Methylphenidate and amphetamine modulate differently the NMDA and AMPA glutamatergic transmission of dopaminergic neurons in the ventral tegmental area.

Behavioral and neurochemical studies suggest that the induction of behavioral sensitization to psychostimulants involves transient changes at the synapses of the ventral tegmental area's dopaminergic neurons (VTA-DA). Differences in the behavioral response to amphetamine (Amph) and methylphenidate (MPD) were observed. In an attempt to understand these behavioral differences at the neuronal level, the dose-response characteristics of these two psychostimulants on electrophysiologically identified VTA-DA neurons at the glutamatergic synapse were investigated. Miniature excitatory post-synaptic currents (mEPSCs) and electrically induced EPSCs were recorded from horizontal midbrain slices of rats that had been pretreated intraperitoneally (i.p.) with saline (control), Amph (2.5, 5.0, 10.0 or 20.0 mg/kg), or MPD (2.5, 5.0, 10.0 or 20.0 mg/kg) 24 h before the recording. Perfusion of Amph through the bath (2.5, 5.0, 10.0 or 20.0 microM) increased the frequency (p<0.01) and the amplitude (p<0.05) of mEPSCs in dose-response characteristics, while MPD perfusion through the bath (2.5, 5.0, 10.0, or 20.0 microM) increased only the frequency (p<0.05) of the mEPSC. Both psychostimulants increased the prefrontal cortex's (PFC) glutamatergic EPSC in the VTA-DA neurons. However, only the higher doses of MPD induced significant effects (p<0.05) on the N-methyl-D-aspartate (NMDA) receptor-mediated EPSC but had no effects on the EPSC mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptors. Contrarily, Amph increased both kinds of mediated EPSC, but mainly the EPSC mediated by AMPA/kainate receptors (p<0.01). These electrophysiological differences could represent the underlying mechanism responsible for the differences of behavioral effects, such as behavioral sensitization, elicited by MPD and Amph.

Dopaminergic ventral tegmental neurons modulated by methylphenidate.

Treatment of psychostimulants leads to the development of behavioral sensitization, an augmented behavioral response to drug re-administration. The induction of behavioral sensitization to psychostimulants such as amphetamine and cocaine occurs at the ventral tegmental area's dopaminergic neurons (VTA-DA). Currently, there is limited experimental data about the physiological properties of methylphenidate (MPD) on VTA-DA neurons. Behavioral and electrophysiological experiments using male rats were performed before and after MPD treatment. The behavioral experiment included dose-response (0.6, 2.5, and 10.0 mg/kg MPD) study to select the most effective dose for the electrophysiological study. Methylphenidate increased locomotion in typical dose response characteristics. Based on this experiment, the 10.0 mg/kg MPD was used in two types of electrophysiological recordings: 1) intracellular recording of neuronal activity performed on horizontal 275-300 microm brain slices and 2) whole-cell patch clamping before and after electrical stimulation to study post-synaptic currents on neurophysiologically identified VTA-DA neurons. Methylphenidate suppressed the neuronal activity of these neurons for 210 +/- 30 sec. Stimulation of the prefrontal cortex afferent fibers to these VTA-DA neurons in the presence of TTX, saclofen, and picrotoxin led to the conclusion that this input is mediated via NMDA and kainate/AMPA receptors and may participate to induce behavioral sensitization to psychostimulants.

Galphimine B modulates synaptic transmission on dopaminergic ventral tegmental area neurons.

