bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptides Homologous to Extracellular Loop Motifs of Connexin 43 Reversibly Abolish Rhythmic Contractile Activity in Rabbit Arteries
Authors: Chaytor A. T. , Evans W. H. , Griffith T. M. , Thornbury K. D.
Published: September 2004

Phenylephrine (10 μM) evoked rises in tension in isolated rings of endothelium‐denuded rabbit superior mesenteric artery. These increases consisted of a tonic component with superimposed rhythmic activity, the frequency of which generally remained constant over time but whose amplitude exhibited cycle‐to‐cycle variability.

The amplitude, but not the frequency, of the rhythmic activity was affected by a series of short peptides possessing sequence homology with extracellular loops 1 and 2 of connexin 43 (Cx43). Oscillatory behaviour was abolished at concentrations of 100–300 μM (IC50 of 20–30 μM), without change in average tone. No synergy was evident between peptides corresuponding to the extracellular loops, and cytoplasmic loop peptides were biologically inactive.

The putative gap junction inhibitor heptanol mimicked the action of the extracellular loop peptides and abolished rhythmic activity at concentrations of 100–300 μM without effects on frequency. However, in marked contrast to the peptides, heptanol completely inhibited the contraction evoked by phenylephrine (IC50, 283 ± 28 μM).

The presence of mRNA encoding Cx32, Cx40 and Cx43 was detected in the rabbit superior mesenteric artery by reverse transcriptase‐polymerase chain reaction. Western blot analysis showed that Cx43 was the major connexin in the endothelium‐denuded vessel wall.

We conclude that intercellular communication between vascular smooth muscle cells via gap junctions is essential for synchronized rhythmic activity in isolated arterial tissue, whereas tonic force development appears to be independent of cell‐cell coupling. The molecular specificity of the peptide probes employed in the study suggests that the smooth muscle relaxant effects of heptanol may be non‐supecific and unrelated to inhibition of gap junctional communication.
Immunoglobulins from motoneurone disease patients enhance glutamate release from rat hippocampal neurones in culture
Authors: Andjus Pavle R. , Stevic‐Marinkovic Zorica , Cherubim Enrico
Published: September 2004

The whole‐cell configuration of the patch‐clamp technique was used to study the effects of immunoglobulins (IgGs) from patients affected by amyotrophic lateral sclerosis (ALS) on spontaneous glutamatergic currents in rat hippocampal cells in culture.

Focal application of ALS IgGs (100 μg ml−1) to hippocampal cells induced a rise in frequency but not in amplitude of spontaneous excitatory postsynaptic currents (SEPSC) which outlasted the period of IgG application. The mean frequency ratio (ALS over control) was 3.2 ± 0.6 (n. 19). No changes in frequency or amplitude of SEPSCs were observed after treatment with IgGs obtained from healthy donors (n=5) or from patients with Alzheimer's disease (n=4).

ALS IgGs also increased the frequency (by a factor of 2.0 ± 0.3) but not the amplitude of miniature excitatory postsynaptic currents (mEPSC) recorded in the presence of TTX (n=19). A rise in frequency of mEPSC was also seen in cells superfused with a calcium‐free solution (n=4).

In the presence of TTX, ALS IgGs did not modify the amplitude or the shape of currents evoked by AMPA (100 μm), recorded at a holding potential of −50 mV.

It is concluded that ALS IgGs enhance both SEPSCs and mEPSCs through a presynaptic type of action. The excessive release of glutamate from nerve endings may be the cause of motoneurone death in ALS patients.
Hypoxia‐induced inhibition of calcium channels in guinea‐pig taenia caeci smooth muscle cells
Authors: Rekalov Vladimir , Juránek Ivo , Máleková L'ubica , Bauer Viktor
Published: September 2004

The effects of hypoxia on whole‐cell current in single smooth muscle cells and on a high K+‐induced contraction of strips of the guinea‐pig taenia caeci were studied.

In physiological salt solution (PSS) and K+‐based pipette solution, hypoxia (PO2= 20 mmHg) reversibly inhibited both the inward Ca2+ current (ICa) and outward Ca2+‐activated K+ current (IK(Ca)) components of the whole‐cell current.

In PSS and Cs+‐based pipette solution, hypoxia reversibly suppressed ICa by 30 ± 5% at 0mV.

When Ba2+ was used as a charge carrier, the IBa was suppressed by hypoxia in a potential‐dependent manner, with the maximum of 40 ± 7% at +10mV. Alterations of concentrations of egta, GDBβS or ATP in the pipette solution did not change the inhibitory effects of hypoxia on ICa and IBa.

In PSS with 2 mm CaCl2 replaced by CoCl2, hypoxia did not affect the Ca2+ influx‐independent potassium current.

In cells voltage clamped at ‐20 mV hypoxia reversibly inhibited the spontaneous transient outward currents.

The response of high K+‐contracted taenia caeci to hypoxia was composed of an initial rapid relaxation followed by a small transient contraction and slow relaxation. The transient contraction was blocked by atropine (1–10 μm), while relaxations were unaffected by atropine and guanethidine (10 μm).

The results show that hypoxia reversibly inhibits ICa and secondarily suppresses IK(Ca) due to decreased Ca2+ influx through Ca2+ channels.

