Marisela Morales, Ph.D. - US grants

The notion of functional interactions between the alpha 7 nicotinic acetylcholine ( alpha 7 nACh) and the cannabinoid systems is emerging from recent in vitro and in vivo studies. Both the alpha 7 nACh receptor and the cannabinoid receptor 1 (CB1) are highly expressed in the hippocampus. To begin addressing possible anatomical interactions between the alpha 7 nACh and the cannabinoid systems in the hippocampus, we investigated the distribution of neurons expressing alpha 7 nACh mRNA in relation to those containing CB1 mRNA. By in situ hybridization we found that the alpha 7 nACh mRNA is diffusely expressed in principal neurons and is highly expressed in a subset of interneurons. We observed that the pattern of distribution of hippocampal interneurons co-expressing transcripts encoding alpha 7 nACh and glutamate decarboxylase (GAD; synthesizing enzyme of gamma-aminobutyric acid, GABA) closely resembles the one displayed by interneurons expressing CB1 mRNA. By double in situ hybridization we established that the majority of hippocampal interneurons expressing alpha 7 nACh mRNA have high levels of CB1 mRNA. As CB1 interneurons contain cholecystokinin (CCK), we investigated the degree of cellular co-expression of alpha 7 nACh mRNA and CCK, and found that the cellular co-existence of alpha 7 nACh and CCK varies within the different layers of the hippocampus.[unreadable] In summary, we established that most of the hippocampal alpha 7 nACh expressing interneurons are endowed with the CB1 receptor. We found that these alpha 7 nACh/CB1 interneurons are the major subpopulation of hippocampal interneurons expressing CB1 mRNA. The alpha 7 nACh expressing interneurons represent half of the total population of CCK containing neurons in the hippocampus. Since it is well established that the vast majority of hippocampal interneurons expressing CB1 mRNA have serotonin type 3 (5-HT3) receptors, we conclude that these hippocampal alpha 7 nACh/ 5HT3/ CB1/CCK interneurons correspond to those previously postulated to relay inputs from diverse cortical and subcortical regions about emotional, motivational, and physiological states.

Interactions between stress and the mesocorticolimbic dopamine system have been suggested from behavioral and electrophysiological studies. As corticotropin releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing dopamine (DA) in the ventral tegmental area (VTA). We first investigated subcellular distribution of CRF in VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF-immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. Of functional importance, we established that the CRF asymmetric synapses are glutamatergic, as the CRF-immunolabeled terminals in these synapses co-expressed the vesicular glutamate transporter 2, and that CRF symmetric synapses are GABAergic, as the CRF-immunolabeled terminals in these synapses co-expressed glutamic acid decarboxilase. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH, a marker for DA neurons). We found that the majority of synapses between CRF-immunoreactive terminals and TH neurons are asymmetric (glutamatergic), and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic dopamine system.[unreadable] The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic dopamine system.

The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or gamma-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely coexpressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic.[unreadable] To investigate whether VTA glutamatergic neurons establish local synapses, we marked axon terminals of VTA neurons by local injection of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHAL) and used double immunolabeling and electron microcopic analysis to determine if PHAL-positive terminals within the VTA contained VGluT2. PHAL-positive terminals established either asymmetric or symmetric synapses, predominantly on dendrites, more than half of all PHAL-positive terminals that made recognizable synapses contained VGluT2. Double labeled PHAL/VGluT2 terminals established predominantly asymmetric synapses while most PHAL-positive terminals lacking VGluT2 formed symmetric synapses. To determine whether DAergic neurons receive glutamatergic inputs from local neurons, we used triple fluorescence immunolabeling with antibodies against PHAL, VGluT2 and tyrosine hydroxylase (TH, a marker for DAergic neurons). By confocal fluorescence microscopy we observed double labeled PHAL/ VGluT2 varicosities contacting TH-positive dendrites. By electron microscopy, we further determined that double labeled PHAL/VGluT2 axon terminals formed synaptic contacts on dendrites of both TH-immunoreactive and TH-negative cells. In whole cell recordings of VTA neurons we observed that blocking local action potential activity significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-DAergic neurons. In summary, we provide anatomical and electrophysiological evidence that local glutamatergic neurons establish intrinsic synapses within the VTA.

The pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) provide cholinergic afferents to several brain areas. This cholinergic complex has been suggested to play a role in sleep, waking, motor function, learning and reward. To have a better understanding of the neurochemical organization of the PPTg/LDTg we characterized the phenotype of PPTg/LDTg neurons by determining in these cells the expression of transcripts encoding choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or the vesicular glutamate transporters (vGluT1, vGluT2 and vGluT3). Within the PPTg/LDTg complex we found neurons expressing ChAT, vGluT2 or GAD transcripts, these neuronal phenotypes were intermingled, but not homogeneously distributed within the PPTg or LDTg. Previous studies suggested the presence of either glutamate or GABA immunolabeling in a large number of PPTg/LDTg cholinergic neurons, leading to the widespread notion that PPTg/LDTg cholinergic neurons co-release acetylcholine together with either glutamate or GABA. To assess the glutamatergic or GABAergic nature of the PPTg/LDTg cholinergic neurons we combined in situ hybridization (to detect vGluT2 or GAD transcripts) and immunohistochemistry (to detect ChAT), and found that over 95% of all PPTg/LDTg cholinergic neurons lack transcripts encoding either vGluT2 mRNA or GAD mRNA. As the vast majority of PPTg/LDTg cholinergic neurons lack transcripts encoding essential proteins for the vesicular transport of glutamate or for the synthesis of GABA, co-release of acetylcholine with either glutamate or GABA is unlikely to be a major factor in the interactions between acetylcholine, glutamate, and GABA at the postsynaptic site.

Corticotropin releasing factor (CRF) and CRF-related peptides mediate stress responses, in part, by their interactions with the CRF-binding protein (CRF-BP). The CRF-BP plays a role in dopamine (DA) and glutamate neurotransmission within the ventral tegmental area (VTA). Here we investigated whether specific local neurons could be a source of CRF-BP in the VTA. By in situ hybridization we detected cellular expression of CRF-BP mRNA in the VTA, but not in neighboring substantia nigra compacta (SNC) or substantia nigra reticulata. The selective expression of CRF-BP mRNA proved to be useful in demarcating boundaries between the SNC and the VTA. By double in situ hybridization we determined that VTA neurons with CRF-BP mRNA co-expressed transcripts encoding either tyrosine hydroxylase (TH, a maker for DAergic neurons) or glutamic acid decarboxylase (GAD, synthesizing enzyme of ?-amino butyric acid, GABA). We found that 25% of the total population of TH expressing neurons contained CRF-BP mRNA, providing support to the notion that discrete subpopulations of dopamine neurons are present in the VTA. We determined that within the total population of neurons expressing CRF-BP mRNA 70% co-expressed CRF-BP mRNA TH mRNA, and only 25% co-expressed GAD mRNA. [unreadable] As far as we are aware, we provide the first anatomical evidence that a molecule, CRF-BP, is encoded by DAergic neurons of the VTA, but not by those of the SNC. Based on the observation that the majority of VTA neurons expressing CRF-BP mRNA are DAergic, we propose that in the VTA interactions of CRF-BP with CRF, or with CRF-related peptides, are likely to be predominately mediated by DAergic neurons.

It has been suggested that TH-VGluT2 neurons corelease dopamine and glutamate, however, it is unclear whether these neurons have proteins necessary for the synthesis (aromatic L-amino acid decarboxylase, AADC) or transport (vesicular monoamine transporter, VMAT2 or dopamine transporter, DAT) of dopamine. By in situ hybridization-immunohistochemistry, we found that all TH neurons in the A10 region had AADC, but not all had VMAT2, DAT or D2 receptors (D2R). To determine whether TH-VGluT2 neurons account for TH neurons lacking these dopamine markers, we implemented an anatomical mirror technique, and found that not all TH-VGluT2 neurons lacked VMAT2, DAT or D2R. Next, by quantitative RT-PCR of individual micro-dissected TH neurons, we discovered 2 classes of TH-VGluT2 and 3 classes of TH-only neurons with different latero-medial distribution. Some of the TH-VGluT2 neurons had both VMAT2 and DAT (TH-VGluT2 Class 1) and others lacked both transporters (TH-VGluT2 Class 2). Most of the TH-only neurons contained VMAT2 and DAT (TH-only Class 1), a few had DAT without VMAT2 (TH-only Class 2), and others lacked both VMAT2 and DAT (TH-only Class 3). We concluded that (a) the majority of TH neurons lacking DAT are TH-VGluT2 neurons, (b) very few TH-only neurons express DAT without VMAT2, and (c) TH-VGluT2 neurons lacking DAT also lack VMAT2. Thus we provide evidence that the A10 region contains dopamine neurons with differential compartmentalization and unique signaling properties.

The rostral ventromedial medulla (RVM) constitutes the efferent component of pain-control systems that descend from the brain to the spinal cord. Considerable evidence has emerged regarding participation of this system in persistent pain conditions such as inflammation and neuropathy. The RVM normally exerts an inhibitory influence on dorsal horn neurons However, persistent noxious stimulation triggers time-related alterations in RVM synaptic activity. In inflammatory pain models, descending facilitation transiently increases reducing the net effect of inhibition. Over time, descending inhibition increases resulting in decreased nocifensive behavior. After nerve injury, RVM plasticity leads to facilitation of spinal cord nociceptive output, exacerbation of primary hyperalgesia and enhanced sensory input from adjacent regions (secondary hyperalgesia). AMPA receptor activation in the RVM has been shown to inhibit spinal nociceptive transmission and nocifensive behavior. Increased AMPA receptor function in the RVM is implicated in the activity-dependent plasticity that occurs in response to persistent pain produced by tissue inflammation. Targeting and synaptic clustering of AMPA receptors is essential for efficient excitatory transmission. Neuronal pentraxin 1 (NP1) is a member of the pentraxin family of proteins that is expressed exclusively in neurons and facilitates AMPA receptor clustering. Given the postulated role of NP1 in excitatory synaptic transmission and AMPA receptor systems in pain processing, we have used gene deletion and viral-mediated transfer techniques to examine whether manipulations that target this protein can affect the expression of persistent pain. To determine the contribution of NP1 to nerve-injury evoked pain, we assessed mechanical hypersensitivity in wild type and NP1 knock out mice following spared nerve injury. Nerve injury led to marked mechanical hypersensitivity of the injured limb. This enhanced nociception is significantly inhibited in NP1 knockout mice. In order to probe the specific involvement of RVM NP1 in mediating the attenuated response of NP1 knockout mice, we infused a lentiviral vector which drives expression of functional NP1 protein directly into the RVM. Selective rescue of RVM NP1 expression in knockout mice restores allodynia produced by nerve injury. Consistent with the data obtained in NP1 knockout mice, silencing NP1 expression in the RVM of rats prior to nerve injury inhibits allodynia. Furthermore, it decreases mechanical hyperalgesia. These findings are consistent with the observation that descending facilitatory systems arising in the RVM are necessary for the maintenance of neuropathic pain and identify NP1 in the RVM as a critical element in the descending facilitation of nerve-injury evoked pain. Together, these data suggest that targeting NP1 may be a novel therapeutic strategy for reversing persistent pain of diverse etiologies. On-going studies examining the role of NP1 in other conditions including those associated with AIDS antiretroviral therapy and peripheral inflammation indicate a key role of this protein in the development of persistent pain. In addition to the RVM, prefrontal cortical areas, including anterior cingulate, prelimbic and orbitofrontal cortex have been implicated in the cognitive and affective manifestations of chronic pain. We are also investigating functional changes in these brain areas in rodents using in vivo microdialysis and the contribution of these areas to the nociceptive and affective components of neuropathic pain. We are using the spared nerve injury as a neuropathic pain model and conditioned place preference/aversion and ultrasonic vocalizations as a measure of the affective component of pain and von frey thresholds and the pin-prick test to evaluate mechanical nociception.