Galphimine B (GB) is a bioactive compound isolated from the plant Galphimia glauca Cav. (Malpighiaceae) and has been shown to have central nervous system depressant properties. In an earlier study, it was reported that both systemic and local administration of GB modified the extracellular spontaneous spiking activity in the ventral tegmental area (VTA) neurons. In the present study we analyzed the synaptic effects of this compound on dopaminergic neurons. Recordings were made in brain slices using intracellular and patch-clamp techniques, in dopaminergic (DA) VTA neurons. Spontaneous miniature excitatory postsynaptic currents (mEPSCs), excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic potentials (IPSPs) were recorded in control situation and after bath infusions of several concentrations of GB (1 microM-5 mM), GABA (1 nM-100 microM) and the GABA A blockers, picrotoxin (100 microM) and bicuculline (10 microM), and GABA B blocker saclofen (200 microM). GB administration reduced the frequency (p < 0.05) but not the amplitude of mESPCs. However, GABA (IC 50 = 645 nM) and GB (IC 50 = 174.5 microM) infusion significantly reduced the amplitude of stimuli induced EPSCs, Bicuculline (10 microM) co-administration only reduced GABA effects and did not modify the GB depressant action. Finally, isolated GABAergic IPSPs were modified by the addition of picrotoxin, but GB had no effect on these evoked synaptic responses. The present results indicate that GB modifies synaptic activity on dopaminergic VTA neurons by a non-GABAergic mechanism.

The role of intrinsic and agonist-activated conductances in determining the firing patterns of preoptic area neurons in the guinea pig.

Whole-cell and intracellular recordings were made in coronal hypothalamic slices prepared from ovariectomized female guinea pigs. 62% of preoptic area (POA) neurons fired action potentials in a bursting manner, and exhibited a significantly greater afterhyperpolarization (AHP) than did non-bursting POA neurons. The majority (70%) of POA neurons (n=76) displayed a time-dependent inward rectification (I(h)) that was blocked by CsCl (3 mM) or by ZD 7288 (30 microM). In addition, 51% of the cells expressed a low-threshold spike (LTS) associated with a transient inward current (I(T)) that was blocked by NiCl(2) (200 microM). A smaller percentage of POA neurons (29%) expressed a transient outward, A-type K(+) current that was antagonized by a high concentration of 4-aminopyridine (3 mM). Moreover, POA neurons responded to bath application of the mu-opioid receptor agonist DAMGO (93%) or the GABA(B) receptor agonist baclofen (83%) with a membrane hyperpolarization or an outward current. These responses were accompanied by a decrease in input resistance or an increase in conductance, respectively, and were attenuated by BaCl(2) (100 microM). In addition, the reversal potential for these responses closely approximated the Nernst equilibrium potential for K(+). These results suggest that POA neurons endogenously express to varying degrees an AHP, an I(h), an I(T) and an A-type K(+) current. The vast majority of these neurons also are inhibited upon mu-opioid or GABA(B) receptor stimulation via the activation of an inwardly-rectifying K(+) conductance. Such intrinsic and transmitter-activated conductances likely serve as important determinants of the firing patterns of POA neurons.

Galphimine-B modifies electrical activity of ventral tegmental area neurons in rats.

Galphimine-B (G-B) is a bioactive compound isolated from the plant Galphimia glauca Cav. (Malpighiaceae) with central nervous system depressant properties previously described. In the present study, extracellular spiking activity records in either somatosensorial cortex or ventral tegmental area (VTA) neurons, were performed in rats after i.p. or local administration of G-B. None of the cortical neurons displayed significant changes induced by any of the applied doses. In VTA cells, two patterns of electrical discharge were recorded, bursting (57%) and nonbursting (43%) types. Systemic administration of G-B induced excitatory effects in neurons with a bursting firing pattern and mixed responses on nonbursting units. When this compound was applied locally by microiontophoresis, most of the bursting and nonbursting spiking neurons showed a firing depression and only a few of the nonbursting neurons showed an increment of discharge frequency. These results are important since VTA is a major dopaminergic center responsible for the innervation of the prefrontal cortex, nucleus accumbens and entorhinal region. These areas are targets for the action of antipsychotic drugs.

Apamin blocks the direct relaxant effect of melatonin on rat ileal smooth muscle.