It is suggested that inhibition of ICa was responsible for the rapid relaxation, whereas transient contraction may have been due to release of acetylcholine from nerve terminals upon hypoxia.
Aminergic Modulation of Glycine Release in a Spinal Network Controlling Swimming in Xenopus Laevis
Authors: McDearmid Jonathan R. , Scrymgeour‐Wedderburn John F. , Sillar Keith T.
Published: September 2004

Neuromodulators can effect changes in neural network function by strengthening or weakening synapses between neurons via presynaptic control of transmitter release. We have examined the effects of two biogenic amines on inhibitory connections of a spinal rhythm generator in Xenopus tadpoles.

Glycinergic inhibitory potentials occurring mid‐cycle in motoneurons during swimming activity are reduced by 5‐hydroxytryptamine (5‐HT; serotonin) and enhanced by noradrenaline (NA). These opposing effects on inhibitory synaptic strength are mediated presynaptically where 5‐HT decreases and NA increases the probability of glycine release from inhibitory terminals.

The amines also have contrasting effects on swimming: 5‐HT increased motor burst durations while NA reduced swimming frequency. Aminergic modulation of glycinergic transmission may thus control fundamental parameters of swimming and force the spinal network to generate opposite extremes of its spectrum of possible outputs.
The effects of calcium buffering and cyclic AMP on mechano‐electrical transduction in turtle auditory hair cells
Authors: Ricci A. J. , Fettiplace R.
Published: September 2004

The effects of intracellular Ca2+ buffering on hair cell mechanotransduction were studied in an intact cochlear epithelium where the endolymphatic and perilymphatic surfaces could be separately perfused with different Ca2+ solutions.

The speed and extent of transducer adaptation increased as the concentration in the patch electrode of the Ca2+ buffer BAPTA was lowered. In 0.1 mm BAPTA or less, the transducer adapted almost completely, with a mean time constant of 0.8 ms.

For a fixed internal BAPTA concentration, the transducer conductance varied with hair cell location, increasing towards the high‐frequency end of the cochlea, and the time constant of adaptation decreased proportionally. At a given cochlear location, hair cells with larger transducer conductances displayed faster adaptation. We suggest that transducer adaptation accounts for a variable high‐pass filter observed in the acoustic tuning curve.

The effects of perfusion of 50 μm Ca2+ endolymph depended on the BAPTA concentration of the electrode: with 3 mm BAPTA, adaptation was abolished, but in most recordings with 0.01 or 0.1 mm BAPTA, rapid adaptation was retained. The current–displacement curve was also shifted less the lower the intracellular BAPTA concentration. Cells in the high‐frequency half of the papilla retained adaptation at a higher BAPTA concentration.

Treatment with the cAMP agonist, 8‐bromo‐cAMP, or with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine, caused a rightward shift in the current–displacement curve which was independent of the internal BAPTA concentration.

We conclude that the free Ca2+ and cyclic nucleotide concentrations of the hair bundle modulate the position of the activation curve of the transducer. The factors which may be important for the correct functioning of adaptation in vivo are discussed.
Inhibition of spontaneous EPSCs and IPSCs by presynaptic GABAB receptors on rat supraoptic magnocellular neurons
Authors: Kabashima Narutoshi , Shibuya Izumi , Ibrahim Nurhadi , Ueta Yoichi , Yamashita Hiroshi
Published: September 2004

The function of presynaptic GABA receptors in the regulation of transmitter release in supraoptic nucleus (SON) magnocellular neurons was investigated by recording spontaneous postsynaptic currents from rat magnocellular SON neurons in a slice preparation (150μM thick, 1.8 mm in diameter) using the whole‐cell patch‐clamp technique.

Both the spontaneous EPSCs and IPSCs were TTX resistant. The EPSCs were abolished by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), whereas the IPSCs were abolished by picrotoxin, suggesting that the EPSCs and IPSCs are synaptic inputs from glutamatergic and GABAergic neurons, respectively.

The selective GABAB agonist, baclofen, reduced the frequency of both the EPSCs and IPSCs without affecting the amplitude. The time constant of the decay phase of both the EPSCs and IPSCs remained unchanged after baclofen application.

The reduction of the frequency of the synaptic currents by baclofen was dose dependent (10 nM to 100 μm) and the EC50 values were 5.8 and 8.5 μm for the EPSCs and IPSCs, respectively.

The effect of baclofen (10μM) was antagonized by the selective GABAB antagonist, 2‐hydroxy‐saclofen (2OH‐saclofen), at 300 μm.

When given alone, 2OH‐saclofen (100 μm) increased the frequency of both the EPSCs and IPSCs without affecting their amplitude, suggesting that endogenously released GABA in the slice acts on presynaptic GABAB receptors.

The GABAA agonist, muscimol, reduced the frequency of EPSCs, and picrotoxin increased the frequency of the EPSCs, suggesting that GABAA receptors also participate in the presynaptic inhibition of glutamate release.

Taken together, these data suggest that GABAB receptors are present on the presynaptic terminals of both GABA and glutamate neurons in the SON, and that these presynaptic GABAB receptors play an important role in the regulation of the neuronal activity in SON magnocellular neurons.
Stimulation of α1‐adrenoceptors and Protein Kinase C‐mediated Activation of Ecto‐5′‐Nucleotidase in Rat Hearts in vivo
Authors: Sato Toshiaki , Obata Toshio , Yamanaka Yasumitsu , Arita Makoto
Published: September 2004

To determine whether protein kinase C (PKC)‐mediated activation of ecto‐5′‐nucleotidase would increase interstitial adenosine concentrations in the rat heart in vivo, we made use of the microdialysis technique and a flexibly mounted probe, which was implanted in the left ventricular myocardium and perfused with Tyrode solution.

