Abnormal γ-aminobutyric acid neurotransmission in a Kcnq2 model of early onset epilepsy.

OBJECTIVE: Mutations of the KCNQ2 gene, which encodes the Kv 7.2 subunit of voltage-gated M-type potassium channels, have been associated with epilepsy in the neonatal period. This developmental stage is unique in that the neurotransmitter gamma aminobutyric acid (GABA), which is inhibitory in adults, triggers excitatory action due to a reversed chloride gradient. METHODS: To examine whether KCNQ2-related neuronal hyperexcitability involves neonatally excitatory GABA, we examined 1-week-old knockin mice expressing the Kv 7.2 variant p.Tyr284Cys (Y284C). RESULTS: Brain slice electrophysiology revealed elevated CA1 hippocampal GABAergic interneuron activity with respect to presynaptic firing and postsynaptic current frequency. Blockade with the GABAA receptor antagonist bicuculline decreased ictal-like bursting in brain slices with lowered divalent ion concentration, which is consistent with GABA mediating an excitatory function that contributes to the hyperexcitability observed in mutant animals. SIGNIFICANCE: We conclude that excitatory GABA contributes to the phenotype in these animals, which raises the question of whether this special type of neurotransmission has broader importance in neonatal epilepsy than is currently recognized.

Acute p38-mediated inhibition of NMDA-induced outward currents in hippocampal CA1 neurons by interleukin-1beta.

Interleukin-1beta (IL-1beta) is a potent pro-inflammatory cytokine that is primarily produced by microglia in the brain. IL-1beta inhibits N-methyl-d-aspartate (NMDA)-induced outward currents (I(NMDA-OUT)) through IL-1 type I receptor (IL-1RI) in hippocampal CA1 neurons (Zhang, R., Yamada, J., Hayashi, Y., Wu, Z, Koyama, S., Nakanishi, H., 2008. Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons, Biochem. Biophys. Res. Commun. 372, 816-820). Although IL-1RI is associated with mitogen-activated protein kinases, their involvement in the effect of IL-1beta on I(NMDA-OUT) remains unclear. In the present study, we demonstrate that IL-1beta caused activation of p38 mitogen-activated protein kinase and that the p38 inhibitor SB203580 significantly blocked the effect of IL-1beta on I(NMDA-OUT) in hippocampal CA1 neurons. Furthermore, the intracellular perfusion of active recombinant p38alpha significantly decreased the mean amplitude of I(NMDA-OUT). In neurons prepared from inflamed hippocampus, the mean amplitude of I(NMDA-OUT) was significantly reduced. In the inflamed hippocampus, IL-1beta and IL-1RI were expressed mainly in microglia and neurons, respectively. These results suggest that IL-1beta increases the excitability of hippocampal CA1 neurons in the p38-dependent inhibition of I(NMDA-OUT).

Effects of the serotonin 1A, 2A, 2C, 3A, and 3B and serotonin transporter gene polymorphisms on the occurrence of paroxetine discontinuation syndrome.

Paroxetine discontinuation symptoms can at times be severe enough to reduce the quality of life. However, it is currently not possible to predict the occurrence of discontinuation syndrome before the initiation or discontinuation of paroxetine treatment. In this study, we investigated the effects of genetic polymorphisms in the serotonin 1A, 2A, 2C, 3A, and 3B receptor, the serotonin transporter, and the cytochrome P450 2D6 (CYP2D6) genes on the occurrence of paroxetine discontinuation syndrome. A consecutive series of 56 Japanese patients who had a diagnosis of major depressive or anxiety disorder according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, were treated with paroxetine. Paroxetine discontinuation syndrome was found in 35.7% of the patients by direct interview. Patients who stopped taking paroxetine abruptly experienced paroxetine discontinuation syndrome significantly more often than patients who had a tapering off of the dosage of medication. Patients who had the -1019C allele experienced paroxetine discontinuation syndrome more frequently than patients who had the -1019G homozygote (nominal P = 0.0423) of the serotonin 1A receptor gene. However, this result did not remain significant after the Bonferroni correction for multiple comparisons. The findings suggest that the abrupt stoppage of medication is a major risk factor for the occurrence of paroxetine discontinuation syndrome and that C(-1019)G polymorphism of the serotonin 1A receptor gene may be related to the occurrence of the syndrome.

Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation.

Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.

Electricity use is associated with residents' vital data and lifestyles: observational study using an IT health support system in Japan.

Motivated by developments in information technology, recording personal parameters with health devices is effective in health promotion. Today's indoor individual lifestyles often involve using electrical appliances. We developed a health support system combined with wireless electricity monitoring and investigated whether electricity use is associated with residents' vital data and lifestyles. We recruited 116 participants in February 2013. Their vital and electricity use data were collected daily. They completed a self-administered questionnaire. Among participants living alone, electricity from 20 February to 11 March 2013 was negatively associated with high-density lipoprotein (HDL) (P = 0.008) and positively associated with low-density lipoprotein (LDL) (P = 0.007) and neutral fat (P = 0.020) levels. Among all participants, electricity use was negatively associated with vegetable intake (P = 0.044) and step count (P = 0.040). Temperature sensitivity in winter was negatively associated with the LDL/HDL ratio for both men and women. For men, temperature sensitivity in winter was negatively related with alcohol intake; for women, it was positively related to body fat percentage and abdominal circumference and negatively correlated to vegetable intake. Temperature sensitivity in summer was positively associated with vegetable intake for men and women. In conclusion, electricity use was related to vital data and lifestyles and influenced by temperature.

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons.

There is increasing evidence that a functional interaction exists between interleukin-1beta (IL-1beta) and N-methyl-D-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1beta on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1beta (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca(2+)-activated K(+) (K(Ca)) channels. IL-1beta (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1beta significantly inhibited ChTX-sensitive K(Ca) currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1beta. These results strongly suggest that IL-1beta increases the neuronal excitability by inhibition of ChTX-sensitive K(Ca) channels activated by Ca(2+) influx through both NMDA receptors and voltage-gated Ca(2+) channels.

Activation of mu-opioid receptor selectively potentiates NMDA-induced outward currents in rat locus coeruleus neurons.

We investigated the opioid modulation of N-methyl-d-aspartic acid (NMDA) receptor-mediated response in dissociated LC neurons using nystatin-perforated patch recording. In Mg(2+)-free extracellular solution, NMDA induced an inward current (I(NMDA-IN)) and a subsequent outward current (I(NMDA-OUT)) at a holding potential of -40 mV. A selective mu-opioid receptor agonist, d-Ala(2),N-MePhe(4),Gly(5)-ol-enkephalin (DAMGO), potentiated I(NMDA-OUT) in a concentration-dependent manner with the half-maximal effective concentration of 0.7 microM, while DAMGO (0.1-10 microM) did not affect I(NMDA-IN). Under the condition of I(NMDA-OUT) blockade by the use of Cs-based pipette solution or by buffering intracellular Ca(2+) with a high concentration of EGTA and zero Ca(2+), DAMGO did not change I(NMDA-IN). The DAMGO potentiation of I(NMDA-OUT) was prevented by naloxone, an opioid receptor antagonist. In addition, the DAMGO potentiation of I(NMDA-OUT) was prevented by treatment with staurosporine, a broad spectrum protein kinase inhibitor. In conclusion, mu-opioid receptor activation selectively potentiated I(NMDA-OUT) via intracellular signal pathway without affecting I(NMDA-IN) in LC neurons. We suggest the inhibitory opioid effect through NMDA receptor-mediated response in LC neurons.

Leptin attenuates D2 receptor-mediated inhibition of putative ventral tegmental area dopaminergic neurons.

Obesity causes hyperleptinemia. We have previously shown that D2 receptor-mediated inhibition of ventral tegmental area (VTA) dopaminergic neurons is attenuated in diet-induced mice with obesity. Consequently, we hypothesized that high concentrations of serum leptin during obesity might modulate D2 receptor-mediated effects on VTA dopaminergic neurons. To investigate our hypothesis, we examined leptin effects on D2 receptor-mediated inhibition of putative VTA dopaminergic neurons from lean mice using electrophysiological techniques. Leptin (100 nmol/L) directly inhibited spontaneous firing in 71% of putative VTA dopaminergic neurons (leptin-responsive), whereas the remaining 29% of neurons were leptin-nonresponsive. In 41% of leptin-responsive neurons, leptin attenuated the reduced firing rate produced by quinpirole (100 nmol/L), whereas the remaining 59% of neurons exhibited no effect of leptin. In leptin-nonresponsive neurons, no significant leptin-induced effect was observed on reduced firing rate produced by quinpirole. In leptin-responsive neurons with positive leptin-induced attenuation of quinpirole effects, leptin-induced attenuation persisted for >20 min, whereas no such persistent attenuation was observed in other types of neurons. In conclusion, leptin attenuates D2 receptor-mediated inhibition in a subpopulation of putative VTA dopaminergic neurons. We suggest that leptin directly decreases, and indirectly increases, excitability of VTA dopaminergic neurons. In turn, this may contribute to a change in feeding behavior through the mesolimbic dopaminergic system during the development of obesity.

