Comparison of the electrophysiological and immunohistochemical properties of acutely dissociated and 1-day cultured rat trigeminal ganglion neurons.

To clarify whether changes to the cellular properties of sensory neurons occur after a brief culture, we compared the electrophysiological and immunohistochemical properties of rat trigeminal ganglion neurons. We compared these neurons after acute dissociation and after a 1-day culture under serum-free and neurotrophin-free conditions. In whole-cell patch-clamp recordings, the 1-day cultured neurons required a lower current threshold to induce an action potential in both small- and medium-sized neurons. Furthermore, the input resistance was higher in the medium-sized neurons after a 1-day culture compared to the acutely dissociated medium-sized neurons. Immunofluorescent studies demonstrated that both the translocation of the activating transcription factor 3 (ATF3) into the nucleus and the expression of a low threshold Na(+) channel (Na(v)1.3) were upregulated after 1-day of culture. However, in the acutely dissociated neurons, ATF3 translocation occurred at low levels, and Na(v)1.3 was not expressed. These electrophysiological and immunohistochemical changes after 1-day of culture were very similar to the reported changes that occur after nerve injury. Our study demonstrated that injury-like characteristics appear to be manifested in the 1-day cultured sensory neurons, which do not occur in acutely dissociated neurons. Overall, our results are relevant and will help when interpreting the results of studies examining dissociated sensory neurons in pain research.

Capacitance increases of dissociated tilapia prolactin cells in response to hyposmotic and depolarizing stimuli.

Prolactin (PRL) is the major hormonal mediator of adaptation to hyposmotic conditions. In tilapia (Oreochromis mossambicus), PRL cells are segregated to the rostral pars distalis of the anterior pituitary facilitating the nearly pure culture of dissociated PRL cells. Membrane capacitance (C(m)) was recorded at 1Hz or higher for tens of minutes as a surrogate monitor of PRL secretion by exocytosis from cells under perforated patch clamp. The study compares secretory responses to trains of depolarizing clamps (100 at 2.5 Hz, from -70 to +10 mV for 100 ms) to the physiological stimulus, exposure to hyposmotic medium, here a switch from 350 to 300 mOsm saline ([Ca²âº] 15 mM). Two-thirds of cells tested with each stimulus responded. In response to depolarizing clamps, C(m) increased linearly at an average rate of 7.2 fF/s. The increase was also linear in response to hyposmotic perfusion, but the average rate was 0.68 fF/s. Response to depolarization was reversibly blocked in Ca²âº-omitted saline, or in saline with 30 µM Cd²âº. It was unaffected by 0.1 µM tetrodotoxin. By contrast, responses were reduced but not absent during perfusion of hyposmotic saline with Ca²âº-omitted; 30 µM Cd²âº appeared to enhance the hyposmotic response. BAPTA-AM eliminated responses to both stimuli, confirming that secretion was dependent on increases of intracellular [Ca²âº]. Together with previous observations from this laboratory of [Ca²âº](i) with simultaneous collection and immunoassay of perfusate for PRL, we conclude that depolarization and hyposmotic stimuli initiate secretion by independent mechanisms.

Seizure-like thalamocortical rhythms initiate in the deep layers of the cortex in a co-culture model.

The oscillatory rhythms underlying many physiological and pathological states, including absence seizures, require both the thalamus and cortices for full expression. A co-culture preparation combining cortical and thalamic explants provides a unique model for investigating how such oscillations initiate and spread. Here we investigated the dynamics of synchronized thalamocortical activity by simultaneous measurement of field-potential recordings and rapid imaging of Ca(2+) transients by fluorescence methods. Spontaneous sustained hypersynchronized "seizure-like" oscillations required reciprocal cortico-thalamocortical connections. Isolated cortical explants can independently develop brief discharges, while thalamic explants alone were unable to do so. Rapid imaging of Ca(2+) transients demonstrated deep-layer cortical initiation of oscillatory network activity in both connected and isolated explants. Further, cortical explants derived from a rat model of genetic absence epilepsy showed increased bursting duration consistent with an excitable cortex. We propose that thalamocortical oscillatory network activity initiates in deep layers of the cortex with reciprocal thalamic interconnections enabling sustained hyper-synchronization.

Isolectin B4binding in populations of rat trigeminal ganglion cells.

We have recently classified dissociated trigeminal ganglion cells into nine types using electrophysiological current signatures. In the present study, we investigated the relationship between isolectin B(4) (IB(4)) binding and the cell types in rat trigeminal ganglion cells. We found that IB(4) was bound to all type 2 cells and more than 70% of cell types 1 and 13; however, it was bound to less than 20% of cell types 7 and 8 and did not bind at all to cell types 3-5 and 9. Thus, each trigeminal ganglion cell type showed high homogeneity in IB(4) binding. These results correspond to reported IB(4) binding profiles in the matched dorsal root ganglion cell types, except for types 5 and 7.

