Prefrontal cortical network activity: Opposite effects of psychedelic hallucinogens and D1/D5 dopamine receptor activation.

The fine-tuning of network activity provides a modulating influence on how information is processed and interpreted in the brain. Here, we use brain slices of rat prefrontal cortex to study how recurrent network activity is affected by neuromodulators known to alter normal cortical function. We previously determined that glutamate spillover and stimulation of extrasynaptic N-methyl-d-aspartic acid (NMDA) receptors are required to support hallucinogen-induced cortical network activity. Since microdialysis studies suggest that psychedelic hallucinogens and dopamine D1/D5 receptor agonists have opposite effects on extracellular glutamate in prefrontal cortex, we hypothesized that these two families of psychoactive drugs would have opposite effects on cortical network activity. We found that network activity can be enhanced by 2,5-dimethoxy-4-iodoamphetamine (DOI) (a psychedelic hallucinogen that is a partial agonist of 5-HT(2A/2C) receptors) and suppressed by the selective D1/D5 agonist SKF 38393. This suppression could be mimicked by direct activation of adenylyl cyclase with forskolin or by addition of a cAMP analog. These findings are consistent with previous work showing that activation of adenylyl cyclase can upregulate neuronal glutamate transporters, thereby decreasing synaptic spillover of glutamate. Consistent with this hypothesis, a low concentration of the glutamate transporter inhibitor threo-beta-benzoylaspartic acid (TBOA) restored electrically-evoked recurrent activity in the presence of a selective D1/D5 agonist, whereas recurrent activity in the presence of a low level of the GABA(A) antagonist bicuculline was not resistant to suppression by the D1/D5 agonist. The tempering of network UP states by D1/D5 receptor activation may have implications for the proposed use of D1/D5 agonists in the treatment of schizophrenia.

The role of Kv1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex.

Serotonin 5-HT(2A) receptors have been implicated in psychiatric illness and the psychotomimetic effects of hallucinogens. In brain slices, focal stimulation of 5-HT(2A) receptors in rat prefrontal cortex results in dramatically increased glutamate release onto layer V pyramidal neurons, as measured by an increase in "spontaneous" (nonelectrically evoked) EPSCs. This glutamate release is blocked by tetrodotoxin (TTX) and is thought to involve local spiking in thalamocortical axon terminals; however, the detailed mechanism has remained unclear. Here, we investigate parallels in EPSCs induced by either serotonin or the potassium channel blockers 4-aminopyridine (4-AP) or alpha-dendrotoxin (DTX). DTX, a selective blocker of Kv1.1-, Kv1.2-, and Kv1.6-containing potassium channels, has been shown to release glutamate in cortical synaptosomes, presumably by inhibiting a subthreshold-activated, slowly inactivating potassium conductance. By comparing DTX with other potassium channel blockers, we found that the ability to induce EPSCs in cortical pyramidal neurons depends on affinity for Kv1.2 subunits. DTX-induced EPSCs are similar to 5-HT-induced EPSCs in terms of sensitivity to TTX and omega-agatoxin-IVA (a blocker of P-type calcium channels) and laminar selectivity. The involvement of thalamocortical terminals in DTX-induced EPSCs was confirmed by suppression of these EPSCs by micro-opiates and thalamic lesions. More directly, DTX-induced EPSCs substantially occlude those induced by 5-HT, suggesting a common mechanism of action. No occlusion by DTX was seen when EPSCs were induced by a nicotinic mechanism. These results indicate that blockade of Kv1.2-containing potassium channels is part of the mechanism underlying 5-HT-induced glutamate release from thalamocortical terminals.

Neurotrophin-3 modulates noradrenergic neuron function and opiate withdrawal.

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

Adenosine preferentially suppresses serotonin2A receptor-enhanced excitatory postsynaptic currents in layer V neurons of the rat medial prefrontal cortex.

