ABT-418: discriminative stimulus properties and effect on ventral tegmental cell activity.

Previous studies have established that ABT-418 [(S)-3-methyl-5-(1 methyl-2-pyrrolidinyl)isoxazole hydrochloride] is a novel neuronal nicotinic acetylcholine receptor (nAChR) ligand with cognitive enhancing and anxiolytic-like activity 3- to 10-fold more potent than (-)-nicotine in rodents. A series of experiments was conducted to determine the discriminative stimulus properties of ABT-418 in comparison with (-)-nicotine, and to determine the relative potencies of these compounds on ventral tegmental area (VTA) neurons. While rats were able to discriminate (-)-nicotine 1.9 mumol/kg in 39 days, they were not able to discriminate 1.9 or 6.2 mumol/kg ABT-418 from a saline solution during 50 days of training. In rats trained to discriminate 1.9 mumol/kg (-)-nicotine, a reduced generalization was induced by ABT-418 at 1.9 and 6.2 mumol/kg, an effect completely blocked by the cholinergic channel blocker mecamylamine (15 mumol/kg, IP). However, in extensively trained rats, intraperitoneal or subcutaneous injections of ABT-418 induced 78-82% generalization at the 6.2 mumol/kg dose. The predominant metabolites of (-)-nicotine and ABT-418 (continine and A-87770, respectively) were devoid of any effect in nicotine-trained rats. The reduced potency of ABT-418 in nicotine-trained rats is consistent with the electrophysiological findings showing that ABT-418 is 3-fold less potent than (-)-nicotine in activating dopamine-containing neurons in the VTA area.

Intracellular recording from putative dopamine-containing neurons in the ventral tegmental area of Tsai in a brain slice preparation.

Coronal slices of rat mesencephalon containing the ventral tegmental area of Tsai (VTA) and the substantia nigra were prepared. Stable intracellular recordings were obtained from presumed dopamine (DA)-containing neurons in the VTA. Both silent and spontaneously active cells were encountered; spontaneously active neurons fired in an extremely regular pacemaker-like fashion. These neurons had resting membrane potentials ranging from -45 to -75 mV and input resistances ranging from 80-400 M omega. DA-containing neurons in the VTA demonstrated marked anomalous rectification in response to hyperpolarizing current pulses. Application of DA or the GABAB agonist, baclofen, to the bathing medium produced suppression of spontaneous firing, sometimes accompanied by membrane hyperpolarization. Neuronal input resistance was not changed consistently by DA and was generally reduced by baclofen.

Antinociception induced by local injections of carbachol into the nucleus raphe magnus in rats: alteration by intrathecal injection of monoaminergic antagonists.

Electrical stimulation of neurons located in the nucleus raphe magnus (NRM) produces antinociception which appears to result from inhibition of spinothalamic tract neurons located in the spinal cord dorsal horn. Iontophoretic application of acetylcholine also activates NRM neurons and microinjection of cholinergic agonists such as carbachol into the NRM produces a profound, long-lasting antinociception. Since the antinociception induced by electrical stimulation of NRM neurons is mediated, at least in part, by bulbospinal serotonergic and noradrenergic neurons, the role of these monoaminergic neurons in mediating the antinociception induced by microinjecting carbachol in the NRM was examined in the present study. To this end, various antagonists of serotonin and norepinephrine were injected into the spinal cord subarachnoid space following the induction of antinociception by the local injection of carbachol into the NRM. The serotonergic antagonist methysergide had no effect on carbachol-induced antinociception. However, the alpha 2-noradrenergic antagonist yohimbine attenuated, while the alpha 1-noradrenergic antagonists prazosin and WB4101 increased the effects of carbachol. The non-selective noradrenergic antagonist phentolamine also attenuated the effects of carbachol. These results lead to the suggestion that the antinociception induced by the local injection of carbachol into the NRM is mediated by selective activation of bulbospinal noradrenergic neurons. Furthermore, the antinociception resulting from the activation of these descending noradrenergic neurons appears to be mediated by alpha 2-noradrenergic receptors located in the spinal cord dorsal horn. Finally, the local injection of carbachol into the NRM also appears to activate another population of noradrenergic neurons which produces hyperalgesia mediated by alpha 1-noradrenergic receptors.

Cholecystokinin potentiates dopamine inhibition of mesencephalic dopamine neurons in vitro.