Electrophysiological and chemical properties in subclassified acutely dissociated cells of rat trigeminal ganglion by current signatures.

In the present study, we subclassified acutely dissociated trigeminal ganglion (TRG) cells of rats using a current signature method in whole cell patch-clamp recordings. Using modified criteria for cell classification for the dorsal root ganglion (DRG), TRG cells were subclassified into nine cell types: 1-5, 7-9, and 13. Types 1, 3, and 7 were in the small cell groups (15-24 µm); types 4, 5, and 8-13 were in the medium cell groups (25-38 µm); and type 2 was a mixed group of both cell sizes. Types 1-3, 5, and 7 showed high-input resistance and types 1, 2, and 7 showed more depolarized resting membrane potentials. Types 1, 2, and 5-13 expressed long-duration action potentials (APs), but types 3 and 4 expressed short-duration APs. Sensitivities to capsaicin, protons, and adenosine 5'-triphosphate (ATP) in TRG cell types largely corresponded to DRG cell types. However, different from the matched DRG types, half of TRG type 1 cells were capsaicin insensitive, showing desensitizing proton-induced currents, and types 5, 7, and 9 exhibited slow-desensitizing ATP-induced currents. Types 4, 5, and 8-13 had nicotine sensitivity, but the other cell types were insensitive. These results indicate that the "current signatures" classification is a useful means to separate TRG cells into internally homogeneous subpopulations that were distinct from other cell types. Furthermore, the data suggest some specific differences in the chemical responsiveness of some cell types between the TRG and DRG.

17beta-estradiol activates estrogen receptor beta-signalling and inhibits transient receptor potential vanilloid receptor 1 activation by capsaicin in adult rat nociceptor neurons.

There is mounting evidence that estrogens act directly on the nervous system to affect the severity of pain. Estrogen receptors (ERs) are expressed by sensory neurons, and in trigeminal ganglia, 17beta-estradiol can indirectly enhance nociception by stimulating expression and release of prolactin, which increases phosphorylation of the nociceptor transducer transient receptor potential vanilloid receptor 1 (TRPV1). Here, we show that 17beta-estradiol acts directly on dorsal root ganglion (DRG) sensory neurons to reduce TRPV1 activation by capsaicin. Capsaicin-induced cobalt uptake and the maximum TRPV1 current induced by capsaicin were inhibited when isolated cultured DRGs neurons from adult female rats were exposed to 17beta-estradiol (10-100 nm) overnight. There was no effect of 17beta-estradiol on capsaicin potency, TRPV1 activation by protons (pH 6-4), and P2X currents induced by alpha,beta-methylene-ATP. Diarylpropionitrile (ERbeta agonist) also inhibited capsaicin-induced TRPV1 currents, whereas propylpyrazole triol (ERalpha agonist) and 17alpha-estradiol (inactive analog) were inactive, and 17beta-estradiol conjugated to BSA (membrane-impermeable agonist) caused a small increase. TRPV1 inhibition was antagonized by tamoxifen (1 microm), but ICI182870 (10 microm) was a potent agonist and mimicked 17beta-estradiol. We conclude that TRPV1 in DRG sensory neurons can be inhibited by a nonclassical estrogen-signalling pathway that is downstream of intracellular ERbeta. This affects the vanilloid binding site targeted by capsaicin but not the TRPV1 activation site targeted by protons. These actions could curtail the nociceptive transducer functions of TRPV1 and limit chemically induced nociceptor sensitization during inflammation. They are consistent with clinical reports that female pelvic pain can increase after reductions in circulating estrogens.

Spontaneous release from mossy fiber terminals inhibits Ni2+-sensitive T-type Ca2+ channels of CA3 pyramidal neurons in the rat organotypic hippocampal slice.

Mossy fibers (axons arising from dentate granule cells) form large synaptic contacts exclusively onto the proximal apical dendrites of CA3 pyramidal neurons. They can generate large synaptic currents that occur in close proximity to the soma. These properties mean that active conductance in the proximal apical dendrite could have a disproportionate influence on CA3 pyramidal neuron excitability. Ni(2+)-sensitive T-type Ca(2+) channels are important modulators of dendritic excitability. Here, we use an optical approach to determine the contribution of Ni(2+) (100 microM)-sensitive Ca(2+) channels to action potential (AP) elicited Ca(2+) flux in the soma, proximal apical and distal apical dendrites. At resting membrane potentials Ni(2+)-sensitive Ca(2+) channels do not contribute to the Ca(2+) signal in the proximal apical dendrite, but do contribute in the other cell regions. Spontaneous release from mossy fiber terminals acting on 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive postsynaptic channels underlies a tonic inhibition of Ni(2+)-sensitive channels. Chelating Zn(2+) with CaEDTA blocks CNQX-sensitive changes in Ca(2+) flux implicating a mechanistic role of this ion in T-type Ca(2+) channel block. To test if this inhibition influenced excitability, progressively larger depolarizing pulses were delivered to CA3 pyramidal neurons. CNQX significantly reduced the size of the depolarizing step required to generate APs and increased the absolute number of APs per depolarizing step. This change in AP firing was completely reversed by the addition of Ni(2+). This mechanism may reduce the impact of T-type Ca(2+) channels in a region where large synaptic events are common.

Voltage-gated currents of tilapia prolactin cells.

The first recordings of neuron-like electrical activity from endocrine cells were made from fish pituitary cells. However, patch-clamping studies have predominantly utilized mammalian preparations. This study used whole-cell patch-clamping to characterize voltage-gated ionic currents of anterior pituitary cells of Oreochromis mossambicus in primary culture. Due to their importance for control of hormone secretion we emphasize analysis of calcium currents (I(Ca)), including using peptide toxins diagnostic for mammalian neuronal Ca(2+) channel types. These appear not to have been previously tested on fish endocrine cells. In balanced salines, inward currents consisted of a rapid TTX-sensitive sodium current and a smaller, slower I(Ca); there followed outward potassium currents dominated by delayed, sustained TEA-sensitive K(+) current. About half of cells tested from a holding potential (V(h)) of -90 mV showed early transient K(+) current; most cells showed a small Ca(2+)-mediated outward current. I-V plots of isolated I(Ca) with 15 mM [Ca(2+)](o) showed peak currents (up to 20 pA/pF from V(h) -90 mV) at approximately +10 mV, with approximately 60% I(Ca) for V(h) -50 mV and approximately 30% remaining at V(h) -30 mV. Plots of normalized conductance vs. voltage at several V(h)s were nearly superimposable. Well-sustained I(Ca) with predominantly Ca(2+)-dependent inactivation and inhibition of approximately 30% of total I(Ca) by nifedipine or nimodipine suggests participation of L-type channels. Each of the peptide toxins (omega-conotoxin GVIA, omega-agatoxin IVA, SNX482) alone blocked 36-54% of I(Ca). Inhibition by any of these toxins was additive to inhibition by nifedipine. Combinations of the toxins failed to produce additive effects. I(Ca) of up to 30% of total remained with any combination of inhibitors, but 0.1mM cadmium blocked all I(Ca) rapidly and reversibly. We did not find differences among cells of differing size and hormone content. Thus, I(Ca) is carried by high voltage-activated Ca(2+) channels of at least three types, but the molecular types may differ from those characterized from mammalian neurons.

Noncholinergic actions of atropine on GABAergic synaptic transmission in the subfornical organ of rat slice preparations.

Actions of atropine on GABAergic inhibitory postsynaptic currents to neurons in the subfornical organ, which is a circumventricular organ, were studied by using rat slice preparations with whole-cell clamp recordings. Atropine at 0.01-1 microM antagonized the decreased frequency of Inhibitory postsynaptic currents by carbachol (Xu et al., Am. J. Physiol. Integr. Comp. Physiol. 280, R1657-R1664, 2001). It acted as a muscarinic antagonist at relatively low concentrations. Although the low concentrations of atropine did not change frequency and amplitude of the inhibitory postsynaptic currents, atropine at 10 microM to 1 mM did decrease them in a dose-dependent manner. Glutamatergic excitatory postsynaptic currents were not influenced by atropine at 100 microM. Atropine at 100 microM suppressed GABA- and muscimol-induced outward currents, but not kainic acid-induced inward currents. In addition, decrease of membrane conductance and induction of inward currents by 100 microM of atropine at a holding membrane potential, -51 mV, were found in subfornical organ neurons. From voltage-current curves, a mean reversal potential was estimated to be -65.9 +/- 3.7 mV, near to an equilibrium potential of chloride channels. These imply that atropine at 100 microM suppresses openings of chloride channels. Taken together, it is suggested that, while atropine at low concentrations has an antagonistic action on muscarinic responses, atropine at high concentrations suppresses GABAergic synaptic transmission in subfornical organ neurons. These findings may be of considerable value in understanding the central mechanisms of extraordinary drinking behavior in atropine intoxication.