Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia.

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.

Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits.

According to the prevailing "amyloid cascade hypothesis," genetic dementias such as Alzheimer's disease and familial Danish dementia (FDD) are caused by amyloid deposits that trigger tauopathy, neurodegeneration, and behavioral/cognitive alterations. To efficiently reproduce amyloid lesions, murine models of human dementias invariably use transgenic expression systems. However, recent FDD transgenic models showed that Danish amyloidosis does not cause memory defects, suggesting that other mechanisms cause Danish dementia. We studied an animal knock-in model of FDD (FDD(KI/+)) genetically congruous with human cases. FDD(KI/+) mice present reduced Bri2 levels, impaired synaptic plasticity and severe hippocampal memory deficits. These animals show no cerebral lesions that are reputed characteristics of human dementia, such as tangles or amyloid plaques. Bri2(+/-) mice exhibit synaptic and memory deficits similar to FDD(KI/+) mice, and memory loss of FDD(KI/+) mice is prevented by expression of WT BRI2, indicating that Danish dementia is caused by loss of BRI2 function. Together, the data suggest that clinical dementia in Danish patients occurs via a loss of function mechanism and not as a result of amyloidosis and tauopathy.

Endogenous amyloid-ß is necessary for hippocampal synaptic plasticity and memory.

OBJECTIVE: The goal of this study was to investigate the role of endogenous amyloid-ß peptide (Aß) in healthy brain. METHODS: Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. RESULTS: We found that both antirodent Aß antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aß42, suggesting that endogenously produced Aß is needed for normal LTP and memory. Furthermore, the effect of endogenous Aß on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aß42 production. INTERPRETATION: Endogenous Aß42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aß levels to treat Alzheimer disease.

Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

Calpains are calcium-dependent enzymes that determine the fate of proteins through regulated proteolytic activity. Calpains have been linked to the modulation of memory and are key to the pathogenesis of Alzheimer disease (AD). When abnormally activated, calpains can also initiate degradation of proteins essential for neuronal survival. Here we show that calpain inhibition through E64, a cysteine protease inhibitor, and the highly specific calpain inhibitor BDA-410 restored normal synaptic function both in hippocampal cultures and in hippocampal slices from the APP/PS1 mouse, an animal model of AD. Calpain inhibition also improved spatial-working memory and associative fear memory in APP/PS1 mice. These beneficial effects of the calpain inhibitors were associated with restoration of normal phosphorylation levels of the transcription factor CREB and involved redistribution of the synaptic protein synapsin I. Thus, calpain inhibition may prove useful in the alleviation of memory loss in AD.

Tau is not necessary for amyloid-ß-induced synaptic and memory impairments.

The amyloid hypothesis posits that the amyloid-beta (Aß) protein precedes and requires microtubule-associated protein tau in a sort of trigger-bullet mechanism leading to Alzheimer's disease (AD) pathology. This sequence of events has become dogmatic in the AD field and is used to explain clinical trial failures due to a late start of the intervention when Aß already activated tau. Here, using a multidisciplinary approach combining molecular biological, biochemical, histopathological, electrophysiological, and behavioral methods, we demonstrated that tau suppression did not protect against Aß-induced damage of long-term synaptic plasticity and memory, or from amyloid deposition. Tau suppression could even unravel a defect in basal synaptic transmission in a mouse model of amyloid deposition. Similarly, tau suppression did not protect against exogenous oligomeric tau-induced impairment of long-term synaptic plasticity and memory. The protective effect of tau suppression was, in turn, confined to short-term plasticity and memory. Taken together, our data suggest that therapies downstream of Aß and tau together are more suitable to combat AD than therapies against one or the other alone.

Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.

Amyloid-beta (Abeta) peptides are produced in high amounts during Alzheimer's disease, causing synaptic and memory dysfunction. However, they are also released in lower amounts in normal brains throughout life during synaptic activity. Here we show that low picomolar concentrations of a preparation containing both Abeta(42) monomers and oligomers cause a marked increase of hippocampal long-term potentiation, whereas high nanomolar concentrations lead to the well established reduction of potentiation. Picomolar levels of Abeta(42) also produce a pronounced enhancement of both reference and contextual fear memory. The mechanism of action of picomolar Abeta(42) on both synaptic plasticity and memory involves alpha7-containing nicotinic acetylcholine receptors. These findings strongly support a model for Abeta effects in which low concentrations play a novel positive, modulatory role on neurotransmission and memory, whereas high concentrations play the well known detrimental effect culminating in dementia.

