Protein kinase C regulation of dopamine transporter initiated by nicotinic receptor activation in slices of rat prefrontal cortex

We previously reported that activation of nicotinic receptors causes an enhancement in amphetamine-stimulated release of dopamine via its transporter from slices of prefrontal cortex, but no such enhancement of release from slices of nucleus accumbens or striatum. The nicotinic receptors mediating the enhancement most likely contain alpha4 and beta2 subunits based upon pharmacological characterization. In this study, we sought to characterize the second messenger systems associated with the nicotine-mediated response. Sodium channel involvement was confirmed by the observation that tetrodotoxin blocked nicotine-mediated enhancement, whereas veratridine or elevated K(+) mimicked the enhancement seen with nicotine. Inclusion of EGTA blocked nicotine-mediated enhancement, suggesting that, even though no exogenous Ca(2+) was added, endogenous stores were required for the enhancement. The enhancement by nicotine was also abolished by the L-type voltage-dependent calcium channel (VDCC) antagonist nitrendipine, but not by the N-type VDCC antagonist omega-conotoxin GVIA. Finally, inhibition of protein kinase C also abolished the nicotine-mediated enhancement of amphetamine-stimulated dopamine release, whereas inhibitors of Ca(2+)/calmodulin kinase II did not. These findings establish that nicotine can exert selective effects on dopamine transporter activity in prefrontal cortex, an area involved in cognition and learning.     

Hidden prenatal malnutrition in the rat: role of β₁-adrenoceptors on synaptic plasticity in the frontal cortex

Moderate reduction in the protein content of the mother's diet (hidden malnutrition) does not alter body and brain weights of rat pups at birth, but leads to dysfunction of neocortical noradrenaline systems together with impaired long-term potentiation and visuo-spatial memory performance. As β?-adrenoceptors and downstream protein kinase signaling are critically involved in synaptic long-term potentiation and memory formation, we evaluated the β?-adrenoceptor density and the expression of cyclic-AMP dependent protein kinase, calcium/calmodulin-dependent protein kinase and protein kinase Fyn, in the frontal cortex of prenatally malnourished adult rats. In addition, we also studied if β?-adrenoceptor activation with the selective β? agonist dobutamine could improve deficits of prefrontal cortex long-term potentiation presenting these animals. Prenatally malnourished rats exhibited half of β?-adrenoceptor binding, together with a 51% and 65% reduction of cyclic AMP-dependent protein kinase α and calcium/calmodulin-dependent protein kinase α expression, respectively, as compared with eutrophic animals. Administration of the selective β? agonist dobutamine prior to tetanization completely rescued the ability of the prefrontal cortex to develop and maintain long-term potentiation in the malnourished rats. Results suggest that under-expression of neocortical β?-adrenoceptors and protein kinase signaling in hidden malnourished rats functionally affects the synaptic networks subserving prefrontal cortex long-term potentiation. β?-adrenoceptor activation was sufficient to fully recover neocortical plasticity in the PKA- and calcium/calmodulin-dependent protein kinase II-deficient undernourished rats, possibly by producing extra amounts of cAMP and/or by recruiting alternative signaling cascades.     

Memory trace in prefrontal cortex: theory for the cognitive switch

The dorsolateral prefrontal cortex in human and non-human primates functions as the highest-order executor for the perception-action cycle. According to this view, when perceptual stimuli from the environment are novel or complex, the dorsolateral prefrontal cortex serves to set consciously a goal-directed scheme which broadly determines an action repertory to meet the particular demand from the environment. In this respect, the dorsolateral prefrontal cortex is a short-term activation device with the properties of a cognitive switch', because it couples a particular set of perceptual stimuli to a particular set of actions. Here, I suggest that, in order for the organism to react systematically to the environment, neural traces for the switch function must be stored in the brain. Thus, the highest-order, perception-action interface function of the dorsolateral prefrontal cortex per se depends on permanently stored neural traces in the dorsolateral prefrontal cortex and related structures. Such a memory system may be located functionally between two of the well-documented memory systems in the brain: the declarative memory system and the procedural memory system. Finally, based on available neurophysiological data, the possible mechanisms underlying the formation of cognitive switch traces are proposed.     

Time-dependent postsynaptic AMPA GluR1 receptor recruitment in the cingulate synaptic potentiation

The anterior cingulate cortex (ACC) is critical for brain functions including learning, memory, fear and pain. Long-term synaptic potentiation (LTP), a cellular model for learning and memory, has been reported in the ACC neurons. Unlike LTP in the hippocampus and amygdala, two key structures for memory and fear, little is known about the synaptic mechanism for the expression of LTP in the ACC. Here we use whole-cell patch clamp recordings to demonstrate that cingulate LTP requires the functional recruitment of GluR1 AMPA receptors; and such events are rapid and completed within 5-10 min after LTP induction. Our results demonstrate that the GluR1 subunit is essential for synaptic plasticity in the ACC and may play critical roles under physiological and pathological conditions.     

