Presence of ethanol-sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens.

KEY POINTS: The nucleus accumbens (nAc) is involved in addiction-related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse. ABSTRACT: Alcohol abuse causes major social, economic and health-related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and ß in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)-positive MSNs. Interestingly, the glycine-evoked currents in dissociated DRD1-positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR-mediated tonic current by ethanol suggests that they modulate the excitability of DRD1-positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction-related and compulsive behaviours.

Role of membrane GM1 on early neuronal membrane actions of Aß during onset of Alzheimer's disease.

The ability of beta-amyloid peptide (Aß) to disrupt the plasma membrane through formation of pores and membrane breakage has been previously described. However, the molecular determinants for these effects are largely unknown. In this study, we examined if the association and subsequent membrane perforation induced by Aß was dependent on GM1 levels. Pretreatment of hippocampal neurons with D-PDMP decreased GM1 and Aß clustering at the membrane (Aß fluorescent-punctas/20µm, control=16.2±1.1 vs. D-PDMP=6.4±0.4, p<0.001). Interestingly, membrane perforation with Aß occurred with a slower time course when the GM1 content was diminished (time to establish perforated configuration (TEPC) (min): control=7.8±2 vs. low GM1=12.1±0.5, p<0.01), suggesting that the presence of GM1 in the membrane can modulate the distribution and the membrane perforation by Aß. On the other hand, increasing GM1 facilitated the membrane perforation (TEPC: control=7.8±2 vs. GM1=6.2±1min, p<0.05). Additionally, using Cholera Toxin Subunit-B (CTB) to block the interaction of Aß with GM1 attenuated membrane perforation significantly. Furthermore, pretreatment with CTB decreased the membrane association of Aß (fluorescent-punctas/20µm, Aß: control=14.8±2.5 vs. CTB=8±1.4, p<0.05), suggesting that GM1 also plays a role in both association of Aß with the membrane and in perforation. In addition, blockade of the Aß association with CTB inhibited synaptotoxicity. Taken together, our results strongly suggest that membrane lipid composition can affect the ability of Aß to associate and subsequently perforate the plasma membrane thereby modulating its neurotoxicity in hippocampal neurons.

Rapamycin protects against Aß-induced synaptotoxicity by increasing presynaptic activity in hippocampal neurons.

The mammalian target of rapamycin (mTOR) is involved in the regulation of learning and memory. Recently, rapamycin has been shown to be neuroprotective in models for Alzheimer's disease in an autophagy-dependent manner. Here we show that rapamycin exerts neuroprotection via a novel mechanism that involves presynaptic activation. Rapamycin increases the frequency of miniature excitatory postsynaptic currents and calcium transients of rat hippocampal primary neurons by a mechanism that involves the up regulation of SV2, a presynaptic vesicular protein linked to neurotransmitter release. Under these conditions, rapamycin-treated hippocampal neurons are resistant to the synaptotoxic effect induced by Aß oligomers, suggesting that enhancers of presynaptic activity can be therapeutic agents for Alzheimer's disease.

Reduced sedation and increased ethanol consumption in knock-in mice expressing an ethanol insensitive alpha 2 subunit of the glycine receptor.

Previous studies have shown the presence of several subunits of the inhibitory glycine receptor (GlyR) in the reward system, specifically in medium spiny neurons (MSNs) of the nucleus Accumbens (nAc). It was suggested that GlyR α1 subunits regulate nAc excitability and ethanol consumption. However, little is known about the role of the α2 subunit in the adult brain since it is a subunit highly expressed during early brain development. In this study, we used genetically modified mice with a mutation (KR389-390AA) in the intracellular loop of the GlyR α2 subunit which results in a heteromeric α2ß receptor that is insensitive to ethanol. Using this mouse model denoted knock-in α2 (KI α2), our electrophysiological studies showed that neurons in the adult nAc expressed functional KI GlyRs that were rather insensitive to ethanol when compared with WT GlyRs. In behavioral tests, the KI α2 mice did not show any difference in basal motor coordination, locomotor activity, or conditioned place preference compared with WT littermate controls. In terms of ethanol response, KI α2 male mice recovered faster from the administration of ataxic and sedative doses of ethanol. Furthermore, KI α2 mice consumed higher amounts of ethanol in the first days of the drinking in the dark protocol, as compared with WT mice. These results show that the α2 subunit is important for the potentiation of GlyRs in the adult brain and this might result in reduced sedation and increased ethanol consumption.