Estimated urinary salt excretion by a self-monitoring device is applicable to education of salt restriction.

The objective was to investigate the validity of a self-monitoring device that estimates 24-h urinary salt excretion from overnight urine samples as a tool for education regarding salt restriction. Twenty healthy volunteers consumed test meals for 14 days, with salt content as follows: 10 g (days 1-5); 5 g (days 6-8, 12 and 14); and 13 g (days 9-11 and 13). On days 2-15, urinary salt excretion was estimated from overnight urine samples by a self-monitoring device. Twenty-four-hour urine samples were collected on days 5 and 8 to measure salt excretion directly. Blood pressure was measured in the morning and during sleep on days 1-15. Estimated urinary salt excretion measured by the device showed a correlation with salt intake, and the ratio of estimated urinary salt excretion to salt intake was 0.84±0.10 (days 2-6), 1.27±0.28 (days 7-9), 0.70±0.11 (days 10-12), 1.37±0.22 (day 13), 0.68±0.13 (day 14) and 1.33±0.19 (day 15). The correlation between estimated urinary salt excretion measured by a device and directly measured 24-h urinary salt excretion was significant (r=0.65, P<0.05) during the period of 10 g salt intake, but not during 5 g salt intake. Blood pressure in the morning was not influenced by the change in salt intake, but systolic pressure during sleep showed a significant increase or decrease according to the levels of salt intake. In conclusion, a self-monitoring device, which can estimate 24-h urinary salt excretion from overnight urine samples, is considered to be a practical tool for education regarding salt restriction, although a similar future investigation is needed in older and/or hypertensive subjects.

Hepcidin/ferroportin expression levels involve efficacy of pegylated-interferon plus ribavirin in hepatitis C virus-infected liver.

AIM: To investigate the relationship between the iron-metabolism-related gene expression profiles and efficacy of antiviral therapy in chronic hepatitis C patients. METHODS: The hepatic expression profile of iron-metabolism-related genes was analyzed and its association with virological response to pegylated-interferon plus ribavirin combination therapy was evaluated. A hundred patients with chronic hepatitis C (genotype1b, n = 50; genotype 2, n = 50) were enrolled and retrospectively analyzed. Liver biopsy samples were subjected to quantitative polymerase chain reaction for iron-metabolism-related genes and protein expression (Western blotting analysis) for ferroportin. As a control, normal liver tissue was obtained from 18 living donors of liver transplantation. Serum hepcidin level was measured by sensitive liquid chromatography/electrospray ionization tandem mass spectrometry. RESULTS: Iron overload is associated with liver damage by increasing oxidative stress and hepatitis C virus (HCV) is reported to induce iron accumulation in hepatocytes in vivo. Conversely, iron administration suppresses HCV replication in vitro. Therefore, the association between HCV infection and iron metabolism remains unclear. Compared with controls, patients had significantly higher gene expression for transferrin, iron-regulatory proteins 1 and 2, divalent metal transporter 1, and ferroportin, but similar for transferrin receptors 1 and 2, and hepcidin. When the expression profiles were compared between sustained virological response (SVR) and non-SVR patients, the former showed significantly lower transcription and protein expression of hepcidin and ferroportin. Expression of hepcidin-regulating genes, BMPR1, BMPR2, and hemojuvelin, was significantly increased, whereas BMP2 was decreased in HCV-infected liver. BMPR2 and hemojuvelin expression was significantly lower in the SVR than non-SVR group. HCV infection affects the expression of iron-metabolism-related genes, leading to iron accumulation in hepatocytes. CONCLUSION: Decreased expression of hepcidin and ferroportin in SVR patients indicates the importance of hepatocytic iron retention for viral response during pegylated-interferon plus ribavirin treatment.