The baseline level of dialysate adenosine was 0.51 ± 0.09 μM (n= 16). Perfusion of adenosine 5′‐monophosphate (AMP, 100 μM) through the probe increased the dialysate adenosine concentration markedly to 9.25 ± 0.46μM (n= 15). αβ‐Methyleneadenosine 5′‐diphosphate (AOPCP, 100 μM), an inhibitor of ecto‐5′‐nucleotidase, abolished the AMP‐induced increase in dialysate adenosine, but did not affect the baseline level of adenosine. These observations suggest that the dialysate adenosine obtained during the perfusion with AMP, but not the baseline levels of adenosine, originated from the dephosphorylation of AMP by ecto‐5′‐nucleotidase. Thus, the level of adenosine measured during AMP perfusion gives an index of the activity of ecto‐5′‐nucleotidase in the tissue.

Noradrenaline (10 μm) increased the adenosine concentration measured in the presence of 100 μm AMP (i.e. the activity of ecto‐5′‐nucleotidase) by 38.7 ± 9.6 % (n= 5, P < 0.05), an increase which was inhibited by an antagonist of the α1adrenoceptor (prazosin, 50 μM) or of PKC (chelerythrine, 10 μm). Further application of either the α1‐adrenoceptor agonist methoxamine (100 μM) or the diacylglycerol analogue 1,2‐dioctanoyl‐sw‐glycerol (DOG, 100 μm) also increased the adenosine concentration by 35.1 ± 10.0% (n= 6, P < 0.05) or 40.6 ± 8.3% (n= 5, P < 0.05), respectively.

The presence of okadaic acid (50 μm), an inhibitor of protein phosphatase, enhanced the noradrenaline‐induced increase in adenosine concentration by 112.4 ± 35.9% (n= 4, P < 0.05), to a level significantly (P < 0.05) greater than the increase caused by noradrenaline alone (38.7 ± 9.6%).

These data provide the first evidence that α1‐adrenoceptor stimulation and the subsequent activation of PKC can increase adenosine concentrations in interstitial spaces of ventricular muscle in vivo, through activation of endogenous ecto‐5′‐nucleotidase.
Pressor effect mediated by bradykinin in the paratrigeminal nucleus of the rat
Authors: Lindsey C. J. , Buck H. S. , Fior‐Chadi D. R. , Lapa R. C. R. S.
Published: September 2004

The participation of the paratrigeminal nucleus (Pa5) in the pressor response produced by bradykinin in the dorsolateral medulla of rats was investigated. The microinjection of 6 pmol of bradykinin directly over the paratrigeminal nucleus of unanaesthetized rats produced a significant increase in arterial pressure and a moderate increase in heart rate.

Bradykinin microinjections in different sites surrounding the Pa5 compromising the external cuneate nucleus, the trigeminal nucleus, the lateral and ventral spinal trigeminal tract and the dorsal trigeminal tract rostral and caudal to the Pa5 did not elicit significant pressor responses. In contrast, microinjections in the paratrigeminal nucleus produced pressor effects. Injections in the dorsolateral medulla directly over the paratrigeminal nucleus produced larger responses than when injections were made in the nucleus. Saline injections in the different nuclei did not produce pressor effects.

Neurochemical lesioning of the Pa5, with microinjections of ibotenic acid in the Pa5, abolished the pressor response to bradykinin injected over the lesioned nucleus. The effect was present, however, when bradykinin was injected on the contralateral side to the lesion, over the intact nucleus of the same animal. Pretreatment with capsaicin (injected in the lateral cerebral ventricle), which causes selective degeneration of afferent sensory fibres, did not alter the pressor effect of bradykinin injected over the paratrigeminal nucleus.

Dose‐related responses were produced by different concentrations of bradykinin (0.6–1.8 pmol) microinjected over the nucleus. The bradykinin receptor antagonist HOE 140, injected over the paratrigeminal nucleus 30 min earlier, abolished the pressor response caused by bradykinin.

Low doses of bradykinin injected in or directly over the paratrigeminal nucleus increased arterial pressure and caused a small increase in heart rate by stimulating kinin receptors of the paratrigeminal nucleus in the dorsolateral medulla of awake and unrestrained rats. The pattern of the response was consistent with that of sympathetic stimulation. The paratrigeminal nucleus, which receives primary afferents and projects to the nucleus tractus solitarii and the rostral ventral lateral medulla, may be positioned as relay nucleus possibly connecting sensory input to structures that regulate blood pressure.
Ca2+ current and Ca2+ ‐activated chloride current in isolated smooth muscle cells of the sheep urethra
Authors: Cotton K. D. , Hollywood M. A. , McHale N. G. , Thornbury K. D.
Published: September 2004

Isolated sheep urethral cells were studied using the perforated patch clamp technique (T= 37 °C). Depolarizing steps ranging from ‐40 to ‐10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of ‐80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 μm). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails.

Changing the chloride equilibrium potential (ECl) from 0 to + 27 mV shifted the reversal potential of the tail currents from 1 ± 1 to 27 ± 1 mV (number of cells, n= 5). Chloride channel blockers, niflumic acid (10 μm) and anthracene‐9‐carboxylic acid (9AC, 1 mm), reduced the slow current and tails suggesting that these were Ca2+‐activated CF currents, ICl(Ca).

Caffeine (10 mm) induced currents that reversed at ECl and were blocked by niflumic acid (10 μm).

In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials.

