Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

Mercaptoacetate and fatty acids exert direct and antagonistic effects on nodose neurons via GPR40 fatty acid receptors.

ß-mercaptoacetate (MA) is a drug known to block mitochondrial oxidation of medium- and long-chain fatty acids (FAs) and to stimulate feeding. Because MA-induced feeding is vagally dependent, it has been assumed that the feeding response is mediated by MA's antimetabolic action at a peripheral, vagally innervated site. However, MA's site of action has not yet been identified. Therefore, we used fluorescent calcium measurements in isolated neurons from rat nodose ganglia to determine whether MA has direct effects on vagal sensory neurons. We found that MA alone did not alter cytosolic calcium concentrations in nodose neurons. However, MA (60 µM to 6 mM) significantly decreased calcium responses to both linoleic acid (LA; 10 µM) and caprylic acid (C8; 10 µM) in all neurons responsive to LA and C8. GW9508 (40 µM), an agonist of the FA receptor, G protein-coupled receptor 40 (GPR40), also increased calcium levels almost exclusively in FA-responsive neurons. MA significantly inhibited this response to GW9508. MA did not inhibit calcium responses to serotonin, high K(+), or capsaicin, which do not utilize GPRs, or to CCK, which acts on a different GPR. GPR40 was detected in nodose ganglia by RT-PCR. Results suggest that FAs directly activate vagal sensory neurons via GPR40 and that MA antagonizes this effect. Thus, we propose that MA's nonmetabolic actions on GPR40 membrane receptors, expressed by multiple peripheral tissues in addition to the vagus nerve, may contribute to or mediate MA-induced stimulation of feeding.

Comparative pharmacology of cholecystokinin induced activation of cultured vagal afferent neurons from rats and mice.

Cholecystokinin (CCK) facilitates the process of satiation via activation of vagal afferent neurons innervating the upper gastrointestinal tract. Recent findings indicate CCK acts on these neurons via a ruthenium red (RuR) sensitive pathway that involves members of the vanilloid (V) subfamily of transient receptor potential (TRP) channels. To further test this mechanism, the mouse provides an ideal model in which genetic tools could be applied. However, whether CCK acts by similar mechanism(s) in mice has not been determined. In the present study we explored the actions of CCK on nodose neurons isolated from Sprague Dawley (SD) rat and two strains of mice; C57BL/6 and BalbC using fluorescence-based calcium imaging. With minor exceptions nodose neurons isolated from all species/strains behaved similarly. They all respond to brief depolarization with a large calcium transient. A significant subset of neurons responded to capsaicin (CAP), a TRPV1 agonist, although neurons from C57BL/6 were 10-fold more sensitive to CAP than SD rats or BalbC mice, and a significantly smaller fraction of neurons from BalbC mice responded to CAP. CCK-8 dose-dependently activated a subpopulation of neurons with similar dose dependency, percent responders, and overlap between CCK and CAP responsiveness. In all species/strains CCK-8 induced activation was significantly attenuated (but not completely blocked) by pretreatment with the TRPV channel blocker RuR. Surprisingly, the CCK analogue JMV-180, which is reported to have pure antagonistic properties in rat but mixed agonist/antagonist properties in mice, behaved as a pure antagonist to CCK in both rat and mouse neurons. The pure antagonistic action of JMV-180 in this in vitro preparation suggests that prior reported differential effects of JMV-180 on satiation in rats versus mouse must be mediated by a site other than vagal afferent activation.

Pharmacological investigations of the cellular transduction pathways used by cholecystokinin to activate nodose neurons.