Synaptic and memory dysfunction induced by tau oligomers is rescued by up-regulation of the nitric oxide cascade.

BACKGROUND: Soluble aggregates of oligomeric forms of tau protein (oTau) have been associated with impairment of synaptic plasticity and memory in Alzheimer's disease. However, the molecular mechanisms underlying the synaptic and memory dysfunction induced by elevation of oTau are still unknown. METHODS: This work used a combination of biochemical, electrophysiological and behavioral techniques. Biochemical methods included analysis of phosphorylation of the cAMP-responsive element binding (CREB) protein, a transcriptional factor involved in memory, histone acetylation, and expression immediate early genes c-Fos and Arc. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated both short-term spatial memory and associative memory. These phenomena were examined following oTau elevation. RESULTS: Levels of phospho-CREB, histone 3 acetylation at lysine 27, and immediate early genes c-Fos and Arc, were found to be reduced after oTau elevation during memory formation. These findings led us to explore whether up-regulation of various components of the nitric oxide (NO) signaling pathway impinging onto CREB is capable of rescuing oTau-induced impairment of plasticity, memory, and CREB phosphorylation. The increase of NO levels protected against oTau-induced impairment of LTP through activation of soluble guanylyl cyclase. Similarly, the elevation of cGMP levels and stimulation of the cGMP-dependent protein kinases (PKG) re-established normal LTP after exposure to oTau. Pharmacological inhibition of cGMP degradation through inhibition of phosphodiesterase 5 (PDE5), rescued oTau-induced LTP reduction. These findings could be extrapolated to memory because PKG activation and PDE5 inhibition rescued oTau-induced memory impairment. Finally, PDE5 inhibition re-established normal elevation of CREB phosphorylation and cGMP levels after memory induction in the presence of oTau. CONCLUSIONS: Up-regulation of CREB activation through agents acting on the NO cascade might be beneficial against tau-induced synaptic and memory dysfunctions.

Role of Amyloid-ß and Tau Proteins in Alzheimer's Disease: Confuting the Amyloid Cascade.

The "Amyloid Cascade Hypothesis" has dominated the Alzheimer's disease (AD) field in the last 25 years. It posits that the increase of amyloid-ß (Aß) is the key event in AD that triggers tau pathology followed by neuronal death and eventually, the disease. However, therapeutic approaches aimed at decreasing Aß levels have so far failed, and tau-based clinical trials have not yet produced positive findings. This begs the question of whether the hypothesis is correct. Here we have examined literature on the role of Aß and tau in synaptic dysfunction, memory loss, and seeding and spreading of AD, highlighting important parallelisms between the two proteins in all of these phenomena. We discuss novel findings showing binding of both Aß and tau oligomers to amyloid-ß protein precursor (AßPP), and the requirement for the presence of this protein for both Aß and tau to enter neurons and induce abnormal synaptic function and memory. Most importantly, we propose a novel view of AD pathogenesis in which extracellular oligomers of Aß and tau act in parallel and upstream of AßPP. Such a view will call for a reconsideration of therapeutic approaches directed against Aß and tau, paving the way to an increased interest toward AßPP, both for understanding the pathogenesis of the disease and elaborating new therapeutic strategies.

Effects of topiramate on the prepulse inhibition of the acoustic startle in rats.

The anticonvulsant topiramate (TPM) has been recently proposed as a novel adjuvant therapy for bipolar disorder and schizophrenia, yet its efficacy remains controversial. As both disorders are characterized by gating deficits, we tested the effects of TPM on the behavioral paradigm of prepulse inhibition (PPI) of the acoustic startle response, a validated animal model of sensorimotor gating. TPM (10, 18, 32, 58, 100 mg/kg, intraperitoneal, i.p.) enhanced PPI in rats in a dose-dependent fashion, prevented the PPI reduction mediated by the dopaminergic agonist apomorphine (0.25 mg/kg, subcutaneous, s.c.) and potentiated the effects of the antipsychotic drugs haloperidol (0.05, 0.1 mg/kg, i.p.) and clozapine (2.5, 5 mg/kg, i.p.). Conversely, TPM elicited no significant effect on the PPI disruption mediated by the NMDA receptor antagonist dizocilpine (0.05, 0.1 mg/kg, s.c.) and surprisingly antagonized the attenuation of dizocilpine-induced PPI disruption mediated by clozapine (5 mg/kg, i.p.). Our results suggest that TPM may exert diverse actions on the neural substrates of sensorimotor gating. While the pharmacological mechanisms of such effects are still elusive, our findings might contribute to shed light on some controversies on the therapeutic action of TPM, and point to this drug as a putative novel adjuvant therapy for some clusters of gating disturbances.