Low levels of methyl β‐cyclodextrin disrupt GluA1‐dependent synaptic potentiation but not synaptic depression

Methyl‐β‐cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol‐enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N‐methyl‐d ‐aspartate receptor (NMDA‐R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post‐synaptic current without changing NMDA‐R‐mediated synaptic transmission and the paired‐pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R) and NMDA‐R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2‐lacking AMPA‐R. MβCD successfully decreased NMDA‐R‐mediated long‐term potentiation but did not affect the formation of either NMDA‐R‐mediated or group I metabotropic glutamate receptor‐dependent long‐term depression. MβCD inhibited de‐depression without affecting de‐potentiation. These results suggest that MβCD regulates GluA1‐dependent synaptic potentiation but not synaptic depression in a cholesterol‐independent manner.

     

Protective effects of omega-3 essential fatty acids against formaldehyde-induced neuronal damage in prefrontal cortex of rats

The aim of this study was to examine the neurotoxicity of formaldehyde on prefrontal cortex and the protective effects of omega-3 essential fatty acids against these toxic effects. For this purpose, 21 male Wistar rats were divided into three groups. The rats in group I comprised the controls, while the rats in group II were injected every other day with formaldehyde (FA). The rats in group III received omega-3 fatty acids daily while exposed to formaldehyde. At the end of the 14-day experimental period, all rats were killed by decapitation. The brains of the rats were removed and the prefrontal cortex tissues were obtained from all brain specimens. Some of the prefrontal cortex tissue specimens were used for determination of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels. The remaining prefrontal cortex tissue specimens were used for light microscopic and immunohistochemical evaluation. The levels of SOD and GSH-Px were significantly decreased, and MDA levels were significantly increased in rats treated with formaldehyde compared with those of the controls. Furthermore, in the microscopic examination of this group, formation of apoptotic bodies, pycnotic cells, and apoptotic cells including nuclear fragmentation and membrane budding were observed. However, increased SOD and GSH-Px enzyme activities, and decreased MDA levels were detected in the rats administered omega-3 fatty acids while exposed to formaldehyde. Additionally, cellular damage caused by formaldehyde was decreased, and structural appearance was similar to that of the control rats in this group. The biochemical and histological findings observed in all groups were also confirmed by immunohistochemical evaluation. It was determined that formaldehyde-induced neuronal damage in prefrontal cortex was prevented by administration of omega-3 essential fatty acids.     

Inhibition of stress- or anxiogenic-drug-induced increases in dopamine release in the rat prefrontal cortex by long-term treatment with antidepressant drugs

The effects of long-term treatment with imipramine or mirtazapine, two antidepressant drugs with different mechanisms of action, on the response of cortical dopaminergic neurons to foot-shock stress or to the anxiogenic drug FG7142 were evaluated in freely moving rats. As expected, foot shock induced a marked increase (+ 90%) in the extracellular concentration of dopamine in the prefrontal cortex of control rats. Chronic treatment with imipramine or mirtazapine inhibited or prevented, respectively, the effect of foot-shock stress on cortical dopamine output. Whereas acute administration of the anxiogenic drug FG7142 induced a significant increase (+ 60%) in cortical dopamine output in control rats, chronic treatment with imipramine or mirtazapine completely inhibited this effect. In contrast, the administration of a single dose of either antidepressant 40 min before foot shock, had no effect on the response of the cortical dopaminergic innervation to stress. These results show that long-term treatment with imipramine or mirtazapine inhibits the neurochemical changes elicited by stress or an anxiogenic drug with an efficacy similar to that of acute treatment with benzodiazepines. Given that episodes of anxiety or depression are often preceded by stressful events, modulation by antidepressants of the dopaminergic response to stress might be related to the anxiolytic and antidepressant effects of these drugs.     

Improved spatial learning is associated with increased hippocampal but not prefrontal long‐term potentiation in mGluR4 knockout mice

Although much information about metabotropic glutamate receptors (mGluRs) and their role in normal and pathologic brain function has been accumulated during the last decades, the role of group III mGluRs is still scarcely documented. Here, we examined mGluR4 knockout mice for types of behavior and synaptic plasticity that depend on either the hippocampus or the prefrontal cortex (PFC). We found improved spatial short‐ and long‐term memory in the radial arm maze, which was accompanied by enhanced long‐term potentiation (LTP) in hippocampal CA1 region. In contrast, LTP in the PFC was unchanged when compared with wild‐type controls. Changes in paired‐pulse facilitation that became overt in the presence of the GABA_A antagonist picrotoxin indicated a function of mGluR4 in maintaining the excitation/inhibition balance, which is of crucial importance for information processing in the brain and the deterioration of these processes in neuropsychological disorders such as autism, epilepsy and schizophrenia .     

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors

β-Endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (K_d=31.6±0.2 nM, B_max=37.4±2.2 pmol/mg protein). Immunorphin at concentrations of 10⁻⁹ to 10⁻⁶ M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10–100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Amphetamine‐induced release of dopamine in primate prefrontal cortex and striatum: striking differences in magnitude and timecourse

The psychostimulant amphetamine (AMPH) is frequently used to increase catecholamine levels in attention disorders and positron emission tomography imