bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement of rapid Ca2+ entry and synaptic activation of metabotropic glutamate receptors for the induction of long‐term depression in adult rat hippocampus
Authors: Otani Satoru , Connor John A.
Published: September 2004

During block of γ‐aminobutyric acid‐A‐mediated inhibition, low‐frequency stimulation (2 Hz, 900 pulses) to Schaffer collateral‐CA1 neuron synapses of adult rat hippocampus induced an N‐methyl‐D‐aspartate receptor‐independent, postsynaptic Ca2+‐dependent depression of synaptic strength (long‐term depression; LTD).

Ratio imaging with fura‐2 revealed moderate dendritic [Ca2+] increases (≈500 nM) during only the initial ≈30 s of the 7.5 min stimulation period. Conditioning for 30 s was, however, insufficient to induce LTD.

The [Ca2+] changes were insensitive to the metabotropic glutamate receptor (mGluR) antagonist (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG). MCPG, however, completely blocked LTD when present during conditioning.

The [Ca2+] changes were abolished by postsynaptic hyperpolarization (‐110 mV at the soma). Hyperpolarizing neurons to ‐110 mV during conditioning significantly attenuated LTD induction.

LTD induction was also blocked by the postsynaptic presence of the protein kinase C inhibitor peptide PKC(19‐36).

These results suggest that LTD induction in adult hippocampus by prolonged low‐frequency stimulation depends on both a rapid Ca2+ influx through voltage‐sensitive channels and synaptic stimulation of mGluRs which may be coupled to phospholipase C.
Activation of cAMP‐dependent Cl− currents in guinea‐pig Paneth cells without relevant evidence for CFTR expression
Authors: Tsumura Takehiko , Hazama Akihiro , Miyoshi Takashi , Ueda Shunji , Okada Yasunobu
Published: September 2004

To determine whether Paneth cells exhibit functional expression of cAMP‐activated Cl− currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole‐cell patch clamp and single‐cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea‐pig small intestine.

Prominent activation of Cl− currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP‐activated Cl− current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A.

Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl− channel, such as the ohmic current‐voltage relationship, the anion selectivity with a Type III sequence (Br− > Cl− > I−≫ F−≥ gluconate−), I−‐induced blockage, insensitivity to a stilbene‐derivative Cl− channel blocker, and sensitivity to a carboxylate analogue Cl− channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl− channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl− currents exhibited inactivation kinetics.

Expression of CFTR mRNA could not be detected by RT‐PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole‐crypt level. The presence of a small number of CFTR‐expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry.

Taken together, it appears that guinea‐pig Paneth cells functionally express cAMP‐activated Cl− conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.
Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus
Authors: Pennartz C. M. A. , De Jeu M. T. G. , Geurtsen A. M. S. , Sluiter A. A. , Hermes M. L. H. J.
Published: September 2004

Whole cell patch clamp recordings of neurons in slices of the suprachiasmatic nucleus (SCN) were made in order to assess their electrophysiological and morphological heterogeneity. This assessment was accomplished by (i) quantification of intrinsic membrane properties recorded in current clamp mode, (ii) studying frequency distributions of these properties, (iii) grouping of cells based on visual inspection of data records, and (iv) use of cluster analysis methods.

Marked heterogeneity was found in the resting membrane potential, input resistance, time constant, rate of frequency adaptation, size of rebound depolarization (low‐threshold Ca2+ potential) and regularity of firing. The frequency distribution of these membrane properties deviated significantly from a normal distribution. Other parameters, including spike amplitude and width, amplitude and rising slope of the spike after‐hyperpolarization (AHP) and amplitude of the spike train AHP, showed considerable variability as well but generally obeyed a normal distribution.

Visual inspection of the data led to partitioning of cells into three clusters, viz. cluster I characterized by monophasic spike AHPs and irregular firing in the frequency range from 1.5 to 5.0 Hz; cluster II with biphasic spike AHPs and regular firing in the same range; and cluster III with large rebound depolarizations and biphasic spike AHPs. In a post hoc analysis, these clusters also appeared to differ in other membrane properties. This grouping was confirmed by hierarchical tree clustering and multidimensional scaling.

The light microscopic properties of recorded neurons were studied by biocytin labelling. Neurons had monopolar, bipolar or multipolar branching patterns and were often varicose. Axons sometimes originated from distal dendritic segments and usually branched into multiple collaterals. Many cells with extra‐SCN projections also possessed intranuclear axon collaterals. We found no morphological differences between clusters except that cluster III neurons possessed more axon collaterals than cluster I or II cells.

These results suggest that SCN neurons are heterogeneous in some basic as well as active membrane properties and can be partitioned into at least three clusters. Cluster I and II cells fire spontaneously in a regular and irregular mode, respectively, and sustain prolonged spike trains. In contrast, cluster III cells have low firing rates but may adopt a burst‐like firing mode when receiving appropriate input. While all clusters transmit output to target cells within and outside SCN, cluster III cells in particular are suggested to affect excitability of large numbers of SCN neurons by their extensive local network of axon collaterals.
Autaptic inhibitory currents recorded from interneurones in rat cerebellar slices
Authors: Pouzat Christophe , Marty Alain
Published: September 2004

While the presence of autapses in the brain is indicated by a large body of morphological evidence, the functional role of these structures has remained unclear. To probe for autaptic currents, we have recorded current responses following short somatic depolarizing pulses in Cl−‐loaded interneurones (stellate and basket cells) from rat cerebellar slices (animals aged 27‐39 days).

In ≈20 % of the recordings, fluctuating inward current transients were obtained with a latency of 1.15‐2.45 ms (measured from the peak of the depolarization‐induced Na+ current; n= 10).

These transients were blocked by bicuculline and were sensitive to the extracellular Ca2+ concentration.

Assuming low release probability, as suggested by the high failure rate (0.65‐0.92, n= 10), quantal sizes ranging from 21 to 178 pA (‐70 mV; n= 10) were calculated from a variance analysis of autaptic current amplitudes.

We conclude that ≈20 % of interneurones have a functional autapse. Autaptic currents may inhibit firing of interneurones during high frequency discharges.
Overexpression of nerve growth factor in the heart alters ion channel activity and β‐adrenergic signalling in an adult transgenic mouse
Authors: Heath B. M. , Xia J. , Dong E. , An R. H. , Brooks A. , Liang C.‐S. , Federoff H. J. , Kass R. S.
Published: September 2004

The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied.

There was a 12 % increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90 % repolarization (APD90) was prolonged by 142 % compared with wild‐type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L‐type Ca2+ current.

The effect on Ito and IK(ur) of TEA and 4‐aminopyridine (4‐AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mm 4‐AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157 %.

The isoprenaline‐induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in β‐adrenergic receptor (β‐AR) density, as this was increased in NGF tissue by 55 %. Analysis of β‐AR subtypes showed that this increase was entirely due to an increase in β2‐AR density with no change in β1‐ARs.

The response of the β‐AR‐coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected.

In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in β2‐AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the β‐ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.
Glycine‐activated currents are changed by coincident membrane depolarization in developing rat auditory brainstem neurones
Authors: Backus Kurt H. , Deitmer Joachim W. , Friauf Eckhard
Published: September 2004

During early ontogeny, glycine receptors (GlyRs) exert depolarizing responses which may be of developmental relevance. We have used the gramicidin‐perforated patch technique to elucidate the mechanism of glycine‐activated currents in developing neurones of the rat lateral superior olive (LSO).

When the holding potential was set to −60 mV, perforated‐patch recordings revealed glycine‐induced inward currents in 59 %, outward currents in 5 % and biphasic currents in 34 % of the LSO neurones tested (n= 44). The biphasic currents were characterized by a transient outward phase which was followed by an inward phase.

Ion substitution experiments showed that both Cl− and HCO3− contributed to the glycine‐ induced biphasic current responses.

In the biphasic responses, the reversal potential of the glycine‐induced current (Egly) depended on the response phase. A strong shift of Egly from a mean of −72 mV during the outward phase of the glycine response to a mean of −51 mV during the inward phase was observed, suggesting a shift of an ion gradient.

When the membrane potential was depolarized, ‘tail’ currents were induced in the presence of glycine. An increased duration or amplitude of the evoked depolarizations resulted in a proportional enlargement of these tail currents, indicating that they were produced by a shift of an ion gradient. Since changes of the HCO3− gradient are negligible, because of the carbonic anhydrase activity, we suggest that these tail currents were caused by a shift of the Cl− gradient.

We conclude that Cl− accumulates intracellularly during the activation of GlyRs and, consequently, Egly moves towards more positive values.

Coincident depolarizing stimuli enhanced intracellular Cl− accumulation and the shift of Egly, thereby switching hyperpolarizing to depolarizing action. This change could assist in an activity‐dependent strengthening and refinement of glycinergic synapses during the maturation of inhibitory connectivity.
μ‐Opioid receptor activation inhibits N‐ and P‐type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus
Authors: Soldo Brandi L. , Moises Hylan C.
Published: September 2004

The whole‐cell voltage‐clamp technique was used to examine opioid regulation of Ba2+ currents (IBa) through voltage‐sensitive Ca2+ channels in isolated magnocellular supraoptic neurones (MNCs). The effects of local application of μ‐, δ‐ or κ‐opioid receptor selective agonists were examined on specific components of high voltage‐activated (HVA) IBa, pharmacologically isolated by use of Ca2+ channel‐subtype selective antagonists.

The μ‐opioid receptor selective agonist, DAMGO, suppressed HVA IBa (in 64/71 neurones) in a naloxone‐reversible and concentration‐dependent manner (EC50= 170 nm, Emax= 19.5 %). The DAMGO‐induced inhibition was rapid in onset, associated with kinetic slowing and voltage dependent, being reversed by strong depolarizing prepulses. Low‐voltage activated (LVA) IBa was not modulated by DAMGO.

Administration of κ‐ (U69 593) or δ‐selective (DPDPE) opioid receptor agonists did not affect IBa. However, immunostaining of permeabilized MNCs with an antibody specific for κ1‐opioid receptors revealed the presence of this opioid receptor subtype in a large number of isolated somata.

μ‐Opioid‐induced inhibition in IBa was largely abolished after blockade of N‐type and P‐type channel currents by ω‐conotoxin GVIA (1 μm) and ω‐agatoxin IVA (100 nm), respectively. Quantitation of antagonist effects on DAMGO‐induced reductions in IBa revealed that N‐ and P‐type channels contributed roughly equally to the μ‐opioid sensitive portion of total IBa.