Role of presynaptic 5-HT1A and 5-HT3 receptors in modulation of synaptic GABA transmission in dissociated rat basolateral amygdala neurons.

Serotonin (5-HT) is considered to play a significant role in anxiety-related behaviors in animals through actions on the amygdaloid complex. To evaluate this role from the point of neurotransmitter release regulation, nystatin-perforated patch recording was employed on mechanically dissociated basolateral amygdala neurons containing functional synaptic boutons. GABAAergic miniature inhibitory postsynaptic currents (mIPSCs) were pharmacologically separated. In subsets of neurons, 8-OH-DPAT (1 microM), a specific 5-HT1A agonist, continuously inhibited mIPSC frequency without effects on mIPSC amplitude. By comparison, mCPBG (1 microM), a specific 5-HT3 agonist, transiently facilitated mIPSC frequency without effects on mIPSC amplitude. Together these results suggest the presynaptic existence of both 5-HT receptor subtypes. In these neurons, application of 8-OH-DPAT and its subsequent removal still suppressed mCPBG-induced responses on mIPSCs. This suppression was not caused by a reduction of presynaptic 5-HT3 receptor affinities to mCPBG and was completely eliminated by pretreatment with N-ethylmaleimide, a pertussis toxin sensitive GTP-binding protein inhibitor. In the neurons exhibiting presynaptic modulation with mCPBG but not 8-OH-DPAT, such suppression by exposure to 8-OH-DPAT was not observed. In conclusion, activation of presynaptic 5-HT1A receptors inhibited mIPSC frequency and at the same time suppressed, via a G-protein-mediated mechanism, the transient facilitation of mIPSC frequency produced by activation of presynaptic 5-HT3 receptors.

Obesity attenuates D2 autoreceptor-mediated inhibition of putative ventral tegmental area dopaminergic neurons.

Abstract The ventral tegmental area (VTA) in the midbrain is important for food reward. High-fat containing palatable foods have reinforcing effects and accelerate obesity. We have previously reported that diet-induced obesity selectively decreased the spontaneous activity of VTA GABA neurons, but not dopamine neurons. The spontaneous activity of VTA dopamine neurons is regulated by D2 autoreceptors. In this study, we hypothesized that obesity would affect the excitability of VTA dopamine neurons via D2 autoreceptors. To examine this hypothesis, we compared D2 receptor-mediated responses of VTA dopamine neurons between lean and obese mice. Mice fed on a high-fat (45%) diet and mice fed on a standard diet were used as obese and lean models, respectively. Brain slice preparations were made from these two groups. Spontaneous activity of VTA neurons was recorded by extracellular recording. Putative VTA dopamine neurons were identified by firing inhibition with a D2 receptor agonist quinpirole, and electrophysiological criteria (firing frequency <5 Hz and action potential current duration >1.2 msec). Single-dose application of quinpirole (3-100 nmol/L) exhibited similar firing inhibition of putative VTA dopamine neurons between lean and obese mice. In stepwise application by increasing quinpirole concentrations of 3, 10, 30, and 100 nmol/L subsequently, quinpirole-induced inhibition of firing decreased in putative VTA dopamine neurons of obese mice compared with those of lean mice. In conclusion, high-fat diet-induced obesity attenuated D2 receptor-mediated inhibition of putative VTA dopamine neurons due to the acceleration of D2 receptor desensitization.

Obesity decreases excitability of putative ventral tegmental area GABAergic neurons.

Palatable food has reinforcing effects on feeding and accelerates obesity. Alteration of food-related behavior in obesity may promote maintenance of obesity. The ventral tegmental area (VTA) of the midbrain is important for food reward. However, it is unknown whether activity of VTA neurons is altered in diet-induced obesity. In this study, we examined VTA neuronal activity using an electrophysiological technique in diet-induced obese mice. Male 4-week-old mice were fed a high-fat diet or a standard diet for 5-6 weeks. Mice fed a high-fat diet gained greater body weight with heavier visceral fat compared with those fed a standard diet. Brain slice preparations were obtained from the lean and obese mice. Spontaneous activity of VTA neurons was recorded extracellularly. We found a negative correlation between firing frequency (FF) and action potential (AP) current duration in lean and obese mice VTA neurons. VTA neurons were classified as group-1 neurons (FF <5.0 Hz and AP current duration >1.2 msec) or group-2 neurons (FF ≧5.0 Hz and AP current duration ≦1.2 msec). FF, AP current duration, and firing regularity of VTA group-1 neurons were similar between lean and obese mice. Obese mice VTA group-2 neurons had a lower FF and shorter AP current duration compared with lean mice. In conclusion, obesity minimally affects VTA group-1 neurons, which are presumed to be dopaminergic, but decreases excitability of VTA group-2 neurons, which are presumed to be GABAergic. This differential effect may contribute to the pathophysiology of reward-related feeding in obesity.

