Endogenous cannabinoids in amygdala and hippocampus in post-mortem brains of Cloninger type 1 and 2 alcoholics.

Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system with alcohol dependence, negative-reinforcement learning, and the modulation of stress responses. Specific alterations in brain regions that are related to stress and negative-reinforcement learning have been reported to exist in Cloninger type 1 and type 2 alcoholics. To study possible differences in profiles of EC systems between Cloninger type 1 (n = 9) and type 2 (n = 8) alcoholics and non-alcoholic control subjects (n = 10), we analyzed post-mortem amygdala and hippocampus brain samples for several ECs by quantitative liquid chromatography with triple quadrupole mass-spectrometric detection. A significant difference was found between these 3 groups in terms of EC profiles in the amygdala (p = 0.037). In particular, this difference was prominent for variations in docosahexaenoylethanolamide levels, which were significantly higher in type 1 alcoholics (p = 0.022) when compared to controls. There was also a large negative correlation between anandamide concentration and mGlu1/5 receptor density in the hippocampi of Cloninger type 1 alcoholics (R = -0.88, p = 0.002), which was not seen in Cloninger type 2 alcoholics or in controls. Although preliminary, and from relatively small diagnostic groups, these results suggest that the EC system profile may be altered in the hippocampus and amygdala of Cloninger type 1 alcoholics.

Trans fat diet causes decreased brood size and shortened lifespan in Caenorhabditis elegans delta-6-desaturase mutant fat-3.

Trans-fatty acids (TFAs) enter the diet through industrial processes and can cause adverse human health effects. The present study was aimed to examine the effects of dietary cis- and trans-fatty acids on the model organism Caenorhabditis elegans. Cis- or trans-18:1n9 triglycerides (25 µM) caused no apparent changes in the numbers of viable progeny of wild-type N2 animals. However, in fat-3 mutants lacking delta-6-desaturase, the trans-isomer caused modest decreases in lifespan and progeny after three generations. Long-chain polyunsaturated fatty acids (PUFA) profiles were significantly altered in fat-3 mutants compared to wild type but were not altered after exposure to dietary cis- or trans-18:1n9. Genome-wide expression analysis of fat-3 mutants revealed hundreds of changes. Several genes involved in fat metabolism (acs-2, fat-7, mdt-15) were significantly increased by cis- or trans-18:1n9 without discrimination between isomers. These results provide support for the hypothesis that dietary trans fats are detrimental to development and aging.

Endogenous cannabinoids in post-mortem brains of Cloninger type 1 and 2 alcoholics.

The endogenous cannabinoid (EC) system has been recently implicated in several neuropsychiatric disorders. This study analyzed post-mortem brain regions of Cloninger type 1 (n=9) and 2 (n=8) alcoholics and non-alcoholic controls (n=10) for ECs by quantitative liquid chromatography with triple quadrupole mass spectrometric detection. A significant difference was found in anandamide (AEA) levels in nucleus accumbens (NAcc) between the three groups (p=0.047). AEA levels were significantly lower when compared to controls in both perigenual anterior cingulate (p=0.017) and frontal cortices (p=0.018) of type 1 alcoholics. Similar trends were observed for dihomo-gamma-linolenoyl ethanolamide and docosahexaenoyl ethanolamide, but not for 2-arachidonoylglycerol, palmitoyl ethanolamide, or oleoyl ethanolamide. Although preliminary, and from diagnostic groups with a relatively small number of subjects and substantially different mean ages for each group, these results suggest that the EC system may be hyperactive in type 2 alcoholics and hypoactive in type 1 alcoholics.

Endogenous calcium buffering capacity of substantia nigral dopamine neurons.