Cholecystokinin octapeptide sulfate (CCK-S) is a neuropeptide that is co-localized with dopamine (DA) in some neurons of the ventral tegmental area (VTA). A functional role for this peptide/monoamine co-localization has not been firmly established; however, behavioral and in vivo electrophysiological studies indicate that CCK-S modifies the action of DA in some brain areas. A brain slice preparation of the rat VTA was developed in order to examine primary effects of CCK-S on DA-containing neurons, and to determine whether CCK-S modulates the inhibitory action of DA on these neurons. Spontaneously active DA neurons of the VTA were identified on the basis of their characteristic spike waveforms and firing rate as determined with extracellular recording techniques. These cells were inhibited by perfusion with DA in a dose-dependent, sulpiride-reversible manner. CCK-S produced brief excitatory increases in firing rate in 83% of these cells tested. This excitation was dose-dependent, and the excitatory responses frequently diminished even in the continued presence of CCK-S. Prior administration of CCK-S to these cells markedly potentiated DA-induced inhibition of spontaneous firing; the magnitude of this effect ranged from a 24 to 376% increase in the inhibitory response. This CCK-induced potentiation of DA inhibition was not blocked by low calcium, high magnesium superfusion medium, indicating that this effect is a direct consequence of a postsynaptic action on the VTA neurons from which recordings were made. These results suggest that co-localized CCK-S may significantly affect neuronal sensitivity to synaptically released DA.

Hypoalgesia induced by the local injection of carbachol into the nucleus raphe magnus.

The medial region of the caudal medulla which contains the nucleus raphe magnus and magnocellular reticular formation has been demonstrated to modulate pain perception. Recent reports from this laboratory have shown that neurons in this region are under tonic inhibitory control by noradrenergic neurons. The excitability of neurons in the raphe magnus and adjacent reticular formation may also be controlled by cholinergic neurons since there is evidence that cholinergic terminals are located in the medial region of the caudal medulla. The present study was designed to examine this possibility by microinjecting carbachol, a cholinergic agonist, into the region of the nucleus raphe magnus. The results indicate that the injection of carbachol into the caudal brainstem produces dose-dependent hypoalgesia, i.e. decreased pain sensitivity. This hypoalgesia appears to be mediated by cholinergic muscarinic receptors since it was reversed by the muscarinic antagonist atropine. The cholinergic innervation of the raphe magnus does not appear to be important in the maintenance of nociceptive threshold since injection of atropine alone did not alter pain responses.

Functional significance of the apamin-sensitive conductance in rat locus coeruleus neurons.

The effects of apamin on rat locus coeruleus (LC) neurons were studied in a brain slice preparation with intracellular recording. Bath application of apamin (2-500 nM) reduced the amplitude of an intermediate component of the afterhyperpolarization (AHP) following single spontaneous action potentials, but did not change the size or time-course of fast and slow components of the AHP, spike amplitude or duration. Apamin blocked the early component of the post-stimulus hyperpolarization (PSH) which follows a train of action potentials. The size of the late component of PSH was sometimes augmented by apamin. Apamin increased the number of spikes evoked by a depolarizing current pulse and increased the slope of the spike frequency-current intensity relation. Accommodation of firing during long depolarizing pulses showed a biexponential time-course indicating 2 distinct components. Apamin specifically reduced the contribution of the fast component of accommodation and increased its time constant. These data indicate that the apamin-sensitive conductance is functionally important in accommodation at faster firing rates such as those seen during evoked spike trains in the present experiments, and which may occur in vivo during behavioral arousal and in anxiety or drug withdrawal syndromes.

Ethanol increases the firing rate of dopamine neurons of the rat ventral tegmental area in vitro.

The ventral tegmental area (VTA) is a brain region rich in dopamine-containing neurons. Since most agents which act as substrates for self-administration increase dopaminergic outflow in the mesolimbic or mesocortical areas, the VTA slice preparation may be useful for identifying drugs with potential for abuse. While ethanol (EtOH) is a drug of abuse which has been widely studied, the properties of ethanol which contribute to its abuse potential are not known. We have developed a brain slice preparation of the VTA in order to study the action of EtOH on putative dopamine neurons. Concentrations of EtOH from 20 to 320 mM produce a dose-dependent excitation of the dopamine-type neurons of the VTA. About 89% of neurons which have electrophysiological characteristics established for presumed dopamine-containing neurons were excited by ethanol in the pharmacologically relevant concentration range. This excitation persists in low-calcium, high-magnesium medium, which suggests a direct excitatory action of EtOH on dopamine-type cells in the VTA slice.

Central versus peripheral mediation of responses to adenosine receptor agonists: evidence against a central mode of action.