These results indicate that μ‐opioid receptors are negatively coupled to N‐ and P‐type Ca2+ channels in the somatodendritic regions of MNCs, possibly via a membrane‐delimited G‐protein‐dependent pathway. They also support a scheme in which opioids may act in part to modulate cellular activity and regulate neurosecretory function by their direct action on the neuroendocrine neurones of the hypothalamic supraoptic neucleus.
Synaptic inhibition: its role in suprachiasmatic nucleus neuronal thermosensitivity and temperature compensation in the rat
Authors: Burgoon P. W. , Boulant J. A.
Published: September 2004

Whole‐cell patch clamp recordings of neurones in the suprachiasmatic nucleus (SCN) from rat brain slices were analysed for changes in spontaneous synaptic activity during changes in temperature. While recent studies have identified temperature‐sensitive responses in some SCN neurones, it is not known whether or how thermal information can be communicated through SCN neural networks, particularly since biological clocks such as the SCN are assumed to be temperature compensated.

Synaptic activity was predominantly inhibitory and mediated through GABAA receptor activation. Spontaneous inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) were usually blocked with perifusion of 10–50 μm bicuculline methiodide (BMI). BMI was used to test hypotheses that inhibitory synapses are capable of either enhancing or suppressing the thermosensitivity of SCN neurones.

Temperature had opposite effects on the amplitude of IPSPs and IPSCs. Warming decreased IPSP amplitude but increased IPSC amplitude. This suggests that thermally induced changes in IPSP amplitude are primarily influenced by resistance changes in the postsynaptic membrane. The thermal effect on IPSP amplitude contributed to an enhancement of thermosensitivity in some neurones.

In many SCN neurones, temperature affected the frequency of IPSPs and IPSCs. An increase in IPSP frequency with warming and a decrease in frequency during cooling made several SCN neurones temperature insensitive, allowing these neurones to maintain a relatively constant firing rate during changes in temperature. This temperature‐adjusted change in synaptic frequency provides a mechanism of temperature compensation in the rat SCN.
Inhibitory control of plateau properties in dorsal horn neurones in the turtle spinal cord in vitro
Authors: Russo Raúl E. , Nagy Frédéric , Hounsgaard Jørn
Published: September 2004

The role of inhibition in control of plateau‐generating neurones in the dorsal horn was studied in an in vitro preparation of the spinal cord of the turtle. Ionotropic and metabotropic inhibition was found to condition the expression of plateau potentials.

Blockade of γ‐aminobutyric acid (GABAA) and glycine receptors by their selective antagonists bicuculline (10‐50 μM) and strychnine (5‐20 μM) enhanced the excitatory response to stimulation of the dorsal root and facilitated the expression of plateau potentials.

Bicuculline and strychnine also facilitated the generation of plateau potentials in response to depolarizing current pulses, suggesting the presence of tonic ionotropic inhibitory mechanisms in turtle spinal cord slices.

Activation of GABAB receptors also inhibited plateau‐generating neurones. The selective agonist baclofen (5‐50 μM) inhibited wind‐up of the response to repeated depolarizations induced synaptically or by intracellular current pulses.

Baclofen reduced afferent synaptic input. This effect was not affected by bicuculline or strychnine and was blocked by the selective GABAB receptor antagonist 2‐hydroxysaclofen (2‐OH‐saclofen, 100‐400 μM).

Postsynaptically, baclofen inhibited plateau properties. Activation of GABAB receptors produced a hyperpolarization (7.0 ± 0.5 mV, mean ± s.e.m., n= 29) with an associated decrease in input resistance (22.7 ± 3.1 %, n= 24). These effects were blocked by extracellular Ba2+ (1‐2 mM).

When the baclofen‐induced hyperpolarization and shunt were compensated for by adjusting the bias current and the strength of the stimulus, baclofen still inhibited generation of plateau potentials. Wind‐up and after‐discharges were also inhibited by baclofen. These effects remained in the presence of tetrodotoxin (1 μM) and were antagonized by 2‐OH‐saclofen.

The inhibition of plateau properties was observed even when the baclofen‐induced hyperpolarization and shunt were blocked by Ba2+ and when potassium channels were blocked by Ba2+ (3 mM), tetraethylammonium (TEA, 15 mM) and apamin (0.25‐0.5 μM). The baclofen‐sensitive component of the plateau potential was reduced by nifedipine (10 μM), suggesting a modulation of postsynaptic L‐type Ca2+ channels.

We suggest that inhibitory regulation of plateau properties plays a role in somatosensory processing in the dorsal horn. The inhibitory control of wind‐up and after‐discharges may be particularly significant in physiological and therapeutic control of central sensitization to pain.
The influence of corticofugal feedback on the temporal structure of visual responses of cat thalamic relay cells
Authors: Wörgötter F. , Nelle E. , Li B. , Funke K.
Published: September 2004

Visually driven single‐unit activity was recorded in the dorsal lateral geniculate nucleus (dLGN) of the anaesthetized cat while inactivating or stimulating the corticofugal feedback from area 17/18 by means of cortical cooling or application of GABA (inactivation), or application of glutamate or quisqualate (Glu, Quis; stimulation) to layer VI.

Manipulations of the corticofugal feedback primarily affected the multimodal interspike interval pattern previously reported to be present in the tonic component of visual responses elicited by spot‐like stimuli.