The present study investigated the mechanisms of melatonin-induced inhibition of the ileal smooth muscle contraction. Rat isolated ileal smooth muscle strips were stimulated in an organ bath using carbachol (CAR) or potassium chloride (KCl) depolarization. Under these conditions, melatonin produced a concentration-dependent inhibition of muscle contraction (mean inhibitory concentration, IC50: 17.3 x 10(-6) M), which was not blocked by either tetrodotoxin (10(-6) M), hexamethonium (10(-4) M), or phentolamine (10(-6) M). The inhibitory effect of melatonin during CAR stimulation was blocked in a concentration-dependent manner by the presence of apamin (4.8 x 10(-9) M), a K(+)-channel blocker. By contrast, other K(+)-channel blockers such as 4-aminopyridine (10(-4) M to 5 x 10(-3) M), tetraethylammonium (10(-4) to 10(-1) M), and glibenclamide (10(-5) M) were ineffective. Additionally, the Ca(2+)-channel antagonists nitrendipine (IC50: 2.4 x 10(-9) M) and verapamil (IC50: 1.1 x 10(-7) M) also blocked the inhibitory action of melatonin. These results suggest that melatonin may interact with an apamin-sensitive, possibly Ca(2+)-activated, K+ channel and thus cause an inhibition of ileal smooth muscle contractions.

Interferon modulates glucose-sensitive neurons in the hypothalamus.

Interferon-alpha (IFN) therapy induces feeding suppression that resembles anorexia. The hypothalamic glucose-sensitive neurons engage in feeding behavior. Coronal sections of rat brains, containing both the lateral hypothalamus (LH) and the ventromedial hypothalamus (VMH), as well as single-cell recordings were used to study the interaction between IFN and glucose-sensitive neurons. IFN suppressed the majority (78%) of LH neurons, while reduction in glucose concentration elicited excitation in the majority (85%) of the same neurons. The opposite effects were observed in the VMH, where IFN excited the majority of neurons (61%), and reduction in glucose concentration exerted the opposite effects in 64% of VMH recordings. Concomitant IFN and glucose reduction exhibited only the effects elicited by IFN, regardless of whether the glucose reduction caused excitation (LH) or suppression (VMH). This observation suggests that IFN causes anorexia by modulating the LH and VMH glucose-sensitive neurons.

Melatonin modulates cholinergic transmission by blocking nicotinic channels in the guinea-pig submucous plexus.

Melatonin, a hormone produced and released by the pineal gland is also synthesized by cells of the gastrointestinal wall, where it might be a local regulator of gut functions. In this study, we investigated the possible role of melatonin as a modulator of the enteric nervous system. Intracellular recordings were made in neurons of the submucosal plexus from the guinea-pig ileum to measure the melatonin effects on their electrophysiological properties. Melatonin did not alter the membrane potential, the membrane resistance and the noradrenergic inhibitory postsynaptic potentials. However, melatonin (30-3000 microM) reversibly decreased the amplitude of nicotinic excitatory postynaptic potentials (EPSPs) in a concentration-dependent manner (IC50 = 247 microM). These actions of melatonin were not modified by the presence of idazoxan and atropine indicating that they are not mediated by endogenous release of acetylcholine, noradrenaline, or by direct activation of alpha 2-adrenoceptors or muscarinic receptors. The superfusion of melatonin also blocked the nicotinic depolarizations induced by locally applied acetylcholine, indicating that at least part of its effects are postsynaptic. In voltage-clamp experiments, using the whole-cell configuration, melatonin also inhibited the nicotinic inward currents induced by acetylcholine (IACh) in a concentration-dependent manner (IC50 = 257 microM). Melatonin decreased the maximal IACh but did not affect the potency of acetylcholine to induce this current, indicating a noncompetitive antagonism. This effect was voltage-dependent. Our observations indicate that melatonin inhibits the fast EPSPs by directly and specifically blocking the nicotinic channels. The relative high concentrations of melatonin required to produce such an effect rules this out as one of its humoral actions. Such an effect, however, might be of physiological significance close to the cells that release melatonin in the gastrointestinal wall or in other organs.

Interferon modulates neuronal activity recorded from the hypothalamus, thalamus, hippocampus, amygdala and the somatosensory cortex.