Serotonin induces 'spontaneous' (non-electrically evoked) excitatory postsynaptic currents in layer V pyramidal neurons in the prefrontal cortex. This is likely due to a serotonin2A receptor-mediated focal release of glutamate onto apical dendrites. In addition, activation of the serotonin2A receptor selectively enhances late components of electrically evoked excitatory postsynaptic currents. In this study, using in vitro intracellular and whole-cell recording in rat brain slices, we examined the role of adenosine in modulating serotonin2A-enhanced 'spontaneous' and electrically evoked excitatory postsynaptic currents in layer V pyramidal neurons in the medial prefrontal cortex. Adenosine and N6-cyclopentyladenosine, an A1 adenosine agonist, markedly suppressed the serotonin2A-induced ('spontaneous') excitatory postsynaptic currents. However, adenosine had no effect on spontaneous miniature (tetrodotoxin-insensitive) postsynaptic potentials. Adenosine also blocked the late excitatory postsynaptic currents induced by the serotonin2A/2C agonist R(-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. Surprisingly, in contrast to other regions, adenosine had a relatively small effect on electrically evoked fast excitatory postsynaptic currents. These findings represent a novel demonstration of adenosine's ability to preferentially modulate serotonin2A-mediated synaptic events in the medial prefrontal cortex. As the serotonin2A receptor is closely linked with the effects of atypical antipsychotics and hallucinogens, further understanding of the modulators of this receptor such as adenosine may provide useful therapeutic applications.

Serotonin induces EPSCs preferentially in layer V pyramidal neurons of the frontal cortex in the rat.

The effect of serotonin (5-HT) on the release of glutamate was examined in pyramidal cells in layers II-VI of the frontal cortex. The intracellular recording electrode contained 1% biocytin so the neurons could later be visualized with an avidin-biotin peroxidase method. Pyramidal cells in layer V of the frontal cortex showed the greatest 5-HT-induced increase in both the frequency and amplitude of 'spontaneous' (non-electrically evoked) excitatory post-synaptic currents (EPSCs). A small proportion of neurons in layer II/III showed an increase in EPSC frequency, whereas cells in layer VI showed no significant change in either EPSC frequency or amplitude. The physiological response to 5-HT mirrors the high density of 5-HT(2A) receptors in layer V, as well as the pattern of thalamic projections in frontal cortex. The specific induction of EPSCs in layer V neurons suggests that 5-HT preferentially modulates the output neurons of the frontal cortex.

Dual control of dorsal raphe serotonergic neurons by GABA(B) receptors. Electrophysiological and microdialysis studies.

We assessed the role of GABA(B) receptors in the control of serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN) by using microdialysis in vivo and intra- and extracellular recording in vitro in the rat. The GABA(B) agonist R(+)baclofen (but not the inactive S(-)enantiomer) enhanced the 5-HT output in the DRN (4. 7-fold at 15 mg/kg s.c.) and, to a much lesser extent, striatum of unanesthetized rats. Phaclofen (2 mg/kg s.c.) antagonized the effects of 6 mg/kg R(+)baclofen in dorsal striatum. Using dual-probe microdialysis, R(+)baclofen (0.1-100 microM) applied in the DRN enhanced the local 5-HT output (4.5-fold at 100 microM) but decreased that in striatum at 100 microM. At concentrations higher than 100 microM there was a moderate decrement in the elevation of 5-HT in the DRN. In midbrain slices, bath R(+)baclofen exerted a biphasic effect on DRN 5-HT neurons. Consistent with a reduced striatal 5-HT release when infused in the DRN, R(+)baclofen (0.1-30 microM) induced an outward current in 5-HT neurons (IC(50) = 1.4 microM). Lower R(+)baclofen concentrations (0.01-1 microM) preferentially reduced GABAergic inhibitory postsynaptic currents induced by N-methyl-D-aspartate (20 microM) in 5-HT neurons (IC(50) = 72 nM). Using extracellular recordings, R(+)baclofen (300 nM) enhanced the ability of NMDA to induce firing in a subpopulation of serotonergic neurons. These results are consistent with a preferential activation by a low concentration of R(+)baclofen of presynaptic GABA(B) receptors on GABAergic afferents that could disinhibit 5-HT neurons and increase 5-HT release.

Serotonin model of schizophrenia: emerging role of glutamate mechanisms.