Dopamine (DA)-containing cells from the substantia nigra pars compacta (SNc) play a major role in the initiation of movement. Loss of these cells results in Parkinson's disease (PD). Changes in intracellular calcium ion concentration ([Ca(2+)](i)) elicit several events in DA cells, including spike afterhyperpolarizations (AHPs) and subthreshold oscillations underlying autonomous firing. Continuous Ca(2+) load due to Ca(2+)-dependent rhythmicity has been proposed to cause the death of DA cells in PD and normal aging. Because of the physiological and pathophysiological importance of [Ca(2+)](i) in DA cells, we characterized their intrinsic Ca(2+)-buffering capacity (K(S)) in brain slices. We introduced a fluorescent Ca(2+)-sensitive exogenous buffer (200 microM fura-2) and cells were tracked from break-in until steady state by stimulating with a single action potential (AP) every 30 s and measuring the Ca(2+) transient from the proximal dendrite. DA neurons filled exponentially with a tau of about 5-6 min. [Ca(2+)](i) was assumed to equilibrate between the endogenous Ca(2+) buffer and the exogenous Ca(2+) indicator buffer. Intrinsic buffering was estimated by extrapolating from the linear relationships between the amplitude or time constant of the Ca(2+) transients versus [fura-2]. Extrapolated Ca(2+)-transients in the absence of fura-2 had mean peak amplitudes of 293.7 +/- 65.3 nM and tau = 124 +/- 13 ms (postnatal day 13 [P13] to P17 animals). Intrinsic buffering increased with age in DA neurons. For cells from animals P13-P17, K(S) was estimated to be about 110 (n = 20). In older animals (P25-P32), the estimate was about 179 (n = 10). These relatively low values may reflect the need for rapid Ca(2+) signaling, e.g., to allow activation of sK channels, which shape autonomous oscillations and burst firing. Low intrinsic buffering may also make DA cells vulnerable to Ca(2+)-dependent pathology.

Isolation and characterization of yuremamine, a new phytoindole.

Yuremamine was isolated and characterized from the stem bark of Mimosa tenuiflora. This plant is still used by indigenous peoples in North-eastern Brazil to make yurema, a psychoactive beverage that is used for medico-religious purpose ( jurema preta or vinho da jurema, in Portuguese). The characterization of this novel compound by NMR and mass spectrometry introduces a new class of phytoindoles.

Phytochemical analyses of Banisteriopsis caapi and Psychotria viridis.

A total of 32 Banisteriopsis caapi samples and 36 samples of Psychotria viridis were carefully collected from different plants on the same day from 22 sites throughout Brazil for phytochemical analyses. A broad range in alkaloid distribution was observed in both sample sets. All B. caapi samples had detectable amounts of harmine, harmaline and tetrahydroharmine (THH), while some samples of P. viridis had little or no detectable levels of N,N-dimethyltryptamine (DMT). Leaves of P. viridis were also collected from one plant and analyzed for DMT throughout a 24-hour cycle.

Various alkaloid profiles in decoctions of Banisteriopsis caapi.

Twenty nine decoctions of Banisteriopsis caapi from four different sources and one specimen of B. caapi paste were analyzed for N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmaline and harmine. Other plants were also used in the preparation of these products, typically Psychotria viridis, which provides DMT. There were considerable variations in alkaloid profiles, both within and between sample sources. DMT was not detected in all samples. Additional THH may be formed from both harmine and harmaline during the preparation of these products. The alkaloid composition of one decoction sample did not change significantly after standing at room temperature for 80 days, but the initial acidic pH was neutralized by natural fermentation after 50 days.

Fast and slow metabolizers of Hoasca.

Harmine, a major alkaloid in ayahuasca (hoasca), is a selective and reversible inhibitor of the enzyme monoamine oxidase-A (MAO-A). It is also a selective inhibitor of the human cytochrome P450 isozyme 2D6 (CYP 2D6), which metabolizes harmine to a more hydrophilic derivative for eventual excretion. CYP 2D6 exhibits a wide range of polymorphisms in human populations, and variations in this enzymatic activity could account for differences in effects between individuals who use hoasca. This report broadly describes two subgroups of CYP 2D6 phenotypes--i.e., fast and slow metabolizers of harmine-in 14 experienced male members of the União do Vegetal (UDV) who received a standardized dosage of hoasca. To compensate for metabolic variations in their normal religious practice, the administered dose of hoasca is always determined by the presiding mestre, who is responsible for deciding the actual amount for each individual. This age-old method compensates for metabolic variations between individuals and variations in both the alkaloid profile and strength of the hoasca.

Effects of temperature on calcium transients and Ca2+-dependent afterhyperpolarizations in neocortical pyramidal neurons.