Activation of GABA(B) receptors reverses spontaneous gating deficits in juvenile DBA/2J mice.

RATIONALE: Gamma-amino-butyric acid (GABA)(B) receptors play a key role in the pathophysiology of psychotic disorders. We previously reported that baclofen, the prototypical GABA(B) agonist, elicits antipsychotic-like effects in the rat paradigm of prepulse inhibition (PPI) of the startle, a highly validated animal model of schizophrenia. OBJECTIVES: We studied the role of GABA(B) receptors in the spontaneous PPI deficits displayed by DBA/2J mice. MATERIALS AND METHODS: We tested the effects of baclofen (1.25-5 mg/kg, intraperitoneal [i.p.]) in DBA/2J and C57BL/6J mice, in comparison to the antipsychotic drugs haloperidol (1 mg/kg, i.p.) and clozapine (5 mg/kg, i.p.). Furthermore, we investigated the expression of GABA(B) receptors in the brain of DBA/2J and C57BL/6J mice by quantitative autoradiography. RESULTS: Baclofen dose-dependently restored PPI deficit in DBA/2J mice, in a fashion similar to the antipsychotic clozapine (5 mg/kg, i.p.). This effect was reversed by pretreatment with the GABA(B) antagonist SCH50211 (50 mg/kg, i.p.). In contrast, baclofen did not affect PPI in C57BL/6J mice. Finally, quantitative autoradiographic analyses assessed a lower GABA(B) receptor expression in DBA/2J mice in comparison to C57BL/6J controls in the prefrontal cortex and hippocampus but not in other brain regions. CONCLUSIONS: Our data highlight GABA(B) receptors as an important substrate for sensorimotor gating control in DBA/2J mice and encourage further investigations on the role of GABA(B) receptors in sensorimotor gating, as well as in the pathophysiology of psychotic disturbances.

Levetiracetam attenuates spontaneous spike-and-wave discharges in DBA/2J mice.

Recent evidence highlights levetiracetam (LEV) as an advantageous treatment of absence epilepsy (AE). Thus, we investigated the effects of this drug in DBA/2J mice, a murine model of AE. Similarly to ethosuximide (200 mg/kg, intraperitoneal, i.p.) and sodium valproate (250 mg/kg, i.p.), two classic antiabsence agents, LEV (50-200 mg/kg, i.p.) reduced the occurrence of spike-and-wave discharges, AE's typical electroencephalographic patterns. Our results confirm LEV's efficacy in AE treatment.

LTP and memory impairment caused by extracellular Aß and Tau oligomers is APP-dependent.

The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAß) and Tau (oTau) proteins impairs memory and its electrophysiological surrogate long-term potentiation (LTP), effects that may be mediated by intra-neuronal oligomers uptake. Intrigued by these findings, we investigated whether oAß and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAß, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAß and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAß and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAß- and oTau-induced defects in spatial/associative memory and LTP. Thus, APP might serve as a common therapeutic target against Alzheimer's Disease (AD) and a host of other neurodegenerative diseases characterized by abnormal levels of Aß and/or Tau.

Incorporation and metabolism of c9,t11 and t10,c12 conjugated linoleic acid (CLA) isomers in rat brain.

Conjugated linoleic acid (CLA) has been shown to exert several biological activities in different organs, in particular organs such as adipose and mammary tissue where CLA accumulates preferentially because of its high incorporation into neutral lipids. However, despite numerous studies carried out in different experimental models, both in vivo and in vitro, very little is known about the accumulation and metabolism of CLA in the brain. In this communication we present data showing that the two CLA isomers c9,t11 and t10,c12 are actively incorporated and metabolised in rat brain, and in cultures of astrocytes in vitro with patterns remarkably similar to those previously reported to occur in other tissues and cells. However, beta oxidation of CLA was found to be more efficient in brain than in other tissues, with t10,c12 a better substrate than the c9,t11 isomer. CLA incorporation and metabolism have been linked to antiinflammatory and antiproliferative activities in experimental models. Therefore, CLA activity in brain could have a positive impact on neurological disorders, such as Alzheimer's disease, Parkinson's disease and adrenoleukodystrophy, where an observed increase in inflammatory responses seems to contribute heavily to the pathogenesis.