Cholecystokinin (CCK) directly activates vagal afferent neurons resulting in coordinated gastrointestinal functions and satiation. In vitro, the effects of CCK on dissociated vagal afferent neurons are mediated via activation of the vanilloid family of transient receptor potential (TRPV) cation channels leading to membrane depolarization and an increase in cytosolic calcium. However, the cellular transduction pathway(s) involved in this process between CCK receptors and channel opening have not been identified. To address this question, we monitored CCK-induced cytosolic calcium responses in dissociated nodose neurons from rat in the presence or absence of reagents that interact with various intracellular signaling pathways. We found that the phospholipase C (PLC) inhibitor U-73122 significantly attenuated CCK-induced responses, whereas the inactive analog U-73433 had no effect. Responses to CCK were also cross-desensitized by a brief pretreatment with m-3M3FBS, a PLC stimulator. Together these observations strongly support the participation of PLC in the effects of CCK on vagal afferent neurons. In contrast, pharmacological antagonism of phospholipase A(2), protein kinase A, and phosphatidylinositol 3-kinase revealed that they are not critical in the CCK-induced calcium response in nodose neurons. Further investigations of the cellular pathways downstream of PLC showed that neither protein kinase C (PKC) nor generation of diacylglycerol (DAG) or release of calcium from intracellular stores participates in the response to CCK. These results suggest that alteration of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content by PLC activity mediates CCK-induced calcium response and that this pathway may underlie the vagally-mediated actions of CCK to induce satiation and alter gastrointestinal functions.

Role of transient receptor potential channels in cholecystokinin-induced activation of cultured vagal afferent neurons.

Cholecystokinin (CCK), an endogenous brain-gut peptide, is released after food intake and promotes the process of satiation via activation of the vagus nerve. In vitro, CCK increases cytosolic calcium concentrations and produces membrane depolarization in a subpopulation of vagal afferent neurons. However, the specific mechanisms and ionic conductances that mediate these effects remain unclear. In this study we used calcium imaging, electrophysiological measurements, and single cell PCR analysis on cultured vagal afferent neurons to address this issue directly. A cocktail of blockers of voltage-dependent calcium channels (VDCC) failed to block CCK-induced calcium responses. In addition, SKF96365, a compound that blocks both VDCC and the C family of transient receptor potential (TRP) channels, also failed to prevent responses to CCK. Together these results suggest that CCK-induced calcium influx is not subsequent to the membrane depolarization. Ruthenium red, an inhibitor of the TRPV family and TRPA1, blocked both depolarizing responses to CCK and CCK-induced calcium increases, but had no effect on the KCl-induced calcium response. Selective block of TRPV1 and TRPA1 channels with SB366791 and HC030031, respectively, had minor effects on the CCK-induced response. Application of 2-aminoethoxydiphenyl borate, an activator of select TRPV channels but a blocker of several TRPC channels, either had no effect or enhanced the responses to CCK. Further, results from PCR experiments revealed a significant clustering of TRPV2-5 in neurons expressing CCK1 receptors. These observations demonstrate that CCK-induced increases in cytosolic calcium and membrane depolarization of vagal afferent neurons are likely mediated by TRPV channels, excluding TRPV1.

Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat.

Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicin-sensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPC1 were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-R(A)) and found there was a significant preference for neurons that express TRAAK to also express CCK-R(A), especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target.

Chronic alcohol treatment in rats alters sleep by fragmenting periods of vigilance cycling in the light period with extended wakenings.

Studies have shown that disturbed sleep produced by chronic alcohol abuse in humans can predict relapse drinking after periods of abstinence. How alcohol produces disturbed sleep remains unknown. In this study we used a novel analysis of sleep to examine the effects of alcohol on sleep patterns in rats. This analysis separates waking into multiple components and defines a period labeled vigilance cycling (VC) in which the rat rapidly cycles through various vigilance states. These VC episodes are separated by long duration wake (LDW) periods. We find that 6 weeks of alcohol (6% in a liquid diet) caused fragmentation of extended VC episodes that normally occur in the light period. However, total daily amounts of slow-wave sleep (SWS) and rapid-eye movement sleep (REMS) remained constant. The daily amount of wake, SWS, and REMS remained constant because the alcohol treated rats increased the amount of VC in the dark period, and the sleep nature of VC in the dark period became more intense. In addition, we observed more wake and less REMS early in the light period in alcohol treated rats. All effects completely reversed by day 16 of alcohol withdrawal. Comparison of the effects of chronic alcohol to acute alcohol exposure demonstrated the effects of chronic alcohol are due to adaptation and not the acute presence of alcohol. The effects of chronic alcohol treatment in rats mimic the effects reported in humans (REMS suppression, difficulty falling asleep, and difficulty remaining asleep).