Neuromodulators interact with classically defined neurotransmitters to regulate a variety of biological processes. The aim of the present study was to study whether interferon-alpha (IFN) can be considered as a neuromodulator. Single cell recordings from five CNS structures were recorded before and following three different routes of IFN administration in Sprague-Dawley rats to substantiate that IFN is a neuromodulator. IFN modulated the majority of the hypothalamic (70%), amygdala (76%), hippocampus (75%) and cortical (82%) cells whether the route of administration was within the brain or given peripherally (i.v. or i.p.). The main difference among the three routes of IFN administration on the neuronal activity of these four CNS sites was the onset of the effect. However, the thalamic neurons responded differently. IFN injection within the brain modulated activity of 43% of thalamic neurons, but only 25% and 17% of the neurons when IFN was given i.v. or i.p., respectively. IFN, in general suppressed hypothalamic neuronal activity while accelerating neuronal activity in all the other studied CNS sites. In conclusion, IFN is an endogenous peptide synthesized and released both peripherally and centrally, with the same effects on neuronal activity whether it is given systemically or locally within the brain. This suggests that IFN can be considered as a neuromodulator.

Lateral hypothalamus: site involved in pain modulation.

The present study is an attempt to examine the neuronal circuitry of a supraspinal site engaged in pain modulation. Five physiological measures were postulated as the criteria for defining a central nervous system site engaged in the circuitry of pain modulation. The lateral hypothalamus met these five measures: (i) 81% of the lateral hypothalamus neurons (247/304) responded to noxious stimuli using a single cell recording procedure; (ii) stimulation of the periaqueductal gray-dorsal raphe area or the habenula modulated 98% and 87% of the lateral hypothalamus noxious-evoked activity; (iii) microiontophoretically applied morphine modulated 77% of the lateral hypothalamus noxious evoked activity; (iv) electrical stimulation of the lateral hypothalamus produced behavioral analgesia proportional to the stimulus intensity as assessed by the tail flick assay; and (v) morphine application into the lateral hypothalamus produced behavioral analgesia in a dose-response manner using the tail flick assay. In conclusion, the lateral hypothalamus can be considered one of the pain modulation sites.

Alpha-interferon suppresses food intake and neuronal activity of the lateral hypothalamus.

Alpha-interferon (alpha-IFN) treatment in humans induces anorexic effects. However, the mechanisms and sites of action are unknown. Rats implanted with an intracerebroventricular (i.c.v.) cannula for local injection, and semi-microelectrodes in the lateral hypothalamic (LH) area for neuronal recording were used. The animals were kept in metabolic cages, and food and water intake was measured daily at 7:00 and 19:00 hr for 35 days, including: 5 days before the experiment; 10 days during daily alpha-IFN application (either i.p. 1500 I.U./gbw, or i.c.v. 1500 and 150 I.U./animal) and/or a vehicle control group; and 20 days post drug treatment. The unitary activity recording from the LH area was made before (30 min), during (10 min) and after (200 min) the alpha-IFN applications. alpha-IFN elicited a reversible dose-related decrease of both food intake and body weight. This decrease in food intake following alpha-IFN injections was correlated with a depression of LH neuronal electrical activity. Since direct brain application (i.c.v.) and systemic (i.p.) alpha-IFN treatment elicited identical responses, it is possible to assume that alpha-IFN suppresses food intake by a direct action on CNS sites including the LH neurons.

[Uterine contraction induced by carbachol is inhibited by melatonin]. / La contracción uterina inducida por carbacol es inhibida por melatonina.

It has been suggested that the pineal gland has a specific role in the regulation of reproductive functions. Melatonin, secreted by pineal gland, is involved in the control of mammalian reproduction. Previous investigations have show that melatonin reduced the smooth muscle contraction. The purpose of the present study was to investigate the effect of melatonin on the uterine contraction provoked by carbachol. This effect was studied in isolated uterus were taken from Wistar rats pretreated with diethylstilboestrol. Here, we describe the effects of various concentrations of melatonin was found to inhibit the carbachol-induced uterine contraction. Our results show that CE50 of carbachol increment in present to 10(-9) and 10(-6) M/ml of melatonin. The degree of the inhibitory effect of melatonin to concentration of 10(-9) M/ml is most evident that 10(-6) M/ml. Thus, it is concluded that melatonin has an pharmacological inhibition effect on the contraction uterine provoked by carbachol, act as physiological antagonist.

Effects of acute and chronic toluene inhalation on behavior, monoamine metabolism and specific binding (3H-serotonin and 3H-norepinephrine) of rat brain.