The serotonin (5-HT) hypothesis of schizophrenia arose from early studies on interactions between the hallucinogenic drug LSD (D-lysergic acid diethylamide) and 5-HT in peripheral systems. More recent studies have shown that the two major classes of psychedelic hallucinogens, the indoleamines (e.g., LSD) and phenethylamines (e.g. , mescaline), produce their central effects through a common action upon 5-HT(2) receptors. This review focuses on two brain regions, the locus coeruleus and the cerebral cortex, where the actions of indoleamine and the phenethylamine hallucinogens have been shown to be mediated by 5-HT(2A) receptors; in each case, the hallucinogens (via 5-HT(2A) receptors) have been found to enhance glutamatergic transmission. In the prefrontal cortex, 5-HT(2A)-receptors stimulation increases the release of glutamate, as indicated by a marked increase in the frequency of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) in the apical dendritic region of layer V pyramidal cells; this effect is blocked by inhibitory group II/III metabotropic glutamate agonists acting presynaptically and by an AMPA/kainate glutamate antagonist, acting postsynaptically at non-NMDA glutamate receptors. A major alternative drug model of schizophrenia, previously believed to be entirely distinct from that of the psychedelic hallucinogens, is based on the psychotomimetic properties of antagonists of the NMDA subtype of glutamate receptor (e.g., phencylidine and ketamine). However, recently it has been found that many of the effects of the NMDA antagonists may also (1) involve 5-HT(2A) receptors and (2) be mediated through excess activity at non-NMDA (i.e., AMPA/kainate) glutamate receptors. Moreover, pharmacological manipulations of glutamate transmission (e. g., by inhibitory metabotropic glutamate agonists) provide unexpected parallels between the actions of these two classes of drugs. Given an emerging recognition of the importance of alterations in glutamatergic transmission in the actions of both psychedelic hallucinogens an NMDA antagonists, this review concludes with of implications for the pathophysiology and therapy of schizophrenia.

Chronic morphine increases GABA tone on serotonergic neurons of the dorsal raphe nucleus: association with an up-regulation of the cyclic AMP pathway.

Major adaptations after chronic exposure to morphine include an up-regulation of the adenosine 3',5'-monophosphate pathway. Acute opioids, via mu-opioid receptors, disinhibit midbrain serotonergic neurons by suppressing inhibitory GABAergic transmission in the dorsal raphe nucleus and adjacent periaqueductal gray. This study examined whether chronic morphine induces a compensatory increase in GABA inputs to 5-hydroxytryptamine neurons and whether this was associated with an up-regulation of the adenosine 3',5'-monophosphate pathway. The firing rate of serotonergic neurons was reduced in brain slices from morphine-dependent rats, an effect reversed by the GABA(A) antagonist bicuculline. The reduction in firing rate was accompanied by an increased frequency of spontaneous GABAergic inhibitory postsynaptic currents, indicating increased GABA tone in the slice. The increase in GABA tone in brain slices from dependent rats was associated with increased induction of inhibitory postsynaptic currents by the adenylyl cyclase activator forskolin, suggesting an up-regulation of the adenosine 3',5'-monophosphate pathway. Indeed, chronic morphine increased levels of adenylyl cyclase VIII (but not of adenylyl cyclase I, III or V) immunoreactivity in the dorsal raphe nucleus area. Two adenosine 3',5'-monophosphate-mediated mechanisms for the increase in GABA tone were discerned. The first, which predominated when impulse-flow was blocked by tetrodotoxin, involves protein kinase A since it was sensitive to protein kinase A inhibitors. The second, seen when impulse-flow was intact (i.e. absence of tetrodotoxin), was insensitive to protein kinase A inhibitors but was suppressed by ZD7288, a blocker of hyperpolarizing-activated Ih channels which are directly activated by adenosine 3',5'-monophosphate. We conclude that chronic morphine induces an up-regulation of the adenosine 3',5'-monophosphate pathway in GABAergic inputs to serotonergic cells, resulting in an increase in spontaneous and impulse-flow dependent GABA release. These changes would lead to an increase in GABA tone and subsequently to the reported decrease in serotonergic activity during opiate withdrawal.

Physiological antagonism between 5-hydroxytryptamine(2A) and group II metabotropic glutamate receptors in prefrontal cortex.

In prefrontal cortex, 5-hydroxytryptamine(2A) (5-HT(2A)) receptors have been linked to the action of hallucinogens and atypical antidepressant/antipsychotic drugs. Previously, we have shown in cortical layer V pyramidal cells that a nonselective metabotropic glutamate (mGlu) receptor agonist suppresses the induction of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) via activation of 5-HT(2A) receptors. In this study, we tested the ability of the selective mGlu2/3 agonist (1S,2S,5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and the selective mGlu2/3 antagonist 2S-2-amino-2-(1S, 2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) to modulate serotonin(5-HT)-induced EPSPs and electrically evoked EPSPs by using intracellular recording from layer V pyramidal cells in medial prefrontal cortex. The mGlu2/3 antagonist LY341495 increased the frequency and amplitude of 5-HT-induced EPSCs, suggesting a role for mGlu2/3 receptors in mediating the action of endogenous glutamate on autoreceptors. Conversely, the mGlu2/3 agonist LY354740 was highly effective and potent (EC(50) = 89 nM) in suppressing glutamate release induced by 5-HT(2A) receptor activation in the medial prefrontal cortex, probably via a presynaptic mechanism. The mGlu2/3 antagonist LY341495 potently blocked the suppressant effect of LY354740 on 5-HT-induced EPSCs as well as electrically evoked early EPSPs. Autoradiography with the radioligands [(3)H]LY354740 and [(125)I](+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane showsa striking overlap of the laminar distribution of mGlu2/3 and 5-HT(2A) receptors in the medial prefrontal cortex that is not apparent in other cortical regions. These findings suggest a close coupling between mGlu2/3 and 5-HT(2A) receptors in the prefrontal cortex that may be relevant for novel therapeutic approaches in the treatment of neuropsychiatric syndromes such as depression and schizophrenia.

Molecular control of locus coeruleus neurotransmission.

The locus coeruleus (LC) is the major noradrenergic nucleus in the brain and innervates large segments of the neuraxis. LC neurons are thought to regulate states of attention and vigilance as well as activity of the sympathetic nervous system. These neurons also have been implicated in the actions of stress, antidepressants, and opiates on the brain. Aided in part by the fact that the LC is relatively homogeneous, it has been possible to understand some of the cellular and molecular mechanisms that control their functional state. This review focuses on the role played by the cAMP pathway in regulation of LC neurons, particularly after chronic perturbations. Thus, several components of this intracellular signaling pathway are upregulated in the LC after chronic stress or chronic opiate treatment, but downregulated after chronic antidepressant treatment. LC neurons exhibit a pacemaker activity, which appears to be mediated, at least in part, by a nonspecific cation current that is activated by protein kinase A. As a result, stimuli that upregulate the cAMP pathway after chronic administration (e.g., stress or opiates) increase the excitability of LC neurons, whereas stimuli that downregulate the cAMP pathway (e.g., antidepressants) exert the opposite effect. Such molecular adaptations could contribute to the behavioral plasticity that is associated with these various conditions.

Serotonin and hallucinogens.

This brief review traces the serotonin (5-HT) hypothesis of the action of hallucinogenic drugs from the early 1950s to the present day. There is now converging evidence from biochemical, electrophysiological, and behavioral studies that the two major classes of psychedelic hallucinogens, the indoleamines (e.g., LSD) and the phenethylamines (e.g., mescaline), have a common site of action as partial agonists at 5-HT2A and other 5-HT2 receptors in the central nervous system. The noradrenergic locus coeruleus and the cerebral cortex are among the regions where hallucinogens have prominent effects through their actions upon a 5-HT2A receptors. Recently, we have observed a novel effect of hallucinogens--a 5-HT2A receptor-mediated enhancement of nonsynchronous, late components of glutamatergic excitatory postsynaptic potentials at apical dendrites of layer V cortical pyramidal cells. We propose that an effect of hallucinogens upon glutamatergic transmission in the cerebral cortex may be responsible for the higher-level cognitive, perceptual, and affective distortions produced by these drugs.

Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release.