Novel analysis of sleep patterns in rats separates periods of vigilance cycling from long-duration wake events.

Rats are polyphasic sleepers. However, a formal definition of when one sleep episode ends and another begins has not been put forth. In the present study we examine the distribution of wake episode durations and based on this distribution conclude there are multiple components of wake. If the wake episode exceeds 300 s the wake episode is assigned to long-duration wake (LDW), if the episode is less than 300 s it is assigned to brief wake (BW). Further support for this separation was found in close analysis of the EEG power spectrum in BW versus LDW. We then used LDW episodes to separate one sleep episode from another. We term the sleep episodes vigilance cycling (VC) because the rat is cycling between the vigilance states of BW, slow-wave sleep (SWS), and rapid-eye movement sleep (REMS). We find that the characteristics of VC are different in the light period versus the dark period. We further find that as VC episodes progress, SWS pressure lessens, but the amount of time spent in REMS increases. These findings suggest that VC episodes are regulated and meaningful to the sleep behavior of rats. The use of the concepts of LDW and VC provides additional insights into the description of sleep patterns in rats that may be important in the development of a complete description of sleep behavior in this animal.

Dose-response study of chronic alcohol induced changes in sleep patterns in rats.

The goal of the present study was to determine an optimum exposure regimen for alterations in sleep induced by chronic alcohol treatments in rats. We used two different exposure routes (alcohol in water and alcohol in liquid diet at two different doses in each regimen (6% and 12% alcohol in water and 3% and 6% alcohol in liquid diet)). All treatments were for 6 weeks. We found the effects of the 6% and 12% in water and 3% in liquid diet to be very similar; all three produced increases in slow-wave sleep (SWS) only in the dark period with no changes in rapid-eye-movement sleep (REMS). On the other hand 6% alcohol in liquid diet caused much more dramatic changes, with alterations in both SWS and REMS in both the dark and light periods. These animals spent less time in SWS and REMS during the light period but more time in SWS and REMS in the dark period. Additionally, the variation of slow-wave amplitude (SWA) across day and night in this group of alcoholic animals is blunted with the loss of the peak of SWA at the beginning of light onset compared to the other groups. We conclude that future alcohol treatment regimens used to investigate the effects of alcohol on sleep in adult rats should use an exposure protocol of at least 6 weeks with 6% alcohol in liquid diet.

Modulation of vagal afferent excitation and reduction of food intake by leptin and cholecystokinin.

The gut-peptide, cholecystokinin (CCK), reduces food intake by acting at CCK-1 receptors on vagal afferent neurons, whereas the feeding effects of the adipokine hormone, leptin, are associated primarily with its action on receptors (ObRb) in the hypothalamus. Recently, however, ObRb mRNA has been reported in vagal afferent neurons, some of which also express CCK-1 receptor, suggesting that leptin, alone or in cooperation with CCK, might activate vagal afferent neurons, and influence food intake via a vagal route. To evaluate these possibilities we have been examining the cellular and behavioral effects of leptin and CCK on vagal afferent neurons. In cultured vagal afferent neurons leptin and CCK evoked short latency, transient depolarizations, often leading to action potentials, and increases in cytosolic calcium. There was a much higher prevalence of CCK and leptin sensitivity amongst cultured vagal afferent neurons that innervate stomach or duodenum than there was in the overall vagal afferent population. Furthermore, almost all leptin-responsive gastric and duodenal vagal afferents also were sensitive to CCK. Leptin, infused into the upper GI tract arterial supply, reduced meal size, and enhanced satiation evoked by CCK. These results indicate that vagal afferent neurons are activated by leptin, and that this activation is likely to participate in meal termination, perhaps by enhancing vagal sensitivity to CCK. Our findings are consistent with the view that leptin and CCK exert their influence on food intake by accessing multiple neural systems (viscerosensory, motivational, affective and motor) at multiple points along the neuroaxis.