Sixty‐three per cent of all neurons could be influenced, and temporally localized interspike interval distributions were measured which commonly consisted of one fundamentalinterval peak (leftmost peak) and integer multiples thereof (higher orderpeaks). During blockade of the corticofugal feedback, interspike intervals were redistributed into the higher order peaks in about 70 % of the cases, accompanied by a reduced mean firing rate. During stimulation the reverse effect occurred in 69 % of cases.

Increased synchronization of the EEG (increased power in the δ‐wave range, 1‐4 Hz) had an effect similar to cortex inactivation. The specificity of corticofugal effects was verified by consideration of these EEG effects and by dLGN double recordings with one dLGN cell topographically matched with the cortical inactivation/activation site and the second cell outside this area. Clear effects due to manipulation of the corticofugal feedback were found only for the matched dLGN site.

In addition we observed that the peaks of the interval distributions were narrower during active corticofugal feedback, such that the temporal dispersion of the signal transmission to the cortex was reduced.

The mechanisms underlying this effect were further analysed in a biophysically realistic model demonstrating that the timing of the spikes in the dLGN is improved as soon as the cortical feedback is active. The high degree of convergence/divergence between neurons along the closed feedback loop thereby leads to a temporal averaging effect which reduces the interval dispersion and also introduces synchronization between dLGN cells.

Such a mechanism may thus counteract the deterioration of spike timing accuracy which would otherwise occur as a consequence of synaptic noise and other uncorrelated sources of activity at a given neuron.
Properties of voltage‐activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus
Authors: Shmigol A. V. , Eisner D. A. , Wray Susan
Published: September 2004

The intracellular calcium concentration ([Ca2+]i) was measured at 35 °C using the fluorescent indicator indo‐1 in patch‐clamped, single uterine myocytes from pregnant rats to investigate the relationship between depolarization, Ca2+ current (ICa) and [Ca2+]i.

Membrane depolarization activated ICa and produced a [Ca2+]i transient. The rapid increase in [Ca2+]i occurred at the same time as the inward ICa. Both ICa and the increase in [Ca2+]i were abolished by nifedipine (10 μm).

When the membrane potential was held at ‐80 mV the threshold depolarization for an increase in [Ca2+]i was about ‐55 to ‐50 mV. As the magnitude of the depolarization was increased to about 0 mV there was an increase in the size of both ICa and the increase in [Ca2+]i. As the magnitude of the depolarization was further increased both ICa and the [Ca2+]i increase declined.

When the depolarizing pulses were applied at 3 Hz to mimic normal action potentials then the individual [Ca2+]i transients did not fully relax and a tetanic rise of [Ca2+]i was observed. Under these conditions, there was not a simple relationship between the magnitude of the Ca2+ response and Ca2+ entry. When pairs of depolarizing pulses were applied, the increase in [Ca2+]i produced by the second pulse was larger (in relation to the magnitude of the L‐type Ca2+ current) than that produced by the first pulse. This facilitation was abolished by both ryanodine and cyclopiazonic acid suggesting a role for release from intracellular stores.

We conclude that the L‐type Ca2+ current is the major source of Ca2+ ions entering the cell to produce the [Ca2+]i transient on depolarization. The magnitude of the increase in [Ca2+]i may, however, be amplified by Ca2+‐induced Ca2+ release.
Presynaptic inhibition by dopamine of a discrete component of GABA release in rat substantia nigra pars reticulata
Authors: Miyazaki Takefumi , Lacey Michael G.
Published: September 2004

Whole‐cell patch clamp recordings were made from substantia nigra pars reticulata (SNr) neurones in rat midbrain slices. Monosynaptic IPSCs were evoked by electrical stimulation of the cerebral peduncle in the presence of the glutamate receptor antagonists CNQX (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione) and AP5 (2‐amino‐5‐phosphonopentanoic acid).

IPSCs were predominantly outward at −70 mV (in 124/135 cells), with a reversal potential of −83 mV, a time to peak of 2.6 ms and a decay time constant of 6.5 ms. Faster inward IPSCs were also observed in thirty‐five cells, with a time to peak of 1.0 ms, a decay time constant of 2.3 ms, and a reversal potential of −61 mV. Both IPSCs were sensitive to the GABAA receptor antagonists picrotoxin or bicuculline.

In cells recorded with Cs+‐filled pipettes, the outward IPSC reversal potential was shifted to −76 mV, closer to the estimated Cl− equilibrium potential of −56 mV, while that of the inward IPSC was unchanged at −64 mV.

The outward IPSC was reversibly depressed by up to 100 % by dopamine in a concentration‐dependent manner with an IC50 of 10.5 μm, while the inward IPSC was relatively insensitive.

Dopamine was without effect on cell holding current, or on outward IPSC reversal potential, but it increased paired‐pulse IPSC facilitation, consistent with a presynaptic site of action.

The D1‐like dopamine receptor agonist SKF 38393 (10 μm) depressed the outward IPSC by 43 %, while the D2‐like dopamine receptor agonist quinpirole (10 μm) was without effect.

It is concluded that GABA‐ergic synaptic input onto distal rather than proximal regions of SNr neurones is susceptible to presynaptic inhibition via a D1‐like receptor. These inputs are probably from striato‐nigral fibres, and their inhibition by dopamine is likely to influence the patterning of basal ganglia output.
Expression of functional GABAA receptors in cholecystokinin‐secreting gut neuroendocrine murine STC‐1 cells
Authors: Glassmeier G. , Herzig K.‐H. , Höpfner M. , Lemmer K. , Jansen A. , Scherübl H.
Published: September 2004

Gastrointestinal neuroendocrine (NE) cells synthesize, store and secrete γ‐aminobutyric acid (GABA). Recently, an autocrine‐paracrine function of GABA has been proposed for secretion from NE cells.