Previously, serotonin (5-HT) was found to induce a marked increase in glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) in apical dendrites of layer V pyramidal cells of prefrontal cortex; this effect was mediated by 5-HT2A receptors, a proposed site of action of hallucinogenic and atypical antipsychotic drugs. Unexpectedly, although the effect of 5-HT was Ca2+-dependent and tetrodotoxin-sensitive, it did not appear to involve the activation of excitatory afferent impulse flow. This paradox prompted us to investigate (in rat brain slices) whether 5-HT was acting through an atypical mode of excitatory transmitter release. We found that the frequency of 5-HT-induced spontaneous EPSCs was fully supported by Sr2+ in the absence of added Ca2+, implicating the mechanism of asynchronous transmitter release which has been linked to the high-affinity Ca2+-sensor synaptotagmin III. Although the early, synchronous component of electrically evoked EPSCs was reduced while 5-HT was being applied, late, nonsynchronous components were enhanced during 5-HT washout and also by the 5-HT2 partial agonist 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI); the effect of DOI was blocked by a selective 5-HT2A antagonist (MDL 100,907). This late, nonsynchronous component was distinct from conventional polysynaptic EPSCs evoked in the presence of the GABAA antagonist bicuculline, but resembled asynchronous glutamatergic excitatory postsynaptic potentials (EPSPs) evoked in the presence of Sr2+. An enhancement of asynchronous EPSCs by a specific neurotransmitter receptor has not been reported previously. The possible role of excessive asynchronous transmission in the cerebral cortex in mediating the hallucinogenic effects of 5-HT2A agonists such as DOI is discussed.

5-HT2A receptor or alpha1-adrenoceptor activation induces excitatory postsynaptic currents in layer V pyramidal cells of the medial prefrontal cortex.

We compared 5-hydroxytryptamine (5-HT), norepinephrine and dopamine for their efficacy at increasing excitatory postsynaptic current frequency in layer V pyramidal cells from rat medial prefrontal cortical slices. 5-HT, norepinephrine and dopamine increased the excitatory postsynaptic current frequency by 15.9-, 4.5- and 1.7-fold, respectively. Similar to previous results with 5-HT-induced excitatory postsynaptic currents, blockade of mu-opioid receptors, of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors and fast Na+ channels suppressed the norepinephrine-induced excitatory postsynaptic currents. The norepinephrine-induced, and in most cases, the dopamine-induced increase in excitatory postsynaptic current frequency was blocked by the alpha1-adrenoceptor antagonist prazosin while the alpha2-adrenoceptor antagonist yohimbine did not block either the norepinephrine- or the 5-HT-induced increase in excitatory postsynaptic currents frequency. The potency of three 5-HT2 receptor antagonists with varying selectivity for 5-HT2A/2B/2C receptors tested against the 5-HT-induced increase in excitatory postsynaptic current frequency are in agreement with the affinity of these drugs for the 5-HT2A receptor. These findings suggest that 5-HT2A receptor or alpha1-adrenoceptor activation enhance neurotransmitter release from a similar subset of glutamate terminals that innervate apical dendrites of layer V pyramidal cells.

The electrophysiology of prefrontal serotonin systems: therapeutic implications for mood and psychosis.

A newly described synaptic action of serotonin (5-HT) in the cerebral cortex is reviewed, and implications for mood and psychosis are discussed. Recordings in brain slices show that 5-HT induces a rapid increase in excitatory postsynaptic potentials/currents (EPSPs/EPSCs) in virtually all layer V pyramidal cells of neocortex. This effect is mediated by the 5-HT2A receptor, which has been linked to the action of hallucinogenic and atypical antipsychotic drugs. The increase in EPSCs is seen most prominently in medial prefrontal cortex and other frontal regions where 5-HT2A receptors are enriched. The induction of EPSCs by 5-HT appears to occur through a novel mechanism that does not depend on the activation of afferent impulse flow. Instead, 5-HT appears to act presynaptically, directly or indirectly, to induce a focal release of glutamate from a subpopulation of glutamatergic terminals impinging upon the apical (but not basilar) dendrites of layer V pyramidal cells; a working hypothesis of the transduction pathway (involving asynchronous transmitter release) for this process is presented. Consistent with a focal action upon glutamatergic nerve terminals, the 5-HT-induced EPSPs can be suppressed by presynaptic inhibitory modulators such as mu-opiate or group II/III metabotropic agonists. We suggest that the suppression of 5-HT-induced EPSCs by 5-HT2A antagonists and mu-opiate agonists may underlie certain shared clinical effects of 5-HT2A antagonists and mu-opiate agonists. We suggest further that since presynaptic group II/III metabotropic glutamate agonists suppress 5-HT-induced EPSCs, metabotropic glutamate agonists may also possess antidepressant and/or antipsychotic properties.