Ethanol inhibition of m-current and ethanol-induced direct excitation of ventral tegmental area dopamine neurons.

Ethanol-induced excitation of ventral tegmental area dopamine (DA VTA) neurons is thought to be critical for the reinforcing effects of ethanol. Although ligand-gated ion channels are known to be the targets of ethanol, ethanol modulation of voltage-dependent ion channels of central neurons has not been well studied. We have demonstrated that ethanol excites DA VTA neurons by the reduction of sustained K(+) currents and recently reported that M-current (I(M)) regulates action potential generation through fast and slow afterhyperpolarization phases. In the present study we thus examined whether ethanol inhibition of I(M) contributes to the excitation of DA VTA neurons using nystatin-perforated patch current- and voltage-clamp recordings. Ethanol (20-120 mM) reduced I(M) in a concentration-dependent manner and increased the spontaneous firing frequency of DA VTA neurons. Ethanol-induced increase in spontaneous firing frequency correlated positively with ethanol inhibition of I(M) with a slope value of 1.3. Specific I(M) inhibition by XE991 (0.3-10 microM) increased spontaneous firing frequency which correlated positively with I(M) inhibition with a slope value of 0.5. In the presence of 10 muM XE991, a concentration that produced maximal inhibition of I(M), ethanol still increased the spontaneous firing frequency of DA VTA neurons in a concentration-dependent manner. Thus we conclude that, although ethanol causes inhibition of I(M) and this results in some increase in the firing frequency of DA VTA neurons, another effect of ethanol is primarily responsible for the ethanol-induced increase in firing rate in these neurons.

Characterization of M-current in ventral tegmental area dopamine neurons.

M-current (I(M)) is a voltage-gated potassium current (KCNQ type) that affects neuronal excitability and is modulated by some drugs of abuse. Ventral tegmental area (VTA) dopamine (DA) neurons are important for the reinforcing effects of drugs of abuse. Therefore we studied I(M) in acutely dissociated rat DA VTA neurons with nystatin-perforated patch recording. The standard deactivation protocol was used to measure I(M) during voltage-clamp recording with hyperpolarizing voltage steps to -65 mV (in 10-mV increments) from a holding potential of -25 mV. I(M) amplitude was voltage dependent and maximal current amplitude was detected at -45 mV. The deactivation time constant of I(M) was voltage dependent and became shorter at more negative voltages. The I(M)/KCNQ antagonist XE991 (0.3-30 microM) caused a concentration-dependent reduction in I(M) amplitude with an IC(50) of 0.71 microM. Tetraethylammonium (TEA, 0.3-10 mM) caused a concentration-dependent inhibition of I(M) with an IC(50) of 1.56 mM. In current-clamp recordings, all DA VTA neurons were spontaneously active. Analysis of evoked action potential shape indicated that XE991 (1-10 microM) reduced the fast and slow components of the spike afterhyperpolarization (AHP) without affecting the middle component of the AHP. Action potential amplitude, duration, and threshold were not affected by XE991. In addition, 10 microM XE991 significantly shortened the interspike intervals in evoked spike trains. In conclusion, I(M) is active near threshold in DA VTA neurons, is blocked by XE991 (10 microM) and TEA (10 mM), may contribute to the shape of the AHP, and may decrease excitability of these neurons.

A-type K+ current of dopamine and GABA neurons in the ventral tegmental area.