To search for functional GABAA receptors in NE gut cells, we performed whole‐cell and perforated‐patch‐clamp studies in the intestinal cholecystokinin (CCK)‐secreting NE cell line STC‐1.

Application of GABA evoked currents in STC‐1 cells. These effects were mimicked by muscimol, an agonist of GABAA receptors, and blocked by picrotoxin or bicuculline, antagonists of GABAA receptors. The GABA‐ or muscimol‐activated currents reversed near 0 mV, which under the recording conditions used was consistent with the activation of the GABAA receptor‐Cl− channel complex.

In contrast to the effect on most neurons, GABA as well as muscimol led to a (reversible) depolarization of the membrane potential of STC‐1 cells. Membrane depolarization in turn activated voltage‐gated Ca2+ channels and increased intracellular Ca2+ concentrations in STC‐1 cells.

In accordance with the observed membrane depolarization and activation of voltage‐gated Ca2+ channels, both GABA and muscimol stimulated Ca2+‐dependent CCK release. In contrast, bicuculline inhibited the GABA‐induced secretion of CCK.

Using the reverse transcription‐polymerase chain reaction (RT‐PCR), mRNA of the GABAA receptor subunits α2, α3, α5, β1, β3 and δ could be detected in STC‐1 cells.

In summary, we have shown that the CCK‐secreting gut NE cell line STC‐1 expresses functional GABAA receptors and that GABA stimulates CCK release. Thus, GABA is involved in the fine tuning of CCK secretion from the gut NE cell line STC‐1.
Persistent depolarizing action of GABA in rat Cajal‐Retzius cells
Authors: Mienville Jean‐Marc
Published: September 2004

To characterize membrane properties that might be relevant to the function and fate of Cajal‐Retzius (CR) cells, the pharmacological and physiological effects of GABA acting at GABAA receptors were studied in CR cells from embryonic (E18) and postnatal (P11–13) slices of rat neocortex.

From the embryonic to the postnatal stage, GABA‐induced maximum current almost tripled, the EC50 increased from 38 to 74 μm, and the Hill number increased from 1.4 to 1.9. Muscimol‐elicited currents were qualitatively and quantitatively similar to those produced by GABA.

GABA‐induced changes in the amplitude of large‐conductance Ca2+‐activated K+ channel current recorded on‐cell from E18 CR cells were consistent with depolarization.

GABA‐mediated depolarization of embryonic and postnatal CR cells was studied directly with perforated‐patch recording techniques. Ten micromolar and 1 mm GABA, respectively, depolarized E18 CR cells to −27 ± 1 and −25 ± 3 mV. These same concentrations of GABA depolarized P11 CR cells to −36 ± 3 and −23 ± 3 mV.

In postnatal cortex, GABA (100 μm) increased the firing rate of CR cells 7.3‐fold. By contrast, the firing of hippocampal pyramidal cells from slices of the same age (P12) was totally and reversibly blocked by GABA.

These experiments suggest that contrary to its postnatal inhibitory shift observed in other cells, the depolarizing effect of GABA remains in CR cells from E18 until their virtual disappearance.
Leukemia inhibitory factor induces sympathetic sprouting in intact dorsal root ganglia in the adult rat in vivo
Authors: Thompson Stephen W. N. , Majithia Anooj A.
Published: September 2004

The role of the cytokine leukemia inhibitory factor (LIF) in axotomy‐induced sprouting of postganglionic sympathetic fibres into the dorsal root ganglia was examined in the adult rat.

Immunocytochemistry was used to study the distribution and density of tyrosine hydroxylase‐immunoreactive (TH‐IR) fibres within the lumbar dorsal root ganglia and lumbar spinal nerves 14 days following continuous intrathecal infusion of LIF (0.33 mg ml−1), or 14 days following unilateral peripheral nerve axotomy.

In LIF‐treated animals, numerous pericellular TH‐IR basket‐like structures were observed surrounding sensory neurones, which were absent from controls.

The number of TH‐IR fibres within the L3, L4 and L5 spinal nerves was significantly higher in LIF‐treated animals than in control or saline‐treated animals (P < 0.01, Student's t test).

Unilateral ligation of the L4 spinal nerve or unilateral sciatic nerve ligation was also associated with the formation of TH‐IR baskets around sensory neurones and a significant increase in the number of TH‐IR fibres within the lumbar spinal nerves (P < 0.01, Student's t test).

The percentage of neurones surrounded by TH‐IR baskets within the L3 and L4 dorsal root ganglia following sciatic axotomy was significantly reduced in animals treated continuously for 2 weeks with a monoclonal antibody against the LIF receptor motif, gp130 (0.833 mg ml−1) (P < 0.05, Mann‐Whitney U test). Antibody treatment did not reduce the axotomy‐induced increase in TH‐IR fibres within lumbar spinal nerves.