In neocortical pyramidal neurons, the medium (mAHP) and slow AHP (sAHP) have different relationships with intracellular [Ca2+]. To further explore these differences, we varied bath temperature and compared passive and active membrane properties and Ca2+ transients in response to a single action potential (AP) or trains of APs. We tested whether Ca(2+)-dependent events are more temperature sensitive than voltage-dependent ones, the slow rise time of the sAHP is limited by diffusion, and temperature sensitivity differs between the mAHP and sAHP. The onset and decay kinetics of the sAHP were very temperature sensitive (more so than diffusion). We found that the decay time course of Ca2+ transients was also very temperature sensitive. In contrast, the mAHP (amplitude, time to peak, and exponential decay) and sAHP peak amplitude were moderately sensitive to temperature. The amplitudes of intracellular Ca2+ transients evoked either by a single spike or a train of spikes showed modest temperature sensitivities. Pyramidal neuron input resistance was increased by cooling. With the exception of threshold, which remained unchanged between 22 and 35 degrees C, action potential parameters (amplitude, half-width, maximum rates of rise and fall) were modestly affected by temperature. Collectively, these data suggest that temperature sensitivity was higher for the Ca(2+)-dependent sAHP than for voltage-dependent AP parameters or for the mAHP, diffusion of Ca2+ over distance cannot explain the slow rise of the sAHP in these cells, and the kinetics of the sAHP and mAHP are affected differently by temperature.

Relationships between intracellular calcium and afterhyperpolarizations in neocortical pyramidal neurons.

We examined the effects of recent discharge activity on [Ca2+]i in neocortical pyramidal cells. Our data confirm and extend the observation that there is a linear relationship between plateau [Ca2+]i and firing frequency in soma and proximal apical dendrites. The rise in [Ca2+] activates K+ channels underlying the afterhyperpolarization (AHP), which consists of 2 Ca(2+)-dependent components: the medium AHP (mAHP) and the slow AHP (sAHP). The mAHP is blocked by apamin, indicating involvement of SK-type Ca(2+)-dependent K+ channels. The identity of the apamin-insensitive sAHP channel is unknown. We compared the sAHP and the mAHP with regard to: 1) number and frequency of spikes versus AHP amplitude; 2) number and frequency of spikes versus [Ca2+]i; 3) IAHP versus [Ca2+]i. Our data suggest that sAHP channels require an elevation of [Ca2+]i in the cytoplasm, rather than at the membrane, consistent with a role for a cytoplasmic intermediate between Ca2+ and the K+ channels. The mAHP channels appear to respond to a restricted Ca2+ domain.

Dendritic synchrony and transient dynamics in a coupled oscillator model of the dopaminergic neuron.

Transient increases in spontaneous firing rate of mesencephalic dopaminergic neurons have been suggested to act as a reward prediction error signal. A mechanism previously proposed involves subthreshold calcium-dependent oscillations in all parts of the neuron. In that mechanism, the natural frequency of oscillation varies with diameter of cell processes, so there is a wide variation of natural frequencies on the cell, but strong voltage coupling enforces a single frequency of oscillation under resting conditions. In previous work, mathematical analysis of a simpler system of oscillators showed that the chain of oscillators could produce transient dynamics in which the frequency of the coupled system increased temporarily, as seen in a biophysical model of the dopaminergic neuron. The transient dynamics was shown to be consequence of a slow drift along an invariant subset of phase space, with rate of drift given by a Lyapunov function. In this paper, we show that the same mathematical structure exists for the full biophysical model, giving physiological meaning to the slow drift and the Lyapunov function, which is shown to describe differences in intracellular calcium concentration in different parts of the cell. The duration of transients was long, being comparable to the time constant of calcium disposition. These results indicate that brief changes in input to the dopaminergic neuron can produce long lasting firing rate transients whose form is determined by intrinsic cell properties.

Subjective effects and tolerability of the South American psychoactive beverage Ayahuasca in healthy volunteers.

