rat. Levels of extra cellular dopamine (DA) and its metabolites 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed in nuc. accumbens (rostral and caudal part) using high performance liquid chromatography in combination with electrochemical detection.

(1) In the rostral part of the nuc. accumbens CCK-8 (10 and 100 μM), CCK-33 (100 μM) but not CCK-4 (10 and 100 μM) increased the levels of DA in the perfusate without increasing the extracellular levels of DOPAC and HVA. (2) In the caudal nuc. accumbens CCK-8 and CCK-4 in concentrations of 10 μM and 100 μ M of CCK-33 had no effect on DA release and metabolism, since the extracellular levels of DA, DOPAC and HVA were not changed. (3) In the rostral nuc. accumbens perfusion with 10 μM of neurotensin but not with any other concentration of neurotensin (0.01, 0.1, 1 and 100 μM) increased the levels of DA in the extracellular fluid. (4) In the caudal nuc. accumbens a 40 min perfusion with neutrotensin produced a concentration dependent increase of the levels of DA in the perfusate (peak action at 10 μ M) which in this case was associated with increases in the extracellular levels of DOPAC and HVA. (5) By means of receptor autoradiography using (3-[¹²⁵I]iodotyrosyl³) neurotensin it was found that a 40 min perfusion with this radioligand in the rostral nuc. accumbens reached a total volume of 0.051 mm³. The diffusion of the radioligand was limited to the rostral or caudal part of the nuc. accumbens depending upon the site of placement of the dialysis probe.

The results indicate the existence of cholecystokinin (CCK) receptors in the rostral nuc. accumbens, which are sensitive to CCK-8 and CCK-33 but not to CCK-4, and which facilitate DA release without producing any detectable increase in DA metabolites. In contrast, such receptors do not appear to play a similar role in the regulation of DA release in the caudal nuc. accumbens, where DA terminals contain CCK-like immunoreactivity. Furthermore, the results indicate that neurotensin receptors exist both in the rostral and caudal nuc. accumbens, where they inter alia enhance the release of DA. In the caudal nuc. accumbens these effects of neurotensin are also associated with an increase of DA metabolites, possibly suggesting that in this region neurotensin receptors may also control DA synthesis.     

Measurement of extracellular 5-HT and 5-HIAA in hippocampus of freely moving rats using microdialysis: long-term applications

Extracellular levels of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in the ventral hippocampus of the awake rat using microdialysis. The basal level of 5-HT in hippocampal dialysates was very close to the detection limit of our assay. However, addition of a 5-HT re-uptake blocker, citalopram, to the perfusion medium resulted in a 3-fold rise of 5-HT levels in dialysates and provided a stable baseline. Under these conditions, extracellular levels of 5-HT and 5-HIAA could be measured up to 11 days after dialysis probe implantation. 5-HT measured in dialysate was Ca²⁺-dependent both 24 h and 7 days after surgery. In comparison K⁺-induced depolarization caused a 9-fold increase in 5-HT output 24 h after probe implantation and this effect had disappeared 10 days later, although behavioral activation following K⁺ application was similar 24 h and 11 days after surgery. Systematic administration of -trytophan had no significant effect on 5-HT but increased 5-HIAA levels by 90%. These results suggest that in the presence of citalopram, 5-HT in hippocampal dialysates is derived from serotonergic neurones. The microdialysis method appears most useful for measurement of extracellular 5-HT 24–72 h after probe implantation. Finally, our data indicate that extracellular 5-HIAA mainly reflects intraneuronal metabolism of 5-HT prior to release.     

Gabapentin produces PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to ameliorate pain hypersensitivity in mice with partial nerve ligation. To clarify the supraspinal mechanism of action of gabapentin, whole-cell patch-clamp recordings were performed on locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury or mice subjected to a sham-operation, and the effects of gabapentin in the modulation of synaptic transmission were studied. Bath application of gabapentin (10, 30 and 100 μM) in a concentration-dependent manner reduced the GABA_A receptor-mediated inhibitory post-synaptic currents (IPSCs) in slices prepared from partially nerve-ligated mice, whereas glutamate-mediated excitatory post-synaptic currents were hardly affected. By contrast, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired-pulse ratio, suggesting that gabapentin acts on the pre-synaptic GABAergic nerve terminals in the LC. As the protein kinase A (PKA) inhibitor H-89 but not the protein kinase C inhibitor chelerythrine abolished the inhibitory action of gabapentin on IPSCs, PKA-mediated phosphorylation seems to be important for supraspinal gabapentin responsiveness in neuropathic conditions. Together, gabapentin generates PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission, and thereby removes the inhibitory influence on LC neurons only under neuropathic pain states. These findings provide crucial evidence of how supraspinally acting gabapentin recruits the descending noradrenergic system.     

Retrograde axonal transport specificity in the locus coeruleus neurons after [H]noradrenaline injection into the rat olfactory bulb

Retrograde axonal transport process was investigated in the afferent systems to the rat olfactory bulb, after [³H]noradrenaline ([³H]NA) injection into the olfactory bulb, in order to provide evidence regarding its specificity and the biochemical basis supporting this specificity.

Radioautographs showed that [³H]NA unilaterally injected into the olfactory bulb at a concentration of 10⁻³ M, resulted in labeling of the structures afferent to the olfactory bulb, mainly on the injected side: locus coeruleus (LC), dorsal and central raphes, nucleus of the lateral olfactory tract and piriform cortex. Heavy labeling was observed on the noradrenergic LC cell bodies, whereas the radioautographic reaction was less intense on the other structures. After 10⁻⁴ M injection, the labeling intensity of the LC cell bodies was lower while very rare weakly labeled cell bodies persisted in the dorsal raphe, nucleus of the lateral olfactory tract and piriform cortex. The LC cell bodies were exclusively labeled when the concentration of [³H]NA injection was 10⁻⁵ M. All the other structures were devoid of labeling. It was still possible to detect labeled cell bodies in the LC for a 10⁻⁶ M concentration.

Following bilateral injections of [³H]NA (10⁻³ M) the total radioactivity retrogradely transported to the LC represented about 4 times the total radioactivity measured in the periaqueductal gray substance (as control tissue of the tracer diffusion). Fractional study by ethanol of LC tissue homogenate and liquid scintillation counting of each fraction showed that 60% of the total radioactivity (about 2.5 times the control value) was in the supernatant and 40% (about 20 times the control value) was associated with the precipitate. In the other regions such as the dorsal and central raphes and periaqueductal gray substance, radioactivity was mainly found in the supernatant, except for the dorsal raphe whose precipitate contained a low amount of radioactivity (about 4 times the control value).

Colchicine (an axonal transport inhibitor) bilaterally injected into the medial forebrain bundle and systemic administration of desipramine (a noradrenaline uptake inhibitor) decreased the radioactivity associated with the LC precipitate by 90 and 85% and the LC supernatant radioactivity by 55 and 35%, respectively. These pretreatments did not significantly affect the radioactivity amounts measured in the different fractions of dorsal and central raphes and periaqueductal gray substance. Radioautographic study after colchicine treatment showed a large decrease in the labeling intensity of the LC cell bodies as compared to the non-treated side.

Therefore, we suggest that low concentrations (10⁻⁵ M) of [³H]NA injected in the olfactory bulb determine specific conditions of noradrenergic pathway labeling. This specific labeling after migration could be supported by the radioactive ethanol precipitate which would appear to contain [³H]NA- and/or ³H-derivatives-binding protein. Such a ³H-macromolecular complex, which could represent the specific carrier, may well undergo retrograde transport from the nerve terminals towards the cell bodies.     

Catecholaminergic microcircuitry controlling the output of airway-related vagal preganglionic neurons.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.     

Alpha2-adrenoceptor mediated co-release of dopamine and noradrenaline from noradrenergic neurons in the cerebral cortex

Previous results suggest that extracellular dopamine (DA) in the rat cerebral cortex originates from dopaminergic and noradrenergic terminals. To further clarify this issue, dialysate DA, dihydroxyphenylacetic acid (DOPAC) and noradrenaline (NA) were measured both in the medial prefrontal cortex (mPFC) and in the occipital cortex (OCC), with dense and scarce dopaminergic projections, respectively. Moreover, the effect of the alpha2-adrenoceptor antagonist RS 79948 and the D2-receptor antagonist haloperidol on extracellular DA, DOPAC and NA was investigated. Extracellular DA and DOPAC concentrations in the OCC were 43% and 9%, respectively, those in the mPFC. Haloperidol (0.1 mg/kg i.p.) increased DA and DOPAC (by 35% and 150%, respectively) in the mPFC, but was ineffective in the OCC. In contrast, RS 79948 (1.5 mg/kg i.p.) increased NA, DA and DOPAC, both in the mPFC (by approximately 50%, 60% and 130%, respectively) and the OCC (by approximately 50%, 80% and 200%, respectively). Locally perfused, the DA transporter blocker GBR 12909 (10 micro m) was ineffective in either cortex, whereas desipramine (DMI, 100 micro m) markedly increased extracellular NA and DA in both cortices. The weak haloperidol effect on DA efflux was not enhanced after DA- and NA-transporter blockade, whereas after DMI, RS 79948 markedly increased extracellular NA, and especially DA and DOPAC in both cortices. The results support the hypothesis that most extracellular DA in the cortex is co-released with NA from noradrenergic terminals, such co-release being primarily controlled by alpha2-adrenoceptors.     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

An in vitro microdialysis methodology to study 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity in liver microsomes

Microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) was used to observe in vitro 11beta-hydroxysteroid dehydrogenase type 1 (HSD1) enzyme-catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsomes from dog, monkey, and human. Experimental conditions that would affect the microdialysis sampling approach including probe length, perfusion fluid flow rate, extraction efficiency (E(d)), substrate concentration, and enzyme reaction conditions were evaluated. Dialysates containing high salt concentrations (>150 mM) were directly assayed using LC/MS/MS without additional sample cleanup. The sensitivity (with lower level of quantitation at 0.1 ng/mL) and selectivity of this assay allowed detection of the enzyme reactants at physiologically relevant levels. The interconversion from M+4 cortisone to M+4 cortisol was detected in dog, human, and monkey liver microsomes. Results show species-specific reaction profiles, with a five times higher conversion rate in dog liver microsomes than in human and monkey liver microsomes. Based on M+4 cortisol production rate obtained using a microdialysis infusion of M+4 cortisone to the microsomes coincubated with a proprietary 11beta-HSD1 inhibitor of different concentrations, the degrees of enzyme inhibition were found to be 40 and 85%, consistent with values obtained by a traditional in vitro incubation method. The microdialysis sampling methodology with LC/MS/MS provided extensive information about 11beta-HSD1 activities in microsomes from different mammalian species.     

Chronic Ethanol Exposure Potentiates NMDA Excitotoxicity in Cerebral Cortical Neurons

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μ M ) to the culture dishes for 25 min in Mg²⁺-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 m M ) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 μ M and 50 μ M NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μ M ) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 m M ) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.