These results demonstrate that exogenous application of the axotomy‐associated cytokine LIF is associated with sprouting of uninjured postganglionic sympathetic neurones around sensory neurones within the dorsal root ganglion. It is likely that increased LIF expression following peripheral axotomy plays an important role in the novel sympathetic sprouting observed within sensory ganglia following peripheral nerve injury.
Nitric oxide mediates contraction‐induced attenuation of sympathetic vasoconstriction in rat skeletal muscle
Authors: Thomas Gail D. , Victor Ronald G.
Published: September 2004

Sympathetic vasoconstriction is attenuated by metabolic events in contracting rat skeletal muscle, in part by activation of ATP‐sensitive potassium (KATP) channels. However, the specific metabolites in contracting muscle that open KATPchannels are not known. We therefore asked if contraction‐induced attenuation of sympathetic vasoconstriction is mediated by the endogenous vasodilators nitric oxide (NO), adenosine, or prostaglandins PGI2or PGE2, all of which are putative KATPchannel openers.

In anaesthetized rats, hindlimb contraction alone significantly attenuated the vasoconstrictor responses to lumbar sympathetic nerve stimulation. Inhibition of NO synthase withN‐nitro‐L‐arginine methyl ester (L‐NAME, 5 mg kg−1, i.v.) partially reversed this effect of contraction, resulting in enhanced sympathetic vasoconstriction in contracting hindlimb. Subsequent treatment with the KATPchannel blocker glibenclamide (20 mg kg−1, i.v.) had no further effect on sympathetic vasoconstriction in contracting hindlimb.

This effect of L‐NAME to partially reverse contraction‐induced attenuation of sympathetic vasoconstriction was not replicated by D‐NAME (5 mg kg−1, i.v.) or angiotensin II (12.5 ng kg−1min−1, i.v.), the latter used as a hypertensive control.

Adenosine receptor blockade with 8‐(p‐sulphophenyl)theophylline (10 mg kg−1, i.v.) or cyclooxygenase inhibition with indomethacin (5 mg kg−1, i.v.) had no effect on contraction‐induced attenuation of sympathetic vasoconstriction.

These results suggest that NO plays an important role in the precise regulation of blood flow in exercising skeletal muscles by opposing sympathetic vasoconstriction. Although the underlying mechanism is not known, it may involve NO‐induced activation of vascular KATPchannels.
Excitation‐induced force recovery in potassium‐inhibited rat soleus muscle
Authors: Nielsen Ole Bækgaard , Hilsted Linda , Clausen Torben
Published: September 2004

Excitation markedly stimulates the Na+‐K+ pump in skeletal muscle. The effect of this stimulation on contractility was examined in rat soleus muscles exposed to high extracellular K+ concentration ([K+]o).

At a [K+]o of 10 mm, tetanic force declined to 58 % of the force in standard buffer with 5.9 mm K+. Subsequent direct stimulation of the muscle at 1 min intervals with 30 Hz pulse trains of 2 s duration induced a 97 % recovery of force within 14 min. Force recovery could also be elicited by stimulation via the nerve. In muscles exposed to 12.5 mm K+, 30 Hz pulse trains of 2 s duration at 1 min intervals induced a recovery of force from 16 ± 2 to 62 ± 4 % of the initial control force at a [K+]o of 5.9 mm.

The recovery of force was associated with a decrease in intracellular Na+ and was blocked by ouabain. This indicates that the force recovery was secondary to activation of the Na+‐K+ pump.

Excitation stimulates the release of calcitonin gene‐related peptide (CGRP) from nerves in the muscle. Since CGRP stimulates the Na+‐K+ pump, this may contribute to the excitation‐induced force recovery. Indeed, reducing CGRP content by capsaicin pre‐treatment or prior denervation prevented both the excitation‐induced force recovery and the drop in intracellular Na+.

The data suggest that activation of the Na+‐K+ pump in contracting muscles counterbalances the depressing effect of reductions in the chemical gradients for Na+ and K+ on excitability.
The characterization of butyrate transport across pig and human colonic luminal membrane
Authors: Ritzhaupt Armin , Ellis Antony , Hosie Ken B. , Shirazi‐Beechey Soraya P.
Published: September 2004

Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [14C]butyrate.

The activity of cysteine‐sensitive alkaline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins, markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV were free from contamination by other cellular organelles and basolateral membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein.

The transport of butyrate into the luminal membrane vesicles was enhanced 5‐fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperature dependent, and was stimulated in the presence of an outward‐directed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration showed saturation kinetics with an apparent Km value of 14.8 ± 3.6 mM and a Vmax of 54 ± 14 nmol min−1 (mg protein)−1.

Butyrate transport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvate and L‐lactate). Butyrate uptake was inhibited by several cysteine group modifying reagents such as p‐chloromercuribenzosulphonic acid (pCMBS), p‐chloromercuribenzoate (pCMB), mersalyl acid and HgCl2, but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonate (DIDS) and 4,4′‐dinitrostilbene‐2,2′‐disulphonate (SITS).

The described properties of butyrate transport across the luminal pole of the colon suggest the involvement of a carrier protein, in the form of a pH‐activated anion exchange process. The transporter is distinct from the erythrocyte band‐3 type anion exchanger and may belong to the monocarboxylate‐type transport proteins (MCT1).
Modulation of transient outward current by extracellular protons and Cd2+ in rat and human ventricular myocytes
Authors: Stengl Milan , Carmeliet Edward , Mubagwa Kanigula , Flameng Willem
Published: September 2004

The effects of extracellular acidosis and Cd2+ on the transient outward current (Ito) have been investigated in rat and human ventricular myocytes, using the whole‐cell patch‐clamp technique.