Prenatal exposure to a cannabinoid receptor agonist does not affect sensorimotor gating in rats.

Clinical evidence suggests that prenatal exposure to cannabis may be conducive to long-term neurobehavioral impairments in executive and attentional domains. Such sensorimotor alterations might be related to disorders in gating functions. Hence, the present study was undertaken to assess the effects of long-term prenatal exposure to WIN 55,212-2, a potent cannabinoid receptor agonist, on prepulse inhibition of the acoustic startle reflex, a well-validated paradigm to test sensorimotor gating. In utero exposure to WIN 55,212-2 (0.5, 1 mg/kg, from day 5 to 20 of gestation) failed to alter startle magnitude in rats in comparison with controls. Similarly, prepulse inhibition of the startle was not significantly affected by such treatment, regardless of the age when behavioral testing was carried out (40, 60 or 80 days). Interestingly, prenatal treatment with WIN 55,212-2 (0.5 mg/kg, from day 5 to 20 of gestation) induced no differences in the prepulse inhibition-disrupting effects of apomorphine (0.125, 0.25 mg/kg, s.c.) and dizocilpine (0.05, 0.1 mg/kg, s.c.), suggesting that a prenatal exposure to a cannabinoid receptor agonist is likely unable to affect sensitivity of sensorimotor gating substrates to dopaminergic agonists and NMDA receptor antagonists. Our results show that prenatal exposure to cannabis does not affect reflex reactivity to environmental stimuli, ruling out that the observed impairments in executive functions are to refer to sensorimotor gating alterations.

Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-ß, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-ß elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.

Time-dependent reversal of synaptic plasticity induced by physiological concentrations of oligomeric Aß42: an early index of Alzheimer's disease.

The oligomeric amyloid-ß (Aß) peptide is thought to contribute to the subtle amnesic changes in Alzheimer's disease (AD) by causing synaptic dysfunction. Here, we examined the time course of synaptic changes in mouse hippocampal neurons following exposure to Aß42 at picomolar concentrations, mimicking its physiological levels in the brain. We found opposite effects of the peptide with short exposures in the range of minutes enhancing synaptic plasticity, and longer exposures lasting several hours reducing it. The plasticity reduction was concomitant with an increase in the basal frequency of spontaneous neurotransmitter release, a higher basal number of functional presynaptic release sites, and a redistribution of synaptic proteins including the vesicle-associated proteins synapsin I, synaptophysin, and the post-synaptic glutamate receptor I. These synaptic alterations were mediated by cytoskeletal changes involving actin polymerization and p38 mitogen-activated protein kinase. These in vitro findings were confirmed in vivo with short hippocampal infusions of picomolar Aß enhancing contextual memory and prolonged infusions impairing it. Our findings provide a model for initiation of synaptic dysfunction whereby exposure to physiologic levels of Aß for a prolonged period of time causes microstructural changes at the synapse which result in increased transmitter release, failure of synaptic plasticity, and memory loss.

Kappa opioid receptor activation disrupts prepulse inhibition of the acoustic startle in rats.

BACKGROUND: Compelling evidence indicates that kappa opioid receptor (KOR) agonists produce perceptual distortions in animals and humans, yet the mechanism of action and clinical relevance of such effects remain unclear. Since abnormalities in preattentional functions and informational processing are hypothesized to underlie psychotic disorders, the present study has been designed to assess the role of KOR on sensorimotor gating. METHODS: The effects of the selective KOR agonist U50488 were evaluated on the behavioral paradigm of prepulse inhibition (PPI) of the acoustic startle reflex (ASR). RESULTS: U50488 (1.25, 2.5, and 5 mg/kg, subcutaneous [SC]) induced a dose-dependent reduction of PPI, which was efficiently prevented by the selective KOR antagonist norbinaltorphimine (nor-BNI, 10 mg/kg, SC), as well as by the atypical antipsychotic clozapine (5, 8 mg/kg, intraperitoneal [IP]) but not by the typical antipsychotic haloperidol (.1, .5 mg/kg, IP). Conversely, nor-BNI (10 mg/kg, SC) failed to reverse the PPI disruption mediated by both apomorphine (.25 mg/kg, SC) and dizocilpine (.1 mg/kg, SC). CONCLUSIONS: Our results support a pivotal role of KOR in the regulation of preattentional functions and sensorimotor gating, pointing to these receptors as a possible neurobiological substrate especially relevant to the clusters of psychosis unresponsive to typical antipsychotics.