Changes in neuronal excitability and synaptic transmission in nucleus accumbens in a transgenic Alzheimer's disease mouse model.

Several previous studies showed that hippocampus and cortex are affected in Alzheimer's disease (AD). However, other brain regions have also been found to be affected and could contribute with new critical information to the pathophysiological basis of AD. For example, volumetric studies in humans have shown a significant atrophy of the striatum, particularly in the nucleus Accumbens (nAc). The nAc is a key component of the limbic reward system and it is involved in cognition and emotional behaviors such as pleasure, fear, aggression and motivations, all of which are affected in neurodegenerative diseases such as AD. However, its role in AD has not been extensively studied. Therefore, using an AD mouse model, we investigated if the nAc was affected in 6 months old transgenic 2xTg (APP/PS1) mice. Immunohistochemistry (IHC) analysis in 2xTg mice showed increased intraneuronal Aß accumulation, as well as occasional extracellular amyloid deposits detected through Thioflavin-S staining. Interestingly, the intracellular Aß pathology was associated to an increase in membrane excitability in dissociated medium spiny neurons (MSNs) of the nAc. IHC and western blot analyses showed a decrease in glycine receptors (GlyR) together with a reduction in the pre- and post-synaptic markers SV2 and gephyrin, respectively, which correlated with a decrease in glycinergic miniature synaptic currents in nAc brain slices. Additionally, voltage-clamp recordings in dissociated MSNs showed a decrease in AMPA- and Gly-evoked currents. Overall, these results showed intracellular Aß accumulation together with an increase in excitability and synaptic alterations in this mouse model. These findings provide new information that might help to explain changes in motivation, anhedonia, and learning in the onset of AD pathogenesis.

High ethanol sensitive glycine receptors regulate firing in D1 medium spiny neurons in the nucleus accumbens.

Inhibitory glycine receptors (GlyRs) are widely expressed in spinal cord and brain stem. They are also expressed in the nucleus Accumbens (nAc) where they have been implicated in the release of dopamine from the ventral tegmental area to the nAc in the presence of ethanol. One of the major types of neurons in the nAc are the Dopamine 1 receptor-expressing (D1+) medium spiny neurons (MSNs) that are activated when addictive drugs, like ethanol, are administrated. Thus, D1(+) MSNs are a relevant target for the study of ethanol effects. Here, using electrophysiological recordings, we report that GlyRs in D1(+) MSNs are highly sensitive to ethanol, with potentiation starting at 5 mM (26 ± 5%). Single channel recordings in D1(+) MSNs showed that 10 mM ethanol increased the open probability of the channel (0.22 ± 0.05 versus 0.66 ± 0.16), but did not affect channel conductance (~40 pS). A glycinergic mediated tonic current in D1(+) MSNs was potentiated by 10 and 50 mM ethanol causing a reduction in the excitability of these cells. A 34 ± 7% reduction in action potential firing was observed in these neurons in the presence of 50 mM ethanol. Interestingly, no effects of ethanol were detected in the presence of strychnine or in D1(-) MSNs in the nAc. These results indicate that GlyRs present in D1(+) MSNs are sensitive to low concentrations of ethanol, and that potentiation of this inhibitory current regulates the activation of nAc, acting as a homeostatic signal that would prevent over-activation of the reward system when drugs like ethanol are consumed.

Regulation of glycinergic and GABAergic synaptogenesis by brain-derived neurotrophic factor in developing spinal neurons.

Brain-derived neurotrophic factor (BDNF) effects on the establishment of glycinergic and GABAergic transmissions in mouse spinal neurons were examined using combined electrophysiological and calcium imaging techniques. BDNF (10 ng/ml) caused a significant acceleration in the onset of synaptogenesis without large effects on the survival of these neurons. Amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) associated to activation of glycine and GABA(A) receptors were augmented in neurons cultured with BDNF. The neurotrophin effect was blocked by long term tetrodotoxin (TTX) addition suggesting a dependence on neuronal activity. In addition, BDNF caused a significant increase in glycine- and GABA-evoked current densities that partly explains the increase in synaptic transmission. Presynaptic mechanisms were also involved in BDNF effects since triethylammonium(propyl)-4-(2-(4-dibutylamino-phenyl)vinyl)pyridinium (FM1-43) destaining with high K(+) was augmented in neurons incubated with the neurotrophin. The effects of BDNF were mediated by receptor tyrosine kinase B (TrkB) and mitogen-activated protein kinase kinase (MEK) activation since culturing neurons with either (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'- kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (K252a) or 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) blocked the augmentation in synaptic activity induced by the neurotrophin.