In rat myocytes, exposure to acidic extracellular solution (pH 6.0) shifted both steady‐state activation and inactivation curves to more positive potentials, by 20.5 ± 2.7 mV (mean ± s.e.m.; n= 4) and 19.8 ± 1.2 mV, respectively. Cd2+ also shifted the activation and inactivation curves in a positive direction in a concentration‐dependent manner.

In human myocytes, the steady‐state activation and inactivation curves were located at more positive potentials. The effect of Cd2+ was similar, but acidosis had less effect than in rat myocytes (e.g. pH 6.0 shifted activation by only 7.2 ± 2.2 mV and inactivation by 13.7 ± 0.5 mV; n= 4).

In both species, the effect of acidosis decreased with increasing concentrations of Cd2+ and vice versa, suggesting competition between H+ and Cd2+ for a common binding site.

The data indicate that acidosis and divalent cations influence Ito via a similar mechanism and act competitively in both rat and human myocytes, but that human cells are less sensitive to the effects of acidosis.
Intracellular recording from myenteric neurons of the guinea‐pig ileum that respond to stretch
Authors: Kunze W. A. A. , Furness J. B. , Bertrand P. P. , Bornstein J. C.
Published: September 2004

Isolated longitudinal muscle‐myenteric plexus preparations from guinea‐pig ileum were used to investigate the activity of myenteric neurons when the tissue was stretched in the circumferential direction. Membrane potentials were recorded via flexibly mounted intracellular recording electrodes containing Neurobiotin in 1 M KCl. The preparations were stretched to constant widths (+20 % and +40 % beyond slack width).

Multipolar neurons (Dogiel type II morphology) discharged spontaneous action potentials and proximal process potentials during maintained stretching, three of twenty‐one at +20 % stretch and seven of nine at +40 % stretch. At the maximum extent of stretch tried, +40 % beyond slack tissue width, action potentials in Dogiel type II neurons occurred at 10‐33 Hz. Neurons with other morphologies were all uniaxonal. Some displayed spontaneous fast EPSPs or action potentials, three of forty‐one at +20 % stretch and seven of nineteen at +40 % stretch.

In seven of eight Dogiel type II neurons, action potentials or proximal process potentials persisted when membrane hyperpolarization was imposed via the recording electrode. Action potential discharge was abolished by hyperpolarization in seven of nine uniaxonal neurons; the exceptions were two orally projecting neurons.

Dogiel type II and uniaxonal neurons were classified as rapidly accommodating if they discharged action potentials only at the beginning of a 500 ms intracellular depolarizing pulse and slowly accommodating if they discharged for more than 250 ms. For Dogiel type II neurons, three of thirteen were slowly accommodating at +20 % stretch and two of four at 40 % stretch. For uniaxonal neurons the corresponding data were twelve of twenty‐six and fifteen of nineteen neurons. The slowly accommodating state was associated with increased cell input resistance in uniaxonal neurons.

The spontaneous action potential discharge in Dogiel type II and uniaxonal neurons ceased when the muscle was relaxed pharmacologically by nicardipine (3 μM) or isoprenaline (1 μM), although the applied stretch was maintained. At the same time, evoked spike discharge became rapidly accommodating

We conclude that many Dogiel type II neurons, and possibly some orally projecting uniaxonal neurons, are intrinsic, stretch‐sensitive, primary afferent neurons that respond to muscle tension with sustained action potential discharge.
Characterization of sensory and corticothalamic excitatory inputs to rat thalamocortical neurones in vitro
Authors: Turner J. P. , Salt T. E.
Published: September 2004

Using an in vitro slice preparation of the rat dorsal lateral geniculate nucleus (dLGN), the properties of retinogeniculate and corticothalamic inputs to thalamocortical (TC) neurones were examined in the absence of GABAergic inhibition.

The retinogeniculate EPSP evoked at low frequency (<= 0.1 Hz) consisted of one or two fast‐rising (0.8 ± 0.1 ms), large‐amplitude (10.3 ± 1.6 mV) unitary events, while the corticothalamic EPSP had a graded relationship with stimulus intensity, owing to its slower‐rising (2.9 ± 0.4 ms), smaller‐amplitude (1.3 ± 0.3 mV) estimated unitary components.

The retinogeniculate EPSP exhibited a paired‐pulse depression of 60.3 ± 5.6 % at 10 Hz, while the corticothalamic EPSP exhibited a paired‐pulse facilitation of > 150 %. This frequency‐dependent depression of the retinogeniculate EPSP was maximal after the second stimulus, while the frequency‐dependent facilitation of the corticothalamic EPSP was maximal after the fourth or fifth stimulus, at interstimulus frequencies of 1‐10 Hz.

There was a short‐term enhancement of the <= 0.1 Hz corticothalamic EPSP (64.6 ± 9.2 %), but not the retinogeniculate EPSP, following trains of stimuli at 50 Hz.

The <= 0.1 Hz corticothalamic EPSP was markedly depressed by the non‐NMDA antagonist 1‐(4‐amino‐phenyl)‐4‐methyl‐7,8‐methylene‐dioxy‐5H‐2,3‐benzodiazepine (GYKI 52466), but only modestly by the NMDA antagonist 3‐((RS)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid ((RS)‐CPP), and completely blocked by the co‐application of GYKI 52466, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), (RS)‐CPP and (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine (MK‐801). Likewise, the corticothalamic responses to trains of stimuli (1‐500 Hz) were greatly reduced by this combination of ionotropic glutamate receptor antagonists.