RATIONALE: Ayahuasca is a South American psychoactive beverage that contains the naturally occurring psychedelic agent N,N-dimethyltryptamine (DMT). This "tea" has been used for centuries in religious and medicinal contexts in the rain forest areas of South America and is presently gaining the attention of psychedelic users in North America and Europe. OBJECTIVES: In the present study, the psychological effects and tolerability of ayvahuasca were assessed. METHODS: Three increasing doses of encapsulated freeze-dried ayahuasca (0.5, 0.75, and 1.0 mg DMT/kg body weight) were administered to six healthy male volunteers with prior experience in the use of this tea, in a single-blind crossover placebo-controlled clinical trial. RESULTS: Ayahuasca produced significant dose-dependent increases in five of the six subscales of the Hallucinogen Rating Scale, in the LSD, MBG, and A scales of the Addiction Research Center Inventory, and in the "liking", "good effects" and "high" visual analogue scales. Psychological effects were first noted after 30-60 min, peaked between 60-120 min, and were resolved by 240 min. The tea was well tolerated from a cardiovascular point of view, with a trend toward increase for systolic blood pressure. Modified physical sensations and nausea were the most frequently reported somatic-dysphoric effects. The overall experience was regarded as pleasant and satisfactory by five of the six volunteers, while one volunteer experienced an intensely dysphoric reaction with transient disorientation and anxiety at the medium dose and voluntarily withdrew from the study. CONCLUSIONS: Ayahuasca can be described as inducing changes in the perceptual, affective, cognitive, and somatic spheres, with a combination of stimulatory and visual psychoactive effects of longer duration and milder intensity than those previously reported for intravenously administered DMT.

Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons.

Neostriatal cholinergic interneurons produce spontaneous tonic firing in the absence of synaptic input. Perforated patch recording and whole-cell recording combined with calcium imaging were used in vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined action of a sodium current and the hyperpolarization-activated cation current (I(h)), which together ensured that there was no zero current point in the subthreshold voltage range. Blockade of sodium channels or I(h) established a stable subthreshold resting membrane potential. A tetrodotoxin-sensitive region of negative slope conductance was observed between approximately -60 mV and threshold (approximately -50 mV) and the h-current was activated at all subthreshold voltages. Calcium imaging experiments revealed that there was minimal calcium influx at subthreshold membrane potentials but that action potentials produced elevations of calcium in both the soma and dendrites. Spike-triggered calcium entry shaped the falling phase of the action potential waveform and activated calcium-dependent potassium channels. Blockade of big-conductance channels caused spike broadening. Application of apamin, which blocks small-conductance channels, abolished the slow spike afterhyperpolarization (AHP) and caused a transition to burst firing. In the absence of synaptic input, a range of tonic firing patterns are observed, suggesting that the characteristic spike sequences described for tonically active cholinergic neurons (TANs) recorded in vivo are intrinsic in origin. The pivotal role of the AHP in regulating spike patterning indicates that burst firing of TANs in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.

Coupled oscillator model of the dopaminergic neuron of the substantia nigra.

Calcium imaging using fura-2 and whole cell recording revealed the effective location of the oscillator mechanism on dopaminergic neurons of the substantia nigra, pars compacta, in slices from rats aged 15-20 days. As previously reported, dopaminergic neurons fired in a slow rhythmic single spiking pattern. The underlying membrane potential oscillation survived blockade of sodium currents with TTX and was enhanced by blockade of voltage-sensitive potassium currents with TEA. Calcium levels increased during the subthreshold depolarizing phase of the membrane potential oscillation and peaked at the onset of the hyperpolarizing phase as expected if the pacemaker potential were due to a low-threshold calcium current and the hyperpolarizing phase to calcium-dependent potassium current. Calcium oscillations were synchronous in the dendrites and soma and were greater in the dendrites than in the soma. Average calcium levels in the dendrites overshot steady-state levels and decayed over the course of seconds after the oscillation was resumed after having been halted by hyperpolarizing currents. Average calcium levels in the soma increased slowly, taking many cycles to achieve steady state. Voltage clamp with calcium imaging revealed the voltage dependence of the somatic calcium current without the artifacts of incomplete spatial voltage control. This showed that the calcium current had little or no inactivation and was half-maximal at -40 to -30 mV. The time constant of calcium removal was measured by the return of calcium to resting levels and depended on diameter. The calcium sensitivity of the calcium-dependent potassium current was estimated by plotting the slow tail current against calcium concentration during the decay of calcium to resting levels at -60 mV. A single compartment model of the dopaminergic neuron consisting of a noninactivating low-threshold calcium current, a calcium-dependent potassium current, and a small leak current reproduced most features of the membrane potential oscillations. The same currents much more accurately reproduced the calcium transients when distributed uniformly along a tapering cable in a multicompartment model. This model represented the dopaminergic neuron as a set of electrically coupled oscillators with different natural frequencies. Each frequency was determined by the surface area to volume ratio of the compartment. This model could account for additional features of the dopaminergic neurons seen in slices, such as slow adaptation of oscillation frequency and may produce irregular firing under different coupling conditions.