Historical and current perspectives of neuroactive compounds derived from Latin America.

Plants and invertebrates in Latin America have contributed to a great extent in the use, discovery and development of novel neuroactive tools. Significantly, these neuroactive drugs have proven to be particularly important for our current understanding of the physiology and pharmacology of the nervous system. In addition, these discoveries have helped to build the modern and successful pharmacological business that we know today. For example, curare helped to introduce the use of muscle relaxing agents into modern surgical techniques. The discovery of cocaine from the leaves of Peruvian coca plants was instrumental in the discovery of local anesthetics. The search and discovery for useful neuroactive compounds derived from Latin America has also been ongoing in other areas and new applications for quinine, capsaicin and epibatidine were recently described. Besides these organic compounds, several peptides produced by spiders and other invertebrates to hunt their prey also induce effects in channels and membrane receptors at very low concentrations, indicating their high potency and selectivity. It is likely that new pharmaceutics will be developed from these molecules. The interest to renew the search for new compounds is timely, since largely unexplored lands, such as the Amazon and Patagonia, hold an important number of plants and animals that contain exciting new active compounds. With the introduction of new techniques to isolate, identify and characterize the molecular targets and actions of chemical entities, together with the need for more potent and selective compounds to treat neurological conditions, it is necessary to broaden the current exploratory effort in order to find more beneficial uses.

ATP leakage induces P2XR activation and contributes to acute synaptic excitotoxicity induced by soluble oligomers of ß-amyloid peptide in hippocampal neurons.

Recent studies suggest that the toxic effects of Aß can be attributed to its capability to insert in membranes and form pore-like structures, which are permeable to cations and molecules such as ATP. Our working hypothesis is that Aß increases extracellular ATP causing activation of P2X receptors and potentiating excitatory synaptic activity. We found that soluble oligomers of ß-amyloid peptide increased cytosolic Ca(2+) 4-fold above control (415 ± 28% of control). Also, ATP leakage (157 ± 10% of control) was independent of extracellular Ca(2+), suggesting that ATP traveled from the cytosol through an Aß pore-mediated efflux and not from exocytotic mechanisms. The subsequent activation of P2XR by ATP can contribute to the cytosolic Ca(2+) increase observed with Aß. Additionally, we found that ß-amyloid oligomers bind preferentially to excitatory neurons inducing an increase in excitatory synaptic current frequency (248.1 ± 32.7%) that was blocked by the use of P2XR antagonists such as PPADS (Aß + PPADS: 110.9 ± 18.35%) or Apyrase plus DPCPX (Aß + inhibitors: 98.97 ± 17.4%). Taken together, we suggest that Aß induces excitotoxicity by binding preferentially to excitatory neuron membranes forming a non-selective pore and by increasing intracellular calcium by itself and through P2XR activation by extracellular ATP leading to an augmention in mEPSC activity. All these effects were blocked with a non-specific P2XR antagonist, indicating that part of the neurotoxicity of Aß is mediated by P2XR activation and facilitation of excitatory neurotransmitter release. These findings suggest that P2XR can be considered as a potential new target for the development of drugs or pharmacological tools to treat Alzheimer's disease. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors.

The effect of glycine receptor activation on neurite outgrowth and survival was studied in 5 DIV (days in vitro) spinal neurons. These neurons were depolarized by spontaneous synaptic activity and by glycine, but not by glutamate. These responses were accompanied by increases in intracellular calcium concentration measured with Indo-1 and Fluo-3. Glycine (100 microM, 48 h) increased (46 +/- 6%) the number of primary neurites and total neuritic length. This effect was mediated by synaptic activity and calcium influx because TTX (1 microM) and nimodipine (4 microM) blocked the stimulatory effect of glycine. Neuronal survival, on the other hand, was not affected. This study shows for the first time the modulatory effect of glycine receptors on spinal neuron development.

Phencyclidine blocks two potassium currents in spinal neurons in cell culture.