Activation of D1, but not D2 receptors potentiates dizocilpine-mediated disruption of prepulse inhibition of the startle.

Although substantial evidence has shown interactions between glutamatergic and dopaminergic systems play a cardinal role in the regulation of attentional processes, their involvement in informational filtering has been poorly investigated. Chiefly, little research has focused on functional correlations between the dopaminergic system and the mechanism of action of N-methyl-D-aspartate (NMDA) receptor antagonists on sensorimotor gating. The present study was targeted at evaluating whether the activation of D1 and D2 receptors is able to interact with the disruption of prepulse inhibition (PPI) of startle mediated by dizocilpine, a selective, noncompetitive NMDA receptor antagonist. We tested the effects of SKF 38393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol) (10 mg/kg, s.c.), a selective D1 agonist, and quinpirole (0.3, 0.6 mg/kg, s.c.), a D2 agonist, in rats, per se and in cotreatment with different doses of dizocilpine, ranging from 0.0015 to 0.15 mg/kg (s.c.). Subsequently, the effect of the D1 antagonist SCH 23390 ((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) (0.05, 0.1 mg/kg, s.c.) on PPI disruptions mediated by dizocilpine and by combination of dizocilpine and SKF 38393 was tested. Two further experiments were performed to verify whether the synergic effect of the D1 agonist with dizocilpine was counteracted by effective doses of haloperidol (0.1, 0.5 mg/kg, i.p.) and clozapine (5, 10 mg/kg, i.p.). All experiments were carried out using standard procedures for the assessment of PPI of the acoustic startle reflex. SKF 38393, while unable to impair sensorimotor gating alone, induced PPI disruption in cotreatment with 0.05 and 0.15 mg/kg of dizocilpine, both ineffective per se. Furthermore, this effect was reversed by SCH 23390, but not by haloperidol or clozapine. Conversely, no synergistic effect was exhibited between quinpirole and dizocilpine, at any given dose. These findings suggest that D1, but not D2 receptors, enhance the disruptive effect of dizocilpine on PPI.

Co-release of noradrenaline and dopamine in the cerebral cortex elicited by single train and repeated train stimulation of the locus coeruleus.

BACKGROUND: Previous studies by our group suggest that extracellular dopamine (DA) and noradrenaline (NA) may be co-released from noradrenergic nerve terminals in the cerebral cortex. We recently demonstrated that the concomitant release of DA and NA could be elicited in the cerebral cortex by electrical stimulation of the locus coeruleus (LC). This study analyses the effect of both single train and repeated electrical stimulation of LC on NA and DA release in the medial prefrontal cortex (mPFC), occipital cortex (Occ), and caudate nucleus. To rule out possible stressful effects of electrical stimulation, experiments were performed on chloral hydrate anaesthetised rats. RESULTS: Twenty min electrical stimulation of the LC, with burst type pattern of pulses, increased NA and DA both in the mPFC and in the Occ. NA in both cortices and DA in the mPFC returned to baseline within 20 min after the end of the stimulation period, while DA in the Occ reached a maximum increase during 20 min post-stimulation and remained higher than baseline values at 220 min post-stimulation. Local perfusion with tetrodotoxin (TTX, 10 microM) markedly reduced baseline NA and DA in the mPFC and Occ and totally suppressed the effect of electrical stimulation in both areas. A sequence of five 20 min stimulations at 20 min intervals were delivered to the LC. Each stimulus increased NA to the same extent and duration as the first stimulus, whereas DA remained elevated at the time next stimulus was delivered, so that baseline DA progressively increased in the mPFC and Occ to reach about 130 and 200% the initial level, respectively. In the presence of the NA transport (NAT) blocker desipramine (DMI, 100 microM), multiple LC stimulation still increased extracellular NA and DA levels. Electrical stimulation of the LC increased NA levels in the homolateral caudate nucleus, but failed to modify DA level. CONCLUSION: The results confirm and extend that LC stimulation induces a concomitant release of DA and NA in the mPFC and Occ. The different time-course of LC-induced elevation of DA and NA suggests that their co-release may be differentially controlled.