The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinsons disease, and other neurodegenerative disorders. PTEN deletion or Akt/PKB activation in dopamine neurons of the ventral midbrain results in remarkable hypertrophy of the substantia nigra and VTA. Our initial characterization of a DAT-PTEN KO strain has provided a clear definition of some of the neuroadaptations in the mesolimbic and nigrostriatal systems, and clearly show dopamine neurotransmission is permanently altered in PTEN KO mice. However, while DAT-PTEN KO animals are viable and appear behaviorally competent, an in depth study of behavioral parameters will clarify if the lack of PTEN interferes with essential functions related to the dopamine system in young, adult and aged animals. Studies performed over the past few years have clearly shown that phenotypes caused by specific genetic modifications are strongly influenced by genes unlinked to the target locus. This problem is exacerbated through the use of Cre-LoxP models as two strains, often containing their own (obscure) genetic backgrounds, are crossed through very specific breeding schedules to generate control and experimental animals. Clearly, it becomes important to avoid the use of a mixed genetic background so complex as to preclude any reasonable use of controls and prevent replication by other investigators. To perform complex behavioral studies, we will use a c57bl/6 congenic DAT-PTEN KO mouse line, generated in our lab. A new mechanism in the brain of rats that may mediate the rewarding and reinforcing properties of drugs of abuse has recently been discovered. This mechanism involves the physical interaction between two proteins in midbrain dopamine neurons, the tumor suppressor PTEN and the brain specific receptor for serotonin, the 5-HT2c receptor (5-HT2cR). Blocking the interaction of PTEN with 5-HT2cR prevents the development of conditioned place preference to nicotine and THC, the active component in marijuana. In addition, PTEN has been shown to physically interact with the NR1 subunit of NMDAR in hippocampus, and PTEN downregulation decreases NMDAR surface expression. These studies suggest PTEN in dopamine neurons may directly modulate functions intimately linked to the development of addiction, and dopamine mediated cognition, such as responses to reward and motivation. We will use a congenic PTEN-KO line to analyze in detail the behavioral profile of KO animals in relationship to drug abuse, overall locomotor performance, as well as other dopamine related cognitive functions. We have shown Pten deletion in differentiated DA neurons causes a significant increase in the number and size of surviving neurons in both the mesolimbic and nigrostriatal projecting pathways (see above). Because at the time of Pten deletion DA neurons have already completed mitosis and phenotypic determination, it is unlikely the reported increase in DA neurons is due to an increase in newly formed neurons. It is thus likely PTEN ablation preserves DA neurons that normally would undergo apoptosis due to the lack of target support, by repressing the initiation of apoptotic pathways. We are now intrigued about several aspects induced by PTEN deletion in dopamine cells: Do all dopamine neurons preserved in DAT-PTEN KO animals project to target areas and form functional connections? Can exposure to an enriched environment enhance dopaminergic function in young and aged KO animals? Do the mesolimbic and nigrostriatal dopamine projections remain functional into aging? Is the PTENless aging dopaminergic system more or less resistant to neurotoxic insults applied during different stages of the mouse life span? Are PTENless dopamine neurons prone to form tumors? Obviously, the tremendous adaptations observed in the PTENless dopaminergic system during development, may pose problems for interpretation of results; however, as previous studies have shown Akt/PKB activation in the adult dopaminergic system can also result in remarkable hypertrophy and plasticity of the nigra and VTA, the results obtained in this study may provide valuable insights into how PTEN ablation changes dopamine function at the molecular and behavioral levels, and the long-term consequences of such adaptations. We believe these studies are important, as manipulations of the PTEN pathway are being considered as a possible venue for therapeutic strategies involving the brain.

Habit learning processes are implicated in the transition from recreational drug use to the compulsive drug seeking that characterizes addiction. Lesions of the dorsal striatum, an area necessary for habit learning, attenuate cue controlled drug seeking and nigrostriatal dopamine lesions disrupt habit formation. Moreover, prior repeated psychostimulant administration facilitates habitual responding for food reinforcers suggesting that their self-administration accelerates habit development. Studies assessing the development of habitual drug seeking are limited. Using outcome devaluation procedures by satiation or pairing of oral reinforcers with sickness induced by a drug (e.g. lithium), two laboratories demonstrated habitual drug seeking of orally administered drug reinforcers. However, studies of intravenous drug administration have been hampered by difficulties inherent in adapting standard devaluation procedures for natural reinforcers to intravenous administered drug. In contrast to natural reinforcers, this route of administration is not associated with an obvious consummatory response and psychostimulants such as cocaine have unconditioned, behaviorally activating effects, that can affect responding. To circumvent these issues, we have used a heterogenous chained schedule of intravenous cocaine administration in which habitual cocaine-seeking is tested by devaluing the final link of a drug seeking/taking chained schedule. In this procedure, responding on the designated drug seeking lever provides access to a drug taking lever, rather than to cocaine itself. Responses on the drug taking lever result in a cocaine infusion. The effects of devaluation of the drug taking link (by extinction) is assessed once performance under the chained schedule has stabilized. Decreased responding during the drug seeking link is indicative of behavior that is goal-oriented. Responding that is insensitive to devaluation is evidential of the development of habitual drug-seeking. Other rats underwent the same protocol except that after completing the cocaine seeking test, an additional 36 sessions were conducted to provide an extended drug seeking history. Given the documented role of the dorsolateral striatum in stimulus-response associations, the influence of inactivation of this brain area was assessed in animals with a prolonged history of cocaine seeking and taking. In rats with limited cocaine access, devaluation of the outcome of the drug seeking link by extinction significantly decreased drug seeking indicating that behavior is goal-oriented rather than habitual. Importantly, however, examination of each animals data revealed that whereas drug seeking was clearly attenuated by outcome devaluation in eight animals, responding of six animals after devaluation was greater than 80% of that under the revalued condition, indicating poor sensitivity to devaluation in these animals. Thus, in this subset of animals, cocaine seeking was not goal-oriented but already habitual in nature. With, however, more prolonged drug experience, all animals transitioned to habitual cocaine seeking. Thus, cocaine-seeking became insensitive to outcome devaluation. Moreover, when the dorsolateral striatum, an area implicated in habit learning, was transiently inactivated, outcome devaluation was again effective in decreasing drug-seeking indicating that responding was no longer habitual but had reverted to control by the goal-oriented system. These studies provide direct evidence that cocaine seeking becomes habitual with prolonged drug experience and describe a rodent model with which to study the neural mechanisms underlying the transition from goal-oriented to habitual drug-seeking. On-going studies are using this model to examine the role of the dynorphin/k-opioid receptor system in modulating the transition from goal-directed to habitual cocaine seeking. Preliminary results suggest that kappa opioid receptors within the dorsolateral striatum are necessary for the expression of habitual cocaine seeking. Currently we are comparing the role of kappa and mu opioid receptors in goal directed and habitual cocaine seeking. In addition, we have started to investigate how the propensity to develop habitual cocaine seeking correlates with other behavioral phenotypes implicated in addiction (i.e. impulsivity, compulsivity, escalation of drug intake, reinstatement). We have also begun to characterize the use of confocal fiberoptic microscopy to assess brain activity in vivo. We have used AAV-directed expression of GCamp3, a fluorescent calcium-binding, to measure intracellular calcium transients in VTA neurons. Initial pharmacological characterization show transient responses to NMDA or morphine. We have also observed transient responses to a painful stimulus (tail-pinch).