In conclusion, these cells have both L‐type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.
Regulation of Quantal Secretion by Neurotrophic Factors at Developing Motoneurons in Xenopus Cell Cultures
Authors: Liou Jau‐Cheng , Yang Rong‐Sen , Fu Wen‐Mei
Published: September 2004

The ability of different neurotrophic factors to maintain and regulate synaptic function at the developing motoneuron was studied in Xenopus nerve–muscle co‐cultures. Spontaneous synaptic currents (SSCs) were measured by using whole‐cell voltage‐clamped myocytes.

Compared with natural synapses, motoneurons without contact on a myocyte (naive neurons) released ACh in smaller quantal packets, the amplitude being inversely proportional to the days in culture. The mean SSC amplitudes of naive neurons, which were measured by manipulating a myoball into contact with the myocyte‐free nerve terminals to form a manipulated synapse, were 99.5 ± 6.7 and 48.2 ± 1.9 pA for day‐1 and day‐3 cultures, respectively.

Chronic treatment of day‐1 cultures with brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin‐4 (NT‐4), ciliary neurotrophic factor (CNTF) or glial cell line‐derived neurotrophic factor (GDNF) for 2 days, increased the ACh quantal size of naive motoneurons in a concentration‐dependent manner, whereas insulin‐like growth factor‐1 (IGF‐1) and basic fibroblast growth factor (bFGF) had no effect, even at high concentrations.

The interaction of various neurotrophic factors was examined, using concentrations that gave maximal effects. Combination of CNTF plus BDNF or CNTF plus NT‐3 had synergistic effects in potentiating SSC amplitude of the manipulated synapse of naive neurons, whereas NT‐3 plus BDNF, NT‐3 plus GDNF, BDNF plus GDNF or CNTF plus GDNF had no synergistic action.

Chronic treatment with d‐tubocurarine for 2 days resulted in a reduction of the quantal size of natural synapses. Concomitant treatment with BDNF, NT‐3, GDNF, CNTF but not bFGF or IGF‐1, reconstituted the SSC amplitude.

Taken together, these findings suggest that BDNF, NT‐3, NT‐4, CNTF and GDNF may regulate and maintain the synaptic function of developing motoneurons, and different neurotrophic factors utilizing distinct signalling mechanisms may have synergistic actions.
Thromboxane A2, released by the anti‐ tumour drug irinotecan, is a novel stimulator of Cl− secretion in isolated rat colon
Authors: Sakai Hideki , Sato Takahiro , Hamada Noriko , Yasue Miyuki , Ikari Akira , Kakinoki Bunpei , Takeguchi Noriaki
Published: September 2004

A camptothecin derivative, irinotecan (Cpt‐11), is a topoisomerase I inhibitor and has a strong activity against a broad range of human cancer. One of the side‐effects of this drug is diarrhoea. Here, we tried to determine the mediator of the irinotecan‐induced Cl− secretion which may underlie this diarrhoea, using isolated mucosae of rat distal colon.

Irinotecan increased Cl− secretory current in a concentration‐dependent manner across the mucosa, set between Ussing chambers. Thromboxane A2 (TXA2) has not been reported to date as a physiological stimulant of Cl− secretion in the distal colon. However, the major part of the present irinotecan‐induced current was inhibited by selective thromboxane A2 receptor antagonists (KW‐3635 and ONO‐3708), and a selective thromboxane synthase inhibitor (Y‐20811). In fact, we found that irinotecan stimulated the release of TXA2 in a concentration‐dependent manner from the isolated mucosa into the bathing solutions.

Furthermore, 9,11‐epithio‐11,12‐methano‐thromboxane A2 (STA2), a stable analogue of TXA2, induced Cl− secretion, which was almost completely inhibited by the TXA2 receptor antagonists.

In single cells of isolated crypts, STA2 depolarized the cell and increased the membrane conductance, indicating that STA2 opened the apical Cl− channel of the crypt cells.

We conclude, therefore, that the irinotecan‐induced endogenous TXA2 is a novel stimulant of the Cl− secretion from the crypt cells of distal colon.
Membrane currents underlying the modified electrical activity of guinea‐pig ventricular myocytes exposed to hyperosmotic solution
Authors: Ogura Toshitsugu , You Yongdong , McDonald Terence F.
Published: September 2004

Guinea‐pig ventricular myocytes were superfused with hyperosmotic (sucrose) Tyrode solution (1.2–2.8 times (T) normal osmolality) for up to 40 min. Action potentials were recorded with microelectrodes, and membrane currents with the perforated‐ or ruptured‐patch technique.

Hyperosmotic treatment for 20 min shrunk cell volume and hyperpolarized the membrane. Moderate (1.2–1.5T) treatment caused biphasic changes in action potential configuration (rapid minor shortening quickly followed by lengthening to a stable 110% control duration). Severe (2.2–2.8T) treatment caused triphasic changes (marked early shortening, strong rebound lengthening and subsequent pronounced shortening). At peak lengthening (6–10 min) action potentials (165% control duration) had a hump near –30 mV and slowed terminal repolarization.

In accordance with previous studies, hyperosmotic solution inhibited the delayed rectifier K+current, and enhanced the outward Na+–Ca2+ exchange current (INaCa) at plateau potentials. A novel finding was that hyperosmolality reduced the amplitude of L‐type Ca2+ current (ICa,L) and slowed its rate of inactivation. Experiments on myocytes loaded with indo‐1 suggest that the reduction in ICa,L is due to a rapid elevation of [Ca2+]i.

When impaled myocytes were preloaded with EGTA, severe hyperosmotic treatment induced a rapid monotonic shortening of the action potential to a stable 20% of control duration. Addition of external K+ quickly nulled the hyperpolarization and slowly lengthened the action potential.