We studied the effects of phencyclidine (PCP) on the transient and delayed outward K+ currents recorded from spinal cord neurons grown (10-20 days) in cell culture. Sodium channels were blocked with tetrodotoxin (1 microM) and solutions containing low calcium concentrations in the presence of Mg2+ or Co2+ (5 mM) were used to reduce Ca2+ currents. PCP decreased the amplitude and prolonged the decay phase of the action potentials recorded at a holding potential of -70 mV. PCP (0.1-0.5 mM) was more effective than tetraethylammonium (TEA) or 4-aminopyridine (4-AP) in reducing both transient and delayed currents. The amplitude of the transient current during control experiments was always larger than that of the delayed current. It appeared that 4-AP (5 mM) was more potent in blocking the transient current, while TEA (10 mM) modified the delayed current more effectively. Both currents were also reduced by about 10% when the cell soma was perfused with Co2+. This suggested that a small fraction of the total outward current is a Ca2+-activated K+ current. The PCP-induced blockade of K+ currents in central neurons coupled with the profound synaptic effects of the drug may provide the basis for explaining the psychopathology of this hallucinogenic agent.

Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons.

We have investigated the effects of nerve growth factor (NGF, 2.5 ng/ml for 1-2 weeks) on enriched adult rat dorsal root ganglion (DRG) neurons maintained in cell culture in defined media. Whole-cell recordings in cells cultured in the absence and presence of NGF revealed no significant difference in resting membrane potential and input resistance. However, the threshold for spike generation was significantly lower in untreated cells than in treated cells; -25 +/- 1.1 mV vs -19 +/- 2.2 mV, respectively. The sensitivity of the Na+ spike to tetrodotoxin (TTX, 1 microM) was different in cells cultured in the absence or presence of NGF. For example, spikes were abolished by TTX in 100% of untreated cells, while in NGF-treated cells the spike was abolished in only 41% of the neurons. Chemosensitivity of DRG neurons was also different in the absence and presence of NGF. For example, the percent of neurons in which a current activated by 8-methyl-N-vanillyl-6-nonenamide (capsaicin, 500 nM) was detected, increased from 18% in untreated cells to 55% in NGF-treated cells. NGF did not influence the number of cells surviving. The results indicate that NGF can regulate TTX and capsaicin sensitivity in these adult rat sensory neurons. Our experimental protocol indicates that this effect is not mediated by a factor in the serum or released from non-neuronal cells.

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons.

Using patch-clamp techniques we studied several aspects of intracellular GABA(A) and glycine Cl- current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl- current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 microM PMA reduced the amplitude of the GABA(A) and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 microM okadaic acid, a phosphatase inhibitor, induced potentiation in both currents. The amplitude of GABA(A) and glycine currents recorded with 1 mM internal CaCl2 and 10 mM EGTA (10 nM free Ca2+) decayed by less than 30% of control. Increasing the CaCl2 concentration to 10 mM (34 microM free Ca2+) induced a transient potentiation of the GABA(A) current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca2+ concentration. Activation of G proteins with internal FAl4- induced an inhibition of the GABA(A) current, but potentiated the amplitude of the strychnine-sensitive Cl- current. These results indicate that GABA(A) and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca2+. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.

Differential effects of GABAergic ligands in mouse and rat hippocampal neurons.

Previous electrophysiological studies suggested that GABAA receptors in rat hippocampal neurons might be less sensitive to ethanol than mouse neurons. Therefore, we examined the effects of ethanol (0.5-850 mM) in cultured mouse (C57BL/6) and rat (Sprague-Dawley) neurons. In 35% of the mouse neurons, the Cl- current was potentiated by ethanol starting at 0.5 mM. In all of the rat neurons examined, on the other hand, the current was potentiated by concentrations starting at 200 mM. We also studied the effects of GABA and other GABAergic ligands. GABAA receptors in rat and mouse neurons displayed EC50s for GABA of 9 +/- 0.3 and 17 +/- 0.8 microM, respectively and ethanol did not significantly change these values. The EC50 for diazepam was 92 +/- 3 and 120 +/- 8 nM in rat and mouse, respectively. Pentobarbital enhanced the current with EC50s of 84 +/- 3 and 106 +/- 6 microM in rat and mouse, respectively. The sensitivity for Cl-218,872, which binds preferentially to the Type I benzodiazepine receptor, was similar in all the neurons. RO 15-4513, an inverse partial agonist to the benzodiazepine receptor, was not effective in reversing the potentiation of the Cl- current in rat neurons and only slightly reduced the potentiation in mouse neurons. The receptors in rat neurons were more sensitive to external Zn2+; the current was inhibited by 50% with a concentration of 93 +/- 3 and 244 +/- 9 microM in rat and mouse, respectively. Analysis of mRNA encoding for the gamma 2L receptor subunit showed similar levels in rat and mouse neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of protein kinase C activation on the glycine evoked Cl(-) current in spinal cord neurons.