Glutamate induces the expression and release of tumor necrosis factor-alpha in cultured hypothalamic cells.

Tumor necrosis factor-alpha (TNFalpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFalpha involvement in physiological sleep regulation, e.g., TNFalpha induces sleep and brain levels of TNFalpha increase during prolonged wakefulness. The immediate cause of enhanced TNFalpha production in brain is unknown. We investigated whether glutamate could signal TNFalpha production because glutamate is a neurotransmitter associated with cell activation and wakefulness. We used primary cultures of fetal rat hypothalamic cells to examine the expression and release of TNFalpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFalpha was detected in both the media and cells under basal conditions. Stimulation of the cells with 1 mM glutamate for 2 h produced an increase in media content of TNFalpha, whereas cell content was elevated at earlier time points. Using trypan blue exclusion and MTT assays, there was no evidence of cell toxicity with this stimulation protocol. Immunocytochemical staining revealed that TNFalpha was expressed by approximately 25% of the neurons and approximately 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFalpha. We conclude that TNFalpha is constitutively expressed and released by healthy cultures of hypothalamic cells and that activation of the cells with a non-toxic challenge of glutamate increases TNFalpha production. These findings support the hypothesis that TNFalpha can participate in normal physiological regulation of sleep and feeding.

Cholecystokinin activates both A- and C-type vagal afferent neurons.

Patch-clamp electrophysiological methods were used on dissociated rat nodose neurons maintained in culture to determine whether responses to cholecystokinin (CCK) were associated with capsaicin-resistant (A type) or capsaicin-sensitive (C type) neurons. Nodose neurons were classified as A or C type on the basis of the characteristics of the Na+ current, a hyperpolarization-activated current, and sensitivity to a low concentration of capsaicin to ascertain the presence of vanilloid receptor 1 that has been associated with C-type neurons in sensory ganglia. It was expected that only capsaicin-sensitive C-type neurons would respond to CCK, because most vagally mediated actions of CCK are blocked by capsaicin treatment. However, we found that subpopulations of both A- and C-type neurons responded to CCK (24 and 38%, respectively). Thus some vagally mediated actions of CCK may be mediated by capsaicin insensitive A-type neurons.

Tumor necrosis factor alpha increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons.

Acute behavioral effects of tumor necrosis factor alpha (TNFalpha) have been previously reported, however the cellular basis for these actions are unknown. To address this issue we examined the effects of TNFalpha on AMPA- and depolarization-induced changes in cytosolic Ca(2+) in cultured hippocampal neurons. Single cell Ca(2+) levels were determined with the fluorescent calcium indicator fura-2. TNFalpha caused an up-regulation of AMPA (10 microM)- and depolarization (55 mM KCl)-induced Ca(2+) responses. This effect occurred within a window of concentrations (1 and 10 ng/ml but not 0.1 or 100 ng/ml) and times (3 and 6 h but not 1 and 24 h). The effect was dependent upon protein synthesis (blocked by cycloheximide) and was prevented by the soluble TNF receptor and by a soluble TNF receptor fragment. Treatment with the soluble TNF receptor fragment also caused a decrease in the basal response. The TNFalpha treatment protocols did not appear to produce any toxicity to the neurons. Results are consistent with the hypothesis that TNFalpha regulates proteins known to be involved in neuronal communication (AMPA receptors) and cell regulation (voltage-dependent calcium channels) in a relatively rapid period of time (a few hours). These actions may be related to the behavioral effects produced by TNFalpha that occur within this time frame.

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons.

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.

Angiotensin II increases intracellular calcium concentration in pig endometrial stromal cells through type 1 angiotensin receptors, but does not stimulate phospholipase C activity or prostaglandin F2alpha secretion.