5-Hydroxytryptamine-induced excitatory postsynaptic currents in neocortical layer V pyramidal cells: suppression by mu-opiate receptor activation.

Activation of 5-hydroxytryptamine-2A receptors increases the frequency of excitatory postsynaptic currents through a focal action at apical, but not basilar, dendrites of neocortical layer V pyramidal cells. Since mu-, delta- and kappa-opiate receptors are known to inhibit depolarization-induced glutamate release in cerebrocortical slices, we examined the opiate receptor subtype(s) that suppress(es) 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex and whether this suppression was occurring through a presynaptic or a postsynaptic mechanism. Only opioid agonists that act upon mu-receptors (i.e. [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin, the endogenous mu-selective agonist endomorphin-1 and the non-selective opioid agonist [Met]enkephalin) suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents. The delta-agonist [D-phen(2,5)]enkephalin and the kappa-agonist U50,488 were ineffective. Only the selective mu-antagonist CTOP blocked the suppressant effect of enkephalin, while the selective delta-antagonist naltrindole and the selective kappa-antagonist norbinaltorphimine were ineffective. Since the 5-hydroxytryptamine-induced excitatory postsynaptic currents are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type excitatory amino acid receptors, the failure of mu-agonists to either block postsynaptic AMPA responses or induce outward currents in layer V pyramidal cells suggest that mu-agonists are acting at a presynaptic site to block 5-hydroxytryptamine-induced excitatory postsynaptic currents. Strikingly, a regional selectivity in the suppressant effect of mu-receptor activation on 5-hydroxytryptamine-induced excitatory postsynaptic currents exists, as 300 nM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex by nearly 100%, while in the frontoparietal cortex 1 microM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents by only 58%. This is the first demonstration of a previously unsuspected physiological interaction between 5-hydroxytryptamine-2A and mu-opiate receptors and may be relevant to the relationship between these receptors and both mood and psychotic disorders.

Molecular and cellular basis of addiction.

Drug addiction results from adaptations in specific brain neurons caused by repeated exposure to a drug of abuse. These adaptations combine to produce the complex behaviors that define an addicted state. Progress is being made in identifying such time-dependent, drug-induced adaptations and relating them to specific behavioral features of addiction. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction, such as drug craving and relapse, and to identify specific genes that contribute to individual differences in vulnerability to addiction. Understanding the molecular and cellular basis of addictive states will lead to major changes in how addiction is viewed and ultimately treated.

CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence.

Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response element-binding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gialpha, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.

Neurotensin and the serotonergic system.

The serotonergic system, because of very diffuse projections throughout the central nervous system, has been implicated in numerous functions including nociception, analgesia, sleep-wakefulness and autonomic regulation. Despite an abundant literature indicating the presence of neurotensin-containing (neurotensinergic) neurons, fibres and terminals in most areas containing serotonergic neurons, little is known about the possible relationship between serotonergic and neurotensinergic systems. The purpose of this review is (i) to summarize current knowledge on the anatomical relation between neurotensinergic and serotonergic system, (ii) to summarize current knowledge on the action of neurotensin on serotonergic neurons and (iii) to discuss the possible physiological relevance of this action. Neurotensin-containing cell bodies can be found in the most rostral raphe nuclei. There are neurotensin-containing fibres and terminals in all raphe nuclei. Raphe nuclei have also been shown to contain neurotensin-receptor binding sites. In the dorsal raphe nucleus, neurotensin induces a concentration-dependent increase in the firing rate of a subpopulation of serotonergic neurons. The neurotensin-induced excitation, which is selectively blocked by the non-peptide neurotensin receptor antagonist SR 48692, is observed mainly in the ventral part of the nucleus. Most serotonergic neurons show marked desensitization to neurotensin, even at low concentrations. In intracellular experiments, neurotensin induces an inward current, associated in some cases with a decrease in apparent input conductance, which is occluded by supramaximal concentrations of the alpha 1-adrenoceptor agonist phenylephrine. In rare cases, neurotensin induces an excitation of GABAergic or glutamatergic neurons. Since the neurotensinergic system has also been implicated in nociception, analgesia, sleep-wakefulness, and autonomic regulation, the review discusses the possibility that part of this regulation could involve the activation of the serotonergic system.