We investigated whether the effect of phorbol-12-myristate-13-acetate (PMA) was altered by a kinase inhibitor and by down-regulation of protein kinase C (PKC) in order to determine if glycine receptors in mouse spinal neurons, unlike those in hippocampal and trigeminal neurons, can be inhibited by PKC. To examine the above, electrophysiological and immunofluorescence studies were carried out in mouse spinal neurons kept in culture for up to 3 weeks. The inhibition of the glycine activated current by PMA (1 microM) increased from 12+/-3% during week 1 to 27+/-6% during week 3. The effect of PMA was completely blocked by the PKC selective inhibitor RO 31-8220 (1 microM). After culturing the cells with 1 microM PMA for 24 h, the inhibitory effect of acute application of PMA disappeared altogether, suggesting that the effect of PMA was via PKC. Immunofluorescence studies showed that a short stimulation with PMA translocated the enzyme to the periphery whereas longer term stimulation (24 h) down regulated the PKC signal. These results indicate that activation of PKC by PMA inhibits the glycine receptor in cultured spinal neurons and that its sensitivity changes during neuronal development.

Ethanol potentiates the GABAA-activated Cl- current in mouse hippocampal and cortical neurons.

The effects of ethanol (1-80 mM) on GABA-activated Cl- current (IGABA) were studied in cultured mammalian hippocampal and cortical neurons. Patch-clamp recordings revealed that ethanol potentiated the Cl- current in a concentration-dependent manner (1-40 mM) in the majority of the cells studied. No higher degree of potentiation was found by increasing the concentration of ethanol to 80 mM. This study demonstrates that ethanol can potentiate IGABA in mammalian central neurons.

Ethanol affects the function of a neurotransmitter receptor protein without altering the membrane lipid phase.

Using patch-clamp and fluorescence techniques we found that ethanol (10-200 mM) potentiated strychnine-sensitive glycine receptors without having detectable effects on lipid order parameters in mouse spinal cord neurons. Hepthanol (0.01-1 mM), in contrast, did not affect the glycine current, but it altered the core and surface of spinal neuron membranes as detected by changes in 1,6-diphenyl-1,3,5-hexatriene (DPH) and Laurdan fluorescence parameters. These findings support the idea that ethanol affects these membrane proteins without changing lipid fluidity.

Membrane currents activated in acutely dissociated rat pineal cells during the circadian cycle.

Whole-cell patch-clamp recordings were performed in cells acutely dissociated from adult rat pineal glands in order to determine possible long lasting influences of the circadian rhythm upon voltage-activated membrane currents. Cells were dissociated either during the light or dark period. From a holding potential of -50 mV, pineal cells bathed in a normal solution responded to depolarizing potentials positive to -20 mV with a slowly rising sustained outward current. From a holding potential of -100 mV, a transient outward current was activated with command potentials positive to -50 mV. This current reached a peak within 15 ms and had a significant decay over 160 ms. In most of the cells, inward currents were not observed and no significant differences were found between the properties of the outward currents in cells dissociated during the light or dark period. This suggests that the previously reported circadian activity in the rat pineal gland is not associated with inward current or with long lasting changes in the voltage-activated membrane currents.

TTX-sensitive action potentials and excitability of adult rat sensory neurons cultured in serum- and exogenous nerve growth factor-free medium.

The excitability of adult rat dorsal root ganglion (DRG) neurons cultured in the absence of serum and exogenously added nerve growth factor (NGF) was studied. Current-clamp recordings revealed the presence of tetrodotoxin (TTX)-sensitive action potentials. Voltage-clamp recordings demonstrated the presence of both inward and outward currents. The inward Na+ current had a maximal amplitude near -10 mV and was completely blocked by TTX. A sustained Ca2+ inward current and a slowly activating outward K+ current were also observed. TTX-sensitive and TTX-resistant action potentials have been observed in previous studies in DRG neurons cultured in the presence of serum. By contrast, in the study reported here, only TTX-sensitive action potentials and Na+ currents were found in the neurons cultured in the absence of serum and nerve growth factor.