A-type K(+) current (I(A)) is a rapidly inactivating voltage-dependent potassium current which can regulate the frequency of action potential (AP) generation. Increased firing frequency of ventral tegmental area (VTA) neurons is associated with the reinforcing effects of some drugs of abuse like nicotine and ethanol. In the present study, we classified dopamine (DA) and GABA VTA neurons, and investigated I(A) properties and the physiological role of I(A) in these neurons using conventional whole cell current- and voltage-clamp recording. DA VTA neurons had a mean firing frequency of 3.5 Hz with a long AP duration. GABA VTA neurons had a mean firing frequency of 16.7 Hz with a short AP duration. For I(A) properties, the voltage-dependence of steady-state I(A) activation and inactivation was similar in DA and GABA VTA neurons. I(A) inactivation was significantly faster and became faster at positive voltages in GABA neurons than DA neurons. Recovery from inactivation was significantly faster in DA neurons than GABA neurons. I(A) current density at full recovery was significantly larger in DA neurons than GABA neurons. In DA and GABA VTA neurons, latency to the first AP after the recovery from membrane hyperpolarization (repolarization latency) was measured. Longer repolarization latency was accompanied by larger I(A) current density in DA VTA neurons, compared with GABA VTA neurons. We suggest that I(A) contributes more to the regulation of AP generation in DA VTA neurons than in GABA VTA neurons.

The effects of long chain-length n-alcohols on the firing frequency of dopaminergic neurons of the ventral tegmental area.

The dopaminergic neurons of the ventral tegmental area (DA VTA neurons) have been implicated in the reinforcing properties of drugs of abuse, including ethanol (ethyl alcohol). Ethanol increases the spontaneous firing frequency of DA VTA neurons in vitro, in both brain slices and acutely dissociated neurons, and also in vivo. In many systems, longer n-alkyl alcohols have a more potent effect than ethanol, and the potency is a function of the number of carbons in the alkyl chain. We studied n-alcohols of chain length 1 (methanol) to 5 (pentanol) on the firing rate of DA VTA neurons in brain slice preparations. All of the alcohols studied produced increases in the spontaneous firing frequency in DA VTA neurons; as the chain length increased, lower concentrations of the alcohols were needed to produce the same percentage increase in firing. With very high concentrations of all the alcohols except methanol, we observed apparent depolarization block of firing. In addition, trichloroethanol (TCE), the active metabolite of chloral hydrate, increased the firing frequency of DA VTA neurons, and the EC(40) (concentration to produce a 40% increase in firing rate) of TCE was below that of ethanol. These studies indicate that excitation of VTA dopamine neurons by n-alcohols is related to the chain length of the carbons. This is likely to be a characteristic of the ethanol-sensitive element of DA VTA neurons and may be useful in identifying the element of the membrane that is responsible for ethanol-induced excitation.

Spontaneous activity and properties of two types of principal neurons from the ventral tegmental area of rat.

We investigated the spontaneous activity and properties of freshly isolated ventral tegmental area (VTA) principal neurons by whole cell recording and single-cell RT-PCR. The VTA principal neurons, which were tyrosine hydroxylase-positive and glutamic acid decarboxylase (GAD67)-negative, exhibited low firing frequency and a long action potential (AP) duration. The VTA principal neurons exhibited a calretinin-positive and parvalbumin-negative Ca2+-binding protein mRNA expression pattern. The VTA principal neurons were classified into two subpopulations based on their firing frequency coefficient of variation (CV) at room temperature (21-23 degrees C): irregular-type neurons with a large CV and tonic-type neurons with a small CV. These two firing patterns were also recorded at the temperature of 34 degrees C and in nystatin-perforated patch recording. In VTA principal neurons, the AP afterhyperpolarization (AHP) amplitude contributed to the firing regularity and AHP decay slope contributed to the firing frequency. The AHP amplitude in the irregular-type VTA principal neurons was smaller than that in the tonic-type VTA principal neurons. There was no significant difference in the AHP decay slope between the two-types of VTA principal neurons. Apamin-sensitive small-conductance Ca2+-activated K+ (SK) channels contributed to the AHP and the regular firing of the tonic-type neurons but contributed little to the AHP and firing of the irregular-type neurons. In voltage-clamp tail-current analysis, in both conventional and nystatin-perforated whole cell recording, the apamin-sensitive AHP current density of the tonic-type neurons was significantly larger than that of the irregular-type neurons. We suggest that apamin-sensitive SK current contributes to intrinsic firing differences between the two subpopulations of VTA principal neurons.