Although adenosine and its analogs have potent effects on the peripheral and central nervous systems, the actions of systemically administered adenosine analogs on the central nervous system are poorly understood. We have previously shown that adenosine analogs depress hippocampal-evoked responses (fEPSPs) in a brain slice preparation, and in the present study, we observed that local pressure ejection of adenosine or R-phenylisopropyladenosine (R-PIA) also reduced the fEPSP recorded in situ in a theophylline-reversible manner. However, systemic administration of R-PIA in up to 1000 times the behaviorally active dose failed to attenuate the fEPSP amplitude. Similar observations were made with systemic injections of 2-chloroadenosine and N-ethylcarboxamidoadenosine, both of which are active in behavioral studies following intraperitoneal or intracerebroventricular injection. Autoradiography showed systemically injected [3H]PIA did not enter the brain in significant concentrations, and this observation was confirmed using microdialysis brain perfusion. These results suggest that systemically administered adenosine analogs do not affect synaptic neurotransmission in the hippocampus because they fail to reach the appropriate receptors.

The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study.

The action of cholecystokinin (CCK) on presynaptic function of dopaminergic nerve terminals has been the subject of much debate in the literature. In efforts to resolve some of the reported ambiguities, high speed in vivo electrochemical recordings were carried out in the caudate nucleus and nucleus accumbens of the urethane anesthetized rat, to determine effects of locally applied sulfated (CCK-8S) and unsulfated (CCK-8US) CCK octapeptide. Locally-applied CCK-8S and CCK-8US caused no increase in the baseline electrochemical signals recorded from either brain region. However, locally applied CCK-8S potentiated the potassium-evoked overflow of dopamine (DA) into the extracellular space in both the caudate and nucleus accumbens. In contrast, pressure ejection of CCK-8US produced no significant effects on the potassium-evoked overflow of DA in either structure. These data support a facilitatory effect of CCK-8S on potassium-evoked overflow from DA-containing nerve terminals in the urethane anesthetized rat that is likely mediated through a peripheral type CCK receptor.

Cocaine effects in the ventral tegmental area: evidence for an indirect dopaminergic mechanism of action.

Behavioral studies have implicated central dopaminergic systems, especially the ventral tegmental area of Tsai (VTA), in the mediation of the reinforcing effects of drugs of abuse such as cocaine. A brain slice preparation of the VTA was used to assess the direct effects of cocaine on the spontaneous activity of dopamine-type neurons. When superfused with 1-10 microM cocaine the firing rate of spontaneously active VTA neurons was decreased, with no corresponding change in spike height. While there was a considerable variability in the response to a given concentration of cocaine among the individual units, every cell inhibited by dopamine was also inhibited by cocaine. The local anesthetic lidocaine had variable effects on firing rate, but never potentiated the inhibitory effects of dopamine. Inhibitory responses to either dopamine or cocaine were blocked by the specific D2 dopamine receptor antagonist sulpiride. Small concentrations of cocaine (0.1-0.5 microM), which by themselves had little or no effect on spontaneous activity, potentiated the inhibitory effect of exogenously applied dopamine. Furthermore, the inhibitory action of apomorphine on spontaneous activity in the VTA was not potentiated by cocaine. These observations suggest that in low concentrations, cocaine can act as a dopamine reuptake inhibitor in the VTA, and that the resultant increase in extracellular dopamine acts upon dopamine autoreceptors to inhibit cellular activity.

Reversal of inhibition of putative dopaminergic neurons of the ventral tegmental area: interaction of GABA(B) and D2 receptors.

Neurons of the ventral tegmental area (VTA) are critical in the rewarding and reinforcing properties of drugs of abuse. Desensitization of VTA neurons to moderate extracellular concentrations of dopamine (DA) is dependent on protein kinase C (PKC) and intracellular calcium levels. This desensitization is called DA inhibition reversal, as it requires concurrent activation of D2 and D1-like receptors; activation of D2 receptors alone does not result in desensitization. Activation of other G-protein-linked receptors can substitute for D1 activation. Like D2 receptors, GABA(B) receptors in the VTA are coupled to G-protein-linked potassium channels. In the present study, we examined interactions between a GABA(B) agonist, baclofen, and dopamine agonists, dopamine and quinpirole, to determine whether there was some interaction in the processes of desensitization of GABA(B) and D2 responses. Long-duration administration of baclofen alone produced reversal of the baclofen-induced inhibition indicative of desensitization, and this desensitization persisted for at least 60 min after baclofen washout. Desensitization to baclofen was dependent on PKC. Dopamine inhibition was reduced for 30 min after baclofen-induced desensitization and conversely, the magnitude of baclofen inhibition was reduced for 30 min by long-duration application of dopamine, but not quinpirole. These results indicate that D2 and GABA(B) receptors share some PKC-dependent mechanisms of receptor desensitization.