Although the presence of endometrial receptors for angiotensin (Ang) II has been demonstrated, a specific function for AngII in the uterus has not been identified. Cytosolic free Ca2+ concentration [Ca2+]i, phospholipase C (PLC) activity and prostaglandin (PG) F2alpha secretion in response to AngII and oxytocin (OT) were measured in pig endometrial stromal cells collected 16 days after oestrus. Treatment with 100 nM OT or AngII increased (P<0.001) [Ca2+]i in stromal cells similarly (720 +/- 34 v. 690 +/- 33 pM, respectively). Subsequent administration of OT or AngII to the same cells induced smaller [Ca2+]i increases (25% or 35% of the initial responses, respectively) that occurred only if the second exposure to the same agent took place at least 5 min after the first. When administered sequentially, OT and AngII each induced a full response within 1 min of the previous treatment, regardless of which peptide was applied first. Whereas OT increased PLC activity and PGF2alpha secretion in stromal cells (P<0.01), AngII did not increase either PLC activity or PGF2alpha secretion. Type I AngII (AT1) receptors were present on stromal cells, whereas AT2 receptors were absent. Therefore, the effect of AngII in stromal cells was mediated via AT1 receptors. That AngII increased [Ca2+]i in stromal cells, but did not increase PLC or PGF2alpha secretion, indicates that either AngII releases a pool of Ca2+ through a mechanism that is not mediated by PLC and is not involved in PGF2alpha secretion or that a mechanism for PGF2alpha production other than one involving Ca2+ may exist.

GHRH and IL1beta increase cytoplasmic Ca(2+) levels in cultured hypothalamic GABAergic neurons.

GHRH and IL1beta regulate sleep via the hypothalamus. However, actions of these substances on neurons are poorly understood. In this study, we found both GHRH (100 nM) and IL1beta (1.2 pM) acutely increased cytosolic Ca(2+) in 7.6 and 4.0% of cultured hypothalamic neurons tested, respectively, and 1.2% of neurons responded to both. The neurons that responded were mostly GABAergic (96, 81, and 100% for GHRH, IL1beta, and dual-responsive neurons, respectively).

Diurnal effects of acute and chronic administration of ethanol on sleep in rats.

BACKGROUND: Disturbances in sleep patterns are a complicating factor in recovery from alcoholism. The effects of acute and chronic alcohol treatments on sleep in rats were determined. METHODS: Adult male Sprague-Dawley rats were acclimated to a temperature-controlled chamber, and electromyograms and electroencephalograms (EEGs) were obtained during 23-hr recording sessions. Time spent in rapid eye movement sleep (REMS) and non-REM sleep (NREMS), EEG slow-wave activity (SWA) during NREMS, a spectral analysis of the EEG by fast Fourier transform, and brain temperatures were determined. RESULTS: Acute exposure to alcohol (2.3 and 3.0 g/kg) by gastric intubation at the beginning of dark onset produced an increase in NREMS and a suppression of SWA. Spectral analysis revealed that during the first 4 hr there was a small increase in very-low-frequency bands (0.5-2 Hz), with a suppression of higher-frequency bands. This was followed by a suppression of low-frequency bands. A dose of 3.0 g/kg at light onset caused an increase in NREMS and a suppression of SWA. Spectral analysis revealed a suppression of low-frequency bands throughout the first 12 hr of recording but no change on high-frequency bands with light-onset alcohol. Chronic treatment with alcohol (6% alcohol in a liquid diet with pair-fed isocaloric controls) for 3 weeks produced an increase in NREMS and a decrease in EEG power density in frequency bands above 2 Hz. Chronic alcohol also reduced the circadian variation of REMS, an effect that showed a rebound 1 week after the alcohol treatment was terminated. Two weeks after the alcohol treatment was stopped, NREMS and REMS values returned to baseline. CONCLUSIONS: These results demonstrate differences in the effect of acute alcohol on sleep depending on the time of administration and demonstrate that distinct alterations in sleep patterns are induced by chronic treatments in as little as 3 weeks.