The present study deals with the effect of chronic toluene inhalation (30,000-40,000 ppm in air, 15 min/day for 30 days) that induced abnormal behavior states resembling the serotonin syndrome in rats: resting tremor, hindlimb abduction, Straub tail, head weaving and rigidity. The head weaving latencies were significantly decreased when assessed at 15 and 30 days of exposure to toluene vapors. The sequence pattern signs of serotonin syndrome were changed after 15 and 30 days of exposure, indicating possible cumulative effects and/or tolerance development. There were no changes in concentrations of indolamine and catecholamine compounds in different parts of the rat brain (cerebral cortex, midbrain, brainstem and cerebellum) as influence of chronic toluene exposure. Examination of specific serotonin ((3H)-5HT) to crude synaptic membranes prepared from rat brains and subjected to chronic toluene inhalation revealed a very high increased value in apparent Kd (30.7 +/- 15) with respect to its air control (9.7 +/- 2.3) and baseline control (5.8 +/- 3.2). This difference was highly significant (p < 0.02). There were no changes in apparent Bmax of specific (3H)-5HT binding sites. On the other hand (3H)-NE binding of rat brain studies did not show any difference either in apparent Kd or apparent Bmax. These results indicate that serotonin syndrome may be a consequence of changes of serotonergic mechanism, specifically a reduced affinity in specific (3H)-5HT binding sites.

Melatonin modifies the spontaneous multiunit activity recorded in several brain nuclei of freely behaving rats.

Melatonin, a pineal hormone, released photoperiodically, was administered systemically in rats, previously implanted with semimicroelectrodes into six different brain structures. The multiunit electrical activity of these structures was recorded for 10 min before and 60 min after melatonin administration in unanesthetized, freely moving rats. Different melatonin doses (100, 200, 500, and 1000 micrograms/kg) produced changes in the electrical activity of all tested structures. However, amygdala, rostral hypothalamus and mesencephalic reticular formation showed the most important changes. The main effect induced by melatonin was a dose-related decrease of the spontaneous electrical activity. The significance of these effects is discussed within the context of the behavioral and endocrinological effects of melatonin.

Modification of nociceptively identified neurons in thalamic parafascicularis by chemical stimulation of dorsal raphe with glutamate, morphine, serotonin and focal dorsal raphe electrical stimulation.

The properties of local application of glutamate, morphine and serotonin in the dorsal raphe (DR) area and the effects of DR electrical stimulation on the spontaneous activity and on the nociceptive responses of 135 parafascicularis (PF) neurons were studied. It was observed that local glutamate application within the DR exerts an effect upon the "nociceptive-on" PF neuronal activity similar to that induced by focal electrical stimulation of the DR in intact animals and in animals after dorsal spinal cord section. In addition, local application of morphine and serotonin in the DR area elicits different effects on the spontaneous activity versus the nociceptive responses of PF neurons. These observations suggest that opioids and serotonin at least in part participate in modulation of pathways from DR to PF. This observation is consistent with the hypothesis that the DR ascending path modulates nociceptive input to the PF (at least in part) via activations of both opioid and serotonergic receptors.

Nociceptive responses in nucleus parafascicularis thalami are modulated by dorsal raphe stimulation and microiontophoretic application of morphine and serotonin.

Single-cell experiments were undertaken to examine the hypothesis that serotonin (5-HT) and morphine participate in ascending pain suppression phenomena. The observations demonstrate that: 1) dorsal raphe stimulation (DRS) modulates the spontaneous activity and the noxious-evoked responses of parafasciculus (PF) neurons, and the modulating effects of DRS are altered by either naloxone or methysergide; 2) morphine ejection into the PF alters the spontaneous activity and the noxious-evoked responses of PF neurons, and naloxone prevents morphine effects; and 3) serotonin ejection into the PF alters the spontaneous activity and the noxious-evoked responses of PF neurons and methysergide prevents the serotonin effects. These findings support the hypothesis that opioid and serotonin participate, at least in part, in the control of ascending pain mechanisms.

Noxious and non-noxious responses in the medial thalamus of the rat.