Orofacial pain update. 2.

This article is the second of a two part series, intended to update dentists regarding some of the current issues associated with pain management in the facial region. Part One presented a look at what constitutes orofacial pain and what should be included in a comprehensive evaluation prior to treating facial pain. The second part of this article focuses on levels of education, standard of care issues, specialty status and implications on how care is delivered.

The effects of clomipramine on the excitatory action of ethanol on dopaminergic neurons of the ventral tegmental area in vitro.

Dopaminergic neurons of the ventral tegmental area (VTA) are important in the mediation of the rewarding properties of drugs of abuse such as ethanol. We have demonstrated in electrophysiological experiments in brain slices that serotonin potentiates the excitatory effect of ethanol on putative dopaminergic neurons of the VTA. Inasmuch as serotonin reuptake inhibitors have been shown to reduce ethanol intake in clinical studies, we investigated the effect of two serotonin reuptake inhibitors, clomipramine and zimelidine, on ethanol-induced excitation of VTA neurons recorded in vitro. Although zimelidine did potentiate ethanol-induced excitation in some neurons, on average, the effect of zimelidine was not significant. Significant potentiation of ethanol excitation was seen with 500 nM clomipramine, but not at higher or lower concentrations. In addition, clomipramine also enhanced the potentiation produced by low concentrations of serotonin. The lack of potentiation of ethanol excitation seen with application of higher concentrations of clomipramine could not be explained by inhibition of reuptake of norepinephrine or dopamine, and is probably due to blockade of 5-HT2 receptors by these higher concentrations of clomipramine. These results may have some implications for the development of serotonergic drugs, including serotonin uptake inhibitors, for the treatment of alcoholism.

The effects of ethanol on dopaminergic neurons of the ventral tegmental area studied with intracellular recording in brain slices.

Dopaminergic neurons in the ventral tegmental area of Tsai (VTA) have been implicated in the mediation of the rewarding effects of ethanol and many other drugs of abuse. Our previous extracellular studies in brain slices have demonstrated that ethanol increases the firing rate of dopaminergic neurons in the VTA. In the present intracellular study, ethanol (40-160 mM) increased the spontaneous firing rate of most (77%) VTA neurons. In addition, most (75%) VTA neurons were depolarized by ethanol. Ethanol also changed the shape of the spontaneous action potential in VTA neurons, reducing the amplitude of the spike after-hyperpolarization (in 74% of neurons) and also reducing the amplitude of the depolarizing phase of the action potential (in 86% of neurons tested). Furthermore, analysis of Voltage/Current curves in the presence and absence of ethanol showed that ethanol had little effect on the resistance of the cell membrane at membrane potentials near rest, but enhanced the time-dependent inward rectification activated at more hyperpolarized membrane potentials (Ih). This intracellular study identifies several electrophysiological effects of ethanol that may underlie the ethanol-induced excitation of VTA neurons and, therefore, may be important for the rewarding effects of ethanol.

Serotonin potentiates dopamine inhibition of ventral tegmental area neurons in vitro.

1. The ventral tegmental area (VTA) has been implicated in both the rewarding effects of drugs of abuse and the etiology of schizophrenia. We report here that serotonin (5-HT) potentiates the inhibitory effect of dopamine on dopaminergic VTA neurons. Dopamine (0.5-10 microM) inhibited the spontaneous firing of putative dopamine-containing neurons of the VTA. 5-HT (5-10 microM) itself did not significantly alter the spontaneous firing rate; however, in the presence of 5-HT, the inhibitory potency of dopamine was significantly increased. 2. The inhibitory potency of the dopamine agonist quinpirole was also increased by 5-HT. 3. 5-HT-induced potentiation was also produced by the selective 5-HT2 agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine, and was reversed by the selective 5-HT2 antagonist ketanserin. 4. This novel action of 5-HT on dopaminergic neurons has important implications for the development of drugs to treat schizophrenia, and for the identification of agents that will be useful in treating drug abuse disorders like alcoholism.

Dopaminergic neurons in the ventral tegmental area of C57BL/6J and DBA/2J mice differ in sensitivity to ethanol excitation.