Consistently in the previous studies the ethanol treatment impaired learning and memory, upregulated dynorphins and increased glutamate overflow in the CA3 region. The critical finding of the present study (paper submitted to Biological Psychiatry) was that the highly selective long-acting KOR-antagonist nor-BNI administered after cessation of ethanol exposure reversed cognitive deficits and normalized elevated glutamate levels in ethanol-treated rats. Nor-BNI was effective under both systemic or intra-hippocampal administration suggesting that ethanol actions on glutamate overflow and spatial memory were mediated through the hippocampal dynorphin-KOR-dependent mechanism. No effects of nor-BNI on learning and memory and glutamate levels were evident in nave animals suggesting that this mechanism is not activated under normal conditions. These findings suggest that impairments of spatial learning and memory by binge-like ethanol exposure are mediated through the KORactivation by upregulated dynorphins resulting in elevation in glutamate levels. Selective KORantagonists may correct alcohol-induced pathological processes, thus representing a novel pharmacotherapy for treating of ethanol-related cognitive deficits.

Part 1. We demonstrated that intra-mPFC administration of the selective KOR agonist U69,593 decreased local DA overflow, but failed to modify DAT function in 3H DA uptake studies. Reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino acid levels were also affected by KORs, as evidenced by U69,593 inhibition of the ability of the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate to elevate mPFC glutamate and GABA levels. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of mini EPSPs, an effect occluded by picrotoxin. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but the attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino acid neurotransmission, and are necessary for KOR-mediated aversion (manuscript submitted to the Journal of Neuroscience). Part 2. Previously we demonstrated that exposure to a cocaine-associated environment increases mPFC GABA transmission by enhancing excitatory drive from the BLA and activation of AMPA/KA receptors on mPFC GABA neurons Chefer et al., 2011). In the present study in-vivo microdialysis was used to identify alterations in rat mPFC glutamate, GABA dopamine and norepinephrine overflows in response to cues previously associated with either saline or cocaine in situations of either low or high certainty of cocaine expectations. Neurotransmitter levels and locomotor activity (LMA) were assessed 24 hrs after the last conditioning session. Some animals were stressed by handling immediately prior to testing. Exposure to a cocaine-associated environment following fixed sequences of conditioning trials increased mPFC GABA overflow and decreased Glutamate/GABA ratio. Handling stress, which increased blood corticosterone levels, increased baseline Glutamate and GABA overflow in rats expecting cocaine administration. Introduction of the cocaine cues augmented GABA overflow and decreased the Glutamate/GABA ratio in these rats. When random sequences of conditioning trials were used and the certainty of cocaine expectation therefore was low, the same stress paradigm did not increase basal GABA levels. Contrary to the fixed sequence regimen, glutamate overflow and Glu/GABA ratio was increased by cocaine cues. Saline-associated cues did not alter Glu or GABA during either trial regimen. Conditioned hyperactivity in cocaine environment was observed in high expectations paradigm and following stress in low expectations paradigm. To our knowledge, the present study is the first to demonstrate a neurochemical interaction between cocaine-associated environment, stress and cocaine expectation. The data suggest that changes in mPFC Glu/GABA equilibrium reflect certainty of cocaine administration and stress reveals the level of expectation before actual cue presentation (manuscript in preparation).