In the presence of GYKI 52466, CNQX, (RS)‐CPP and MK‐801, residual corticothalamic responses and slow EPSPs, with a time to peak of 2‐10 s, could be generated following trains of five to fifty stimuli. Neither of these responses were occluded by 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), suggesting they are not mediated via group I and II metabotropic glutamate receptors.
The contribution of pH‐dependent mechanisms to fatigue at different intensities in mammalian single muscle fibres
Authors: Chin E. R. , Allen D. G.
Published: September 2004

The contribution of intracellular pH (pHi) to the failure of Ca2+ release and inhibition of contractile proteins observed during fatigue was assessed in single intact mouse muscle fibres at 22 °C. Fatigue was induced by repeated tetani at intensities designed to induce different levels of intracellular acidosis. Force and either intracellular free Ca2+ concentration ([Ca2+]i; measured using indo‐1) or pHi (measured using SNARF‐1) were recorded in fibres fatigued at two different intensities.

Intensity was varied by the repetition rate of tetani and quantified by the duty cycle (the fraction of time when the muscle was tetanized). Stimulation at the low intensity (duty cycle ∼0.1) reduced force to 30 % of initial values in 206 ± 21 s (60 ± 7 tetani); at the high intensity (duty cycle ∼0.3) force was reduced to 30 % in 42 ± 7 s (43 ± 7 tetani) (P < 0.05; n= 14).

When force was reduced to 30 % of initial values, tetanic [Ca2+]i had fallen from 648 ± 87 to 336 ± 64 nm (48 % decrease) at the low intensity but had only fallen from 722 ± 84 to 468 ± 60 nm (35 % decrease) at the higher intensity (P < 0.05 low vs. high intensity; n= 7).

Fatigue resulted in reductions in Ca2+ sensitivity of the contractile proteins which were greater at the high intensity (pre‐fatigue [Ca2+]i required for 50 % of maximum force (Ca50) = 354 ± 23 nm; post‐fatigue Ca50= 421 ± 48 nm and 524 ± 43 nm for low and high intensities, respectively). Reductions in maximum Ca2+‐activated force (Fmax) were similar at the two intensities (pre‐fatigue Fmax= 328 ± 22 μN; post‐fatigue Fmax= 271 ± 20 and 265 ± 19 μN for low and high intensities, respectively).

Resting pHi was 7.15 ± 0.05. During fatigue at the low intensity, pHi was reduced by 0.12 ± 0.02 pH units and at the high intensity pHi was reduced by 0.34 ± 0.07 pH units (P < 0.05; n= 5).

Our results indicate that the more rapid fall in force at a high intensity is due to a reduction in Ca2+ sensitivity of the contractile proteins, probably related to the greater acidosis. Our data also indicate that the failure of Ca2+ release and reduced maximum Ca2+‐activated force observed during fatigue are not due to reductions in intracellular pH.
Apamin‐ and nitric oxide‐sensitive biphasic non‐adrenergic non‐cholinergic inhibitory junction potentials in the rat anococcygeus muscle
Authors: Selemidis S. , Ziogas J. , Cocks T. M.
Published: September 2004

Changes in membrane potential following electrical field stimulation (EFS; 1, 2 and 5 pulses at 5 Hz, 0.5 ms duration, 60–80 V) of non‐adrenergic non‐cholinergic (NANC) inhibitory nerves in the rat isolated anococcygeus muscle were measured using standard intracellular recording techniques. Resting membrane potential ranged between −60 and −70 mV.

In the presence of guanethidine (30 μm), atropine (1 μm), propranolol (1 μm) and phentolamine (0.05 μm) to establish NANC conditions, the membrane potential depolarized to between −40 and −50 mV. Under these conditions, EFS caused pulse‐dependent, tetrodotoxin (1 μm)‐sensitive biphasic inhibitory junction potentials (IJPs) comprising a fast onset and time‐to‐peak phase followed by a second, slower phase that delayed repolarization. The duration of NANC IJPs ranged between 10 and 20 s.

Inhibition of small‐conductance Ca2+‐activated K+ channels with apamin (0.1 μm) selectively blocked the first fast phase of the NANC IJP, whereas inhibitors of large‐conductance Ca2+‐activated K+ channels (charybdotoxin and iberiotoxin) and ATP‐sensitive K+ channels (glibenclamide) all had no effect on NANC IJPs.

Both the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine (l‐NOARG; 100 μm) and the inhibitor of soluble guanylate cyclase 1‐H‐oxodiazol‐[1,2,4]‐[4,3‐a] quinoxaline‐1‐one (ODQ; 10 μm) had no effect on the first fast phase of the NANC IJP. Each treatment, however, markedly inhibited the slow phase with the duration of the IJP reduced to between 1 and 3 s. The l‐NOARG‐resistant fast phase of the NANC IJP was almost abolished by the subsequent addition of apamin (0.1 μm).

In conclusion, the present study demonstrates unequivocal NANC nerve‐mediated biphasic IJPs in the rat isolated anococcygeus. We propose that nitric oxide (NO), via activation of cGMP‐dependent K+ channels, and a non‐NO inhibitory factor which activates apamin‐sensitive K+ channels contribute to NANC nerve‐evoked IJPs in the rat anococcygeus.