The results suggest that modified electrical activity in osmotically shrunken myocytes is primarily caused by increases in [K+]i, [Na+]i and [Ca2+]i: (i) elevated [K+]i hyperpolarizes the membrane (which may contribute to increased [Na+]i); (ii) elevated [Na+]i shortens all phases of the action potential (increased outward‐directed INaCa); and (iii) elevated [Ca2+]i has antagonistic plateau shortening (inhibition of inward ICa,L) and plateau lengthening (reduced outward INaCa) influences, as well as a strong subplateau lengthening effect (enhanced inward INaCa).
The Biphasic Force–Velocity Relationship in Frog Muscle Fibres and its Evaluation in Terms of Cross‐Bridge Function
Authors: Edman K. A. P. , Månsson A. , Caputo C.
Published: September 2004

The relationship between force and velocity of shortening was studied during fused tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (1.5–3.3°C; sarcomere length, 2.20 mm). Stiffness was measured as the change in force that occurred in response to a 4 kHz length oscillation of the fibre.

The results confirmed the existence of two distinct curvatures of the force–velocity relationship located on either side of a breakpoint in the high‐force, low‐velocity range. Reduction of the isometric force (P0) to 83.4 ± 1.7% (mean ±s.e.m., n= 5) of the control value by dantrolene did not affect the relative shape of the force–velocity relationship. The breakpoint between the two curvatures was located at 75.9 ± 0.9% of P0 and 11.4 ± 0.6% of maximum velocity of shortening (Vmax) in control Ringer solution and at 75.6 ± 0.7% of P0 and 12.2 ± 0.7% of Vmax in the presence of dantrolene. These results provide evidence that the transition between the two curvatures of the forcevelcity relationship is primarily related to the speed of shortening, not to the actual force within the fibre.

The instantaneous stiffness varied with the speed of shortening forming a biphasic relationship with a breakpoint near 0.15 Vmax and 0.8 P0, respectively. The force/stiffness ratio (probably reflecting the average force per cross‐bridge), increased with force during shortening. The increase of the force/stiffness ratio with force was less steep at forces exceeding 0.8 P0 than below this point.

A four‐state cross‐bridge model (described in the Appendix) was used to evaluate the experimental results. The model reproduces with great precision the characteristic features of the force–stiffness–velocity relationships recorded in intact muscle fibres.
Local opioid inhibition and morphine dependence of supraoptic nucleus oxytocin neurones in the rat in vivo
Authors: Ludwig Mike , Brown Colin H. , Russell John A. , Leng Gareth
Published: September 2004

Single neurones of the rat supraoptic nucleus were recorded during microdialysis of naloxone onto the ventral surface of the nucleus in anaesthetized rats. We used this combination of techniques to test whether the acute or chronic effects of systemically or centrally applied opioids upon oxytocin cell activity were due to actions of the opioids within the nucleus itself.

Supraoptic nucleus oxytocin neurones were identified antidromically and by an excitatory response to intravenously injected cholecystokinin. Acute intravenous injection of the κ‐agonist U50488H or the μ‐agonist morphine (1–5 mg kg−1) reduced the firing rate of identified oxytocin neurones by 97.7 ± 4.8% (n= 6) and 94.1 ± 4.1% (n= 7), respectively. The inhibition by each of these opioids was completely reversed after administration by microdialysis (retrodialysis) of the opioid antagonist naloxone (0.1–1.0 μg μl−1 at 2 μl min−1) onto the exposed ventral surface of the supraoptic nucleus.

Retrodialysis of naloxone (0.1‐10.0 μg μl−1) onto the supraoptic nucleus of rats made dependent by intracerebroventricular morphine infusion for 5 days increased the firing rate of oxytocin neurones from 0.9 ± 0.4 to 3.1 ± 0.7 spikes s−1 (P < 0.05, n= 6). This increase in firing rate from basal was 58.5 ± 15.1% of that following subsequent intravenously injected naloxone (5 mg kg−1).

Thus, the acute inhibition of supraoptic nucleus oxytocin neurones which results from systemic administration of opioid agonists primarily occurs within the supraoptic nucleus itself, since the antagonist naloxone was effective when given into the supraoptic nucleus. Furthermore, oxytocin neurones develop morphine dependence by a mechanism which is distinct from an action on their distant afferent inputs. Nevertheless, withdrawal excitation of these afferent inputs may enhance the magnitude of oxytocin neurone withdrawal excitation.
Neurones in the supraoptic nucleus of the rat are regulated by a projection from the suprachiasmatic nucleus
Authors: Cui L.‐N. , Saeb‐Parsy K. , Dyball R. E. J.
Published: September 2004

In the rat, projections from the suprachiasmatic nucleus (SCN) to the supraoptic nucleus (SON) of the hypothalamus were characterized in vivo using extracellular recordings and in slice preparations using both extracellular and whole‐cell patch clamp recording.

Of 117 magnocellular neurones recorded in the SON in vivo, fifteen (13%) displayed a short latency excitation, sixty‐eight (58%) a short latency inhibition, six (5%) were unresponsive and twenty‐eight (24%) gave long latency responses following SON stimulation.

The responses of putative vasopressin cells in the SON to SCN stimulation in vivo (4 out of 61 cells, 7% excited; 49 out of 61 cells, 80% inhibited) were significantly different from those of putative oxytocin cells (10 out of 50 cells, 20% excited and 16 out of 50 cells, 32% inhibited; P < 0.02, test for differences between proportions).