BACKGROUND: The mesolimbic dopamine pathway that originates in the ventral tegmental area (VTA) is important for the rewarding effects of ethanol. Ethanol has been shown to excite dopaminergic neurons of the VTA, both in vivo and in vitro, in rats. Behavioral differences in the rewarding effects of ethanol have been observed between C57BL/6J and DBA/2J mice. The present electrophysiological study examined the effect of ethanol on individual dopaminergic VTA neurons from these two inbred mouse strains. METHODS: Extracellular single unit recordings of spontaneous action potentials were made from dopaminergic VTA neurons in brain slices from either C57BL/6J or DBA/2J mice. Ethanol (10 to 160 mM) was administered in the superfusate and the mean change in firing rate produced by ethanol was measured. RESULTS: There was no significant difference in basal spontaneous firing rate of dopaminergic VTA neurons between these two mouse strains. Ethanol caused a concentration-dependent increase in the firing rate of neurons from both mouse strains. Ethanol excited dopaminergic VTA neurons from DBA/2J mice more potently than those from C57BL/6J mice. CONCLUSIONS: The difference in sensitivity to ethanol excitation of dopaminergic VTA neurons in C57BL/6J and DBA/2J mice may contribute to differences in their behavioral response to ethanol. The fact that a given concentration of ethanol causes greater excitation of dopaminergic VTA (reward) neurons in DBA/2J mice than in C57BL/6J mice could explain why DBA/2J mice show much stronger place preference conditioning with ethanol. The higher voluntary intake of ethanol by C57BL/6J mice may be partly due to the insensitivity of their dopaminergic VTA neurons that requires them to drink a lot of ethanol to achieve sufficient excitation of reward neurons, whereas DBA/2J mice avoid oral ingestion of ethanol, despite its rewarding effect, because of their aversion to its taste.

Cocaine potentiates ethanol-induced excitation of dopaminergic reward neurons in the ventral tegmental area.

The coabuse of cocaine and ethanol is one of the most frequently used substance abuse combinations in the United States. The dopamine (DA) neurons in the ventral tegmental area (VTA) are important in the rewarding mechanism of these two substances. Cocaine is known to block the reuptake of DA and serotonin (5-HT). At concentrations below 1 microM, cocaine preferentially blocks the reuptake of 5-HT compared with DA. We have previously shown that ethanol increases the firing rate of DA neurons in the VTA, and that this excitation is enhanced by 5-HT. Extracellular single-unit recordings were made from VTA dopaminergic neurons in coronal brain slices from young adult Fischer 344 rats. Cocaine (1-10 microM) reduced the spontaneous firing rate in VTA dopaminergic neurons in a concentration-related manner. A lower concentration of cocaine (500 nM), which is a concentration that is pharmacologically relevant in addicts, produced only a very small decrease in the firing rate of VTA neurons but potentiated ethanol excitation of these neurons. Higher concentrations of cocaine (1 microM) did not enhance ethanol excitation. Ethanol-induced excitation was potentiated by the higher concentrations of cocaine (1 and 2 microM) in the presence of the D(2) receptor antagonist sulpiride (1 microM). Furthermore, cocaine potentiation of ethanol-induced excitation was reversed by ketanserin (2 microM), a 5-HT(2) antagonist. The enhanced ethanol excitation of VTA dopaminergic neurons caused by cocaine may partially explain the high incidence of the coabuse of these two substances.

Serotonin potentiates ethanol-induced excitation of ventral tegmental area neurons in brain slices from three different rat strains.

Neurons of the ventral tegmental area of Tsai (VTA) are important in mediation of the rewarding effects of drugs of abuse, including ethanol. We have demonstrated previously that ethanol excites neurons of the VTA recorded in a brain slice preparation. In the present study, we tested the interaction between serotonin and ethanol on putative dopamine neurons of the VTA. As reported previously, ethanol (20-160 mM) excited VTA neurons in a concentration-dependent manner. Serotonin (1-50 microM) produced only minor increases or decreases of the firing rate. In the presence of serotonin, the potency of ethanol was increased significantly. This potentiation of ethanol excitation by serotonin was seen in VTA slices taken from Sprague-Dawley, Fischer 344 and Lewis rats. The effect of 160 mM ethanol on VTA neurons from Fischer 344 rats, for example, was increased from 38.5 +/- 6.1% before serotonin to 77.4 +/- 16.7% after administration of 10 microM serotonin. Two serotonergic agonists, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride and alpha-methylserotonin, also potentiated the ethanol-induced excitation. The action of serotonin to increase the potency of ethanol to excite VTA neurons may be an important factor in the rewarding effects of ethanol, and might be exploited to develop effective pharmacotherapeutic agents for the treatment of alcohol craving.