Single-cell experiments were undertaken to localize and characterize the medial thalamic (MT) neurons which respond to noxious and non-noxious input in the rat. The observations demonstrated that: (1) 61 and 42% of MT neurons respond to noxious (Nox) and non-noxious (NN) stimulation, respectively; (2) MT neurons exhibit 4 cell types according to their pattern of response; Type A units were excited exclusively by Nox stimulation; Type B units were excited exclusively by NN stimulation; Type C units were excited by both (Nox and NN) stimulation, and Type D units exhibited decreases in firing rate following both stimulation modalities; (3) neurons of the parafascicularis nucleus exhibit more noxious responses (Type A units) than other medial thalamic areas.

Dorsal raphe stimulation, 5-HT and morphine microiontophoresis effects on noxious and nonnoxious identified neurons in the medial thalamus of the rat.

In single cell experiments, the characterization of the responses of medial thalamic neurons to noxious and nonnoxious stimulation was made to examine the effects of two substances involved in pain, morphine and 5-HT, and the action of one pain suppressor mechanism, dorsal raphe stimulation. Single cell activity was recorded in urethane anesthetized rats. Tail pinch and tail immersion in hot water were used as nociceptive stimuli. Skin strokes, air puffs and hair brushing were used as nonnociceptive stimuli. Morphine, 5-HT microiontophoresis and dorsal raphe stimulation were performed in all the recorded units. Fifty-eight percent from 61 medial thalamic recorded units responded both to noxious and nonnoxious stimulation; whereas only 18% and 24.6% of the units responded exclusively to noxious and nonnoxious stimulation, respectively. The noxious responding units were located in the most posterior portions of the medial thalamus. Dorsal raphe stimulation and 5-HT ejection prevented the excitation elicited by noxious input. Morphine ejection prevented both the noxious and nonnoxious input in medial thalamus, in a different population as compared to dorsal raphe stimulation or 5-HT ejection. These findings support the existence of a pain ascending mechanism mediated by an opioid-serotonergic interaction in the medial thalamus of the rat.

Lever pressing and active avoidance conditioning after electrolytic lesions of the entopeduncular nucleus in cats.

Several authors have shown that CN participates in the acquisition of motor conditioned responses (MCR), probably as the integrating structure. Most of CN's efferent fibers in the cat end in the entopeduncular nucleus (EPN), therefore the command for the motor pattern could be exerted through EPN, representing part of the efferent link of the neuronal circuitry involved in MCR. Thirty-two cats were trained to avoid actively an electrical stimulus when a series of flashes appeared, and to press a lever to obtain 0.5 ml of milk. After the cats reached the learning criterion for both responses, electrolytic lesions of the entopeduncular nucleus or internal capsule (IC) were made bilaterally. When the cats recovered their normal motor behavior, the conditioned sessions were resumed once a day, for 45-50 days. Both learning responses disappeared (p less than 0.01) in those animals with the largest EPN lesions. In contrast, for small EPN lesions, learned responses were absent only during the first 3 or 4 sessions, and then the level of responses increased each day. However, it never reached that of sham lesioned cats. On the other hand, IC lesioned cats showed no statistical differences with respect to sham lesioned animals. These data support the participation of EPN in the motor circuitry responsible for MCR.

Single injection of three different preparations of alpha-interferon modifies morphine abstinence signs for a prolonged period.

Opiates exert numerous effects on all levels of the central nervous system, with tolerance and physical dependence (addiction) being characteristics of this drug class. The capacity of the immune system to participate in processes primarily considered to be central nervous system phenomena has been suggested recently by several studies demonstrating the ability of various immune-modifiers to attenuate opiate withdrawal severity. Therefore, the immunomodulator agent, interferon was investigated to determine the effect upon the opiate withdrawal signs in an animal model. The degree of morphine dependence is measured by quantifying the various behavioral signs associated with naloxone-induced withdrawal. Three different preparations of human alpha interferon (alpha-IFN) were investigated to determine the duration of their attenuating effect upon the naloxone-induced abstinence syndrome in morphine-addicted rats. All three preparations of alpha-IFN reduced the severity of the opiate withdrawal (i.e., addiction) signs for several weeks. There were differences in the potency and the duration of the effects among the three different preparations of alpha-IFN.