Ligand interaction with the purified serotonin transporter in solution and at the air/water interface.

The purified serotonin transporter (SERT) was spread at the air/water interface and the effects both of its surface density and of the temperature on its interfacial behavior were studied. The recorded isotherms evidenced the existence of a stable monolayer undergoing a lengthy rearrangement. SERT/ligand interactions appeared to be dependent on the nature of the studied molecules. Whereas an unrelated drug (chlorcyclizine) did not bind to the spread SERT, it interacted with its specific ligands. Compared to heterocyclic drugs, for which binding appeared to be concentration-dependent, a 'two-site' mechanism was evidenced for pinoline and imipramine.

Afferent modulation of dopamine neuron firing patterns.

In recent studies examining the modulation of dopamine (DA) cell firing patterns, particular emphasis has been placed on excitatory afferents from the prefrontal cortex and the subthalamic nucleus. A number of inconsistencies in recently published reports, however, do not support the contention that tonic activation of NMDA receptors is the sole determinate of DA neuronal firing patterns. The results of work on the basic mechanism of DA firing and the action of apamin suggest that excitatory projections to DA neurons from cholinergic and glutamatergic neurons in the tegmental pedunculopontine nucleus, and/or inhibitory GABAergic projections, are also involved in modulating DA neuron firing behavior.

Pharmacokinetics of Hoasca alkaloids in healthy humans.

N,N-Dimethyltryptamine (DMT), harmine, harmaline and tetrahydroharmine (THH) are the characteristic alkaloids found in Amazonian sacraments known as hoasca, ayahuasca, and yajè. Such beverages are characterized by the presence of these three harmala alkaloids, where harmine and harmaline reversibly inhibit monoamine oxidase A (MAO-A) while tetrahydroharmine weakly inhibits the uptake of serotonin. Together, both actions increase central and peripheral serotonergic activity while facilitating the psychoactivity of DMT. Though the use of such 'teas' has be known to western science for over 100 years, little is known of their pharmacokinetics. In this study, hoasca was prepared and administered in a ceremonial context. All four alkaloids were measured in the tea and in the plasma of 15 volunteers, subsequent to the ingestion of 2 ml hoasca/kg body weight, using gas (GC) and high pressure liquid chromatographic (HPLC) methods. Pharmacokinetic parameters were calculated and peak times of psychoactivity coincided with high alkaloid concentrations, particularly DMT which had an average Tmax of 107.5 +/- 32.5 min. While DMT parameters correlated with those of harmine, THH showed a pharmacokinetic profile relatively independent of harmine's.

The distribution of histamine and serotonin in the barnacle's nervous system.

The use of antisera directed against conjugates of histamine and serotonin has revealed the locations of neurons labeling for these transmitters in the nervous system of barnacles. Photoreceptors label for histamine but not serotonin and also satisfy a number of other criteria indicating that histamine is their neurotransmitter. Photoreceptors also take up radioactively labeled histamine but not serotonin. Within the barnacle's brain no somata are consistently found that label with antiserum against histamine, but one to three pairs of small cells, depending on species, label with antiserum against serotonin. The most impressive serotonin-like immunoreactivity in the brain, however, is in a pair of large fibers ascending through the circumesophageal connectives and ramifying extensively. Within the ventral ganglion, the only other ganglion in the barnacle, ten pairs of cells label with antiserum against histamine. These neurons are confined to the posterior portion of the ganglion but ramify extensively throughout the ganglion. Antiserum against serotonin labels about 15 cell pairs, depending on species, located throughout the ganglion. The positions of the arbors of many of these cells suggest that these amines have a role in modulating either the motor pathways underlying feeding or the visual pathways responsible for the detection of shadows.

Ayahuasca preparations and serotonin reuptake inhibitors: a potential combination for severe adverse interactions.

The Amazonian psychoactive plant beverage ayahuasca has attracted increasing interest in recent years. Little attention has been given, however, to potentially dangerous interactions with other drugs. In particular, the interaction between the potent monoamine oxidase-inhibiting harmala alkaloids in ayahuasca and the selective serotonin reuptake inhibitor (SSRI) class of antidepressants may induce a serotonin syndrome with potentially grave outcome. Caution is advised when combining ayahuasca with certain pharmaceutical drugs.