Recordings in vitro using patch technology in whole‐cell mode showed both inward and outward currents in SON cells at holding potentials near resting membrane potential following stimulation of the SCN region. The outward currents could be blocked by bicuculline (10 μm; n= 7) and the inward currents were blocked by the non‐NMDA antagonist 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2,3‐dione (5 μm; n= 4).

We conclude that there is a strong projection from the SCN to the SON with both inhibitory (GABAergic) and excitatory (glutamatergic) components which may regulate the daily changes in neurohypophysial hormone secretion.
Detachment of low‐force bridges contributes to the rapid tension transients of skinned rabbit skeletal muscle fibres
Authors: Seow Chun Y. , Shroff Sanjeev G. , Ford Lincoln E.
Published: September 2004

To probe the cross‐bridge cycle and to learn more about the cardioplegic agent BDM (2,3‐butanedione monoxime), its effects on the force–velocity properties and tension transients of skinned rabbit muscle fibres were studied at 1–2 °C and pH 7.0.

Three millimolar BDM decreased isometric force by 50%, velocity by 29%, maximum power by 73%, and stiffness by 25%, so that the relative stiffness (stiffness/force ratio) increased by 50% compared with reference conditions in the absence of BDM.

Tension transients obtained under the reference condition (0 BDM) could be represented by three components whose instantaneous stiffness accounted for the initial (Phase 1) force deviation and whose exponential recoveries caused the rapid, partial (Phase 2) force recovery following the step. The fastest component had non‐linear extension–force properties that accounted for about half the isometric stiffness and it recovered fully. The two slower components had linear extension‐force properties that together accounted for the other half of the sarcomere stiffness. These components recovered only partially following the step, producing the intermediate (T2) level which the force approached during Phase 2.

Matching the force transients obtained under test conditions (3 mm BDM) required three alterations: (1) reducing the amplitude of the two slower components by 50%, in proportion to isometric force, (2) adding a non‐relaxing component and (3) decreasing the amplitude of the rapidly recovering component by 12.5% so that its relative amplitude (amplitude/isometric force) was increased by 75%. The non‐recovering component and the increase in relative amplitude of the rapid component were responsible for the increase in relative stiffness of the fibres produced by BDM. The rapidly recovering component had the same time constant and step‐size‐dependent recovery rates as the fastest of the three mono‐exponential components isolated from the tension transient response under the reference condition. BDM therefore appeared to augment the fastest component of the tension transient under the reference condition.

The results suggest that BDM detains cross‐bridges in low‐force, attached states. Since these bridges are attached, they contribute to sarcomere stiffness. Since they are detained, relaxation or reversal of their immediate responses is probably due to bridge detachment rather than to their undergoing the power stroke. The observation that a portion of the test response matched the fastest component of the reference response when the amplitude of the fastest component was increased suggests that a part of the normal rapid, transient tension recovery following a release step is due to detachment of low‐force bridges moved to negative‐force positions by the step.
Presynaptic GABAB and adenosine A1 receptors regulate synaptic transmission to rat substantia nigra reticulata neurones
Authors: Shen Ke‐Zhong , Johnson Steven W.
Published: September 2004

Patch pipettes were used to record whole‐cell currents under voltage clamp in substantia nigra zona reticulata (SNR) neurones in the rat midbrain slice. Bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA receptors (IPSCs).

Baclofen reduced the amplitude of IPSCs by 48% at its IC50 value of 0.60 mm. The GABAB antagonist CGP 35348 blocked this effect with a Kd value estimated by Schild analysis of 5 mm.

Adenosine reduced IPSCs by 48% at its IC50 value of 56 mm. Adenosine agonists reduced IPSCs with the following rank order of potency: CPA (N6 ‐cyclopentyladenosine) > R‐PIA (R(‐)N6 ‐(2‐phenylisopropyl) adenosine) > CHA (N6 ‐cyclohexyladenosine) = NECA (5’‐N‐ethylcarboxamidoadenosine) > 2‐CADO (2‐chloroadenosine) > adenosine. Schild analysis yielded a Kd value of 0.4 nM for antagonism of CPA by the adenosine A1 receptor antagonist DPCPX (8‐cyclopentyl‐l,3‐dipropylxanthine).

Both baclofen and adenosine reduced the magnitude of paired‐pulse depression of IPSCs, and neither blocked currents evoked by GABA, which was pressure‐ejected from micropipettes.

Glutamate EPSCs were reduced by baclofen (IC50= 0.78 mm) and adenosine (IC50= 57 mm) Schild analysis yielded a Kd value of 11 mm for antagonism of baclofen‐induced inhibition of EPSCs by CGP 35348. DPCPX (1 mm) completely blocked the inhibitory effects of adenosine (100 mm) and CPA (100 nM) on EPSCs. Neither adenosine nor baclofen reduced inward currents evoked by glutamate which was pressure‐ejected from micropipettes.

These results show that presynaptic GABAB and A1 receptors reduce glutamate and GABA release from nerve terminals in the SNR.
Fast (mainly 30–100 Hz) oscillations in the cat cerebellothalamic pathway and their synchronization with cortical potentials
Authors: Timofeev Igor , Steriade Mircea
Published: September 2004

Intracellular recordings from 216 thalamocortical (TC) neurones in the ventrolateral (VL) nucleus of intact‐cortex and decorticated cats under ketamine–xylazine anaesthesia revealed spontaneously occurring fast oscillations (mainly 30–100 Hz) in 86% of investigated cells. The fast depolarizing events consisted of excitatory postsynaptic potentials (EPSPs), giving rise to fast prepotentials (FPPs) in 22% of neurones, which eventually lead to full‐blown action potentials. The frequency of fast events changed by factors of 2–5 in periods as short as 0.3–1.0 s.

The spontaneous oscillations were similar to responses evoked in VL relay neurones by stimuli to the afferent cerebellofugal axons in brachium conjunctivum (BC) and were strikingly reduced or abolished after electrolytic lesion of BC axons.

The amplitude and duration of fast depolarizing events were significantly reduced during the descending phase of the inhibitory postsynaptic potentials (IPSPs) in TC cells, related to spontaneous spindles or evoked by local thalamic stimulation.

Averaged field potentials recorded from motor cortex and triggered by EPSPs and/or action potentials of intracellularly recorded VL cells demonstrated that both spontaneous and BC‐evoked fast depolarizations in VL relay neurones were coherent with fast rhythms in cortical area 4.

These results show that, in addition to the thalamic and cortical generation sites of the fast (so‐called gamma) oscillations, prethalamic relay stations, such as deep cerebellar nuclei, are major contributors to the induction of fast rhythms which depend on the depolarization of thalamic and cortical neurones and which represent a hallmark of brain activation patterns.
Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in xenopus laevis oocytes
Authors: Amasheh Salah , Wenzel Uwe , Weber Wolf‐Michael , Clauss Wolfgang , Daniel Hannelore
Published: September 2004

To gain information on the mode of operation of the renal proton‐coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2.

Using differently charged glycyl‐dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH‐dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2–50 μm) between pH 5.5 and 7.4 and membrane potentials of ±0 to −50 mV.

Maximal substrate‐evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pH0). Potential‐dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages.

The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from –50 to –150 mV substrate affinities decrease 10‐ to 50‐fold whereas apparent Imax increases almost 10‐fold.

Even at saturating H+/H3O+ and dipeptide concentrations the I–V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters.

These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.
Spike‐evoked suppression and burst patterning in dorsal root ganglion neurons of the rat
Authors: Amir Ron , Devor Marshall
Published: September 2004

A low level of spontaneous impulse discharge is generated within dorsal root ganglia (DRGs) in intact animals, and this activity is enhanced following nerve injury. Many physiological stimuli present in vivo are capable of augmenting this ectopic discharge. Whatever their cause, episodes of sharply accelerated DRG firing tend to be followed by ‘after‐suppression’ during which discharge falls below baseline rate. In this study we examined the process of postexcitation suppression of firing rate, and how it shapes spike patterning in primary sensory neurons.

We recorded intracellularly from sensory neurons in excised rat DRGs in vitro. Trains of spikes triggered by intracellular current pulses evoked a prolonged hyperpolarizing shift. This shift appeared to be due to activation of a Ca2+‐dependent K+ conductance (gK(Ca)). Spikes evoked by just‐suprathreshold pulses triggered a hyperpolarizing shift and spike cessation. As the shift decayed, spiking was restored. The net result was bursty (on–off) discharge, a previously unexplained peculiarity of ectopic discharge in some DRG neurons in vivo.

Conditioning nerve tetani delivered to axons of neurons which share the DRG with the impaled neuron evoked transient depolarization (‘cross‐depolarization’). However, when stimulus strength was increased so as to include the axon of the impaled neuron, the net result was a hyperpolarizing shift. Nerve stimulation that straddled the threshold of the axon of the impaled neuron drove it intermittently, but it always drove axons of at least some neighbouring neurons. The result was dynamic modulation of the membrane potential of the impaled neuron as cross‐depolarization and spike‐evoked hyperpolarizing shifts played off against one another. Membrane potential shifted in the hyperpolarizing direction whenever the axon was activated, and shifted in the depolarizing direction whenever it was silent. Dynamic modulation of this sort probably also occurs in vivo when stimuli are drawn over the surface of the skin.
Linear transduction of natural stimuli by dark‐adapted and light‐adapted rods of the salamander, Ambystoma tigrinum
Authors: Vu Tania Q. , McCarthy Sean T. , Owen W. Geoffrey
Published: September 2004

We examined signal, noise and response properties of salamander rod photoreceptors by measuring: (a) the circulating current of rods which were adapted to darkness and to a wide range of backgrounds; (b) contrasts of natural environments; (c) the effect of adaptation on the linear response range of rods; and (d) the behaviour of rods responding to dynamically modulated stimuli having a range of contrasts found in nature.

In the dark, the circulating current contained two noise components analogous to those described in toad. A discrete noise component consisted of events occurring at a rate of 1 event per 32 s (21 °C) and had a variance of 0.036 pA2. A continuous noise component contributed 0.022 pA2 to the dark current, roughly equal to the discrete noise variance.

Exposure to a wide range of steady backgrounds (suppressing up to 80% of the circulating current), elicited a sustained fluctuating photocurrent having a power spectrum which resembled those of single photon responses and was consistent with the linear summation of single photon events; this indicates that the primary source of noise in the current is caused by the light.

Eighty‐nine per cent of the contrasts (C) measured in natural environments had :C: < 50 %, where C= :I–Imean:/:Imean:. The linear response range elicited by brief flashes expanded with brighter backgrounds, well‐encompassing flash contrasts of 100 %.

Dynamically modulated stimuli and incremental flashes having contrasts similar to those in natural scenes elicited small currents which deviated by a few picoamps about the mean and the transfer functions computed from each type of stimulus–response pair closely corresponded to one another. These results indicate that in natural environments, rods behave as linear small‐signal transducers of light.
Oxygen Transport and Utilization during Feeding in the Young Lamb
Authors: Grant Daniel A. , Fewell James E. , Walker Adrian M. , Wilkinson Malcolm H.
Published: September 2004

Five lambs (19–27 days old) were studied to determine the effects of feeding on cardiorespiratory function.

Each lamb was instrumented to record cardiac output, aortic and pulmonary artery pressure and arterial and mixed venous oxyhaemoglobin saturations (Sa,O2 and Sv,O2).

During feeding, arterial haemoglobin desaturated and resaturated sequentially during the periods of sucking and non‐sucking. The nadir of these Sa,O2 desaturations (83 ± 2%, mean ±s.e.m.) was significantly lower than the baseline value (92 ± 2%, P≤ 0.05, ANOVA). Sa,O2 returned to the baseline level between periods of sucking. Sv,O2 also decreased (55 ± 3% baseline, 46 ± 3% sucking, P≤ 0.05) but, in contrast to Sa,O2, it remained significantly lower than baseline levels in the pauses between periods of sucking.

Arterial pressure increased during feeding (94 ± 4 mmHg baseline, 113 ± 6 mmHg feeding, P≤ 0.05), while heart rate and cardiac index did not change.

Total body oxygen consumption rose during the pauses between sucking periods (10.9 ± 1.1 ml O2 min−1 kg−1 baseline, 13.9 ± 1.2 ml O2 min−1 kg−1 non‐sucking, P≤ 0.05) and was provided for by a significant increase in total body oxygen extraction as systemic oxygen transport was unchanged.

Our results reveal that during feeding in young lambs oxygen consumption increases and body stores of oxygen (e.g. Sv,O2) become depleted; this combination may promote rapid arterial desaturation and cyanosis during feeding.
Paradoxical effect of oxygen administration on breathing stability following post‐hyperventilation apnoea in lambs
Authors: Wilkinson M. H. , Berger P. J. , Blanch N. , Brodecky V. , Jones C. A.
Published: September 2004

Oxygen administration is thought to suppress periodic breathing (PB) by reducing carotid body activity, and yet earlier experiments in neonates have shown that PB incidence may be increased following the application of hyperoxia. To clarify this paradox, we studied the changes in the pattern of PB that occur following administration of oxygen in a lamb model of PB.

PB was induced in eleven of seventeen anaesthetized lambs following passive hyper‐ventilation with air. When oxygen was administered during PB, the pattern was first enhanced, as evidenced by a sudden decrease in the ratio of the ventilatory duration to the apnoeic pause duration, and then suppressed, as evidenced by a progressive return to stable breathing which was associated with an increase in minute ventilation.

Five of the six lambs that did not show PB following passive hyperventilation with air could be made to do so if oxygen was substituted for air as the inspired gas following passive hyperventilation.

Five of the eleven lambs that showed PB following hyperventilation with air responded to the application of oxygen during PB by switching to a gross form of episodic breathing consisting of long apnoeic pauses followed by equally long periods of breathing during which minute ventilation fell progressively with time.

We conclude that when applied against a background of arterial hypoxaemia, oxygen has a destabilizing influence on ventilation in that (a) it accentuates the unstable breathing that occurs during PB, (b) it induces PB in lambs that exhibited stable breathing in air, and (c) it may precipitate episodic breathing.
A Comparison of the Ventilatory Response of Sleeping Newborn Lambs to Step and Progressive Hypoxaemia
Authors: Cohen Gary , Malcolm Girvan , Henderson‐Smart David
Published: September 2004

Slight variations in the rate at which hypoxaemia develops may significantly alter the ventilatory response (VR) elicited. Here we have developed a technique to compare the VRs elicited from sleeping newborn lambs by specific (stepversusprogressive), short‐duration (≥ 5 min) episodes of hypoxaemia. The results may help us understand the limitations of using tests which deliver poorly defined stimuli to evaluate the postnatal development of the oxygen chemoreflex.

The VRs of five lambs elicited by a 5 min step or progressive reduction in the arterial oxygen saturation (Sa,O2) during quiet sleep were compared. Minute ventilation (V̇i, face mask) andSa,O2(pulse oximeter) were measured continuously. Alternate step (Sa,O2reduced to 80–85 % within 60 s and maintained for a further 4 min) and progressive tests (progressive reduction inSa,O2to 80 % over 5 min) were administered daily between postnatal days 2–14.

There was a significant difference between the mean VR to stepversusprogressive hypoxaemia. The VR to a step challenge was biphasic (ΔV̇i=+32 ± 5% at 1 min and −1 ± 4% at 5 min; mean ±s.e.m.). Progressive hypoxaemia elicited a more subdued but sustained hyperpnoea (ΔV̇i=+11 ± 2% at 1 min and +11 ± 4% at 5 min). The difference between these two response profiles was statistically significant (P< 0.001).

Mean responses of lambs aged ≤ 5 days (4 ± 0.2 days) and ≥ 9 days (10 ± 0.3 days) were also compared. There was an upward shift in the position of step and progressive response curves of older lambs, reflecting primarily the increased vigour of the initial hyperpnoea elicited by step (ΔV̇iat 1 min =+20 ± 4% at 4 daysvs.+40 ± 11 % at 10 days) as well as progressive (ΔV̇iat 1 min =+6 ± 2% at 4 daysvs.+17 ± 5%at 10 days) hypoxaemia.

Qualitatively different VRs may be elicited from the newborn, depending upon the specific hypoxaemic profile administered. Therefore, to evaluate the significance of VRs elicited in response to classical, steady‐state hypoxia at different postnatal ages properly, the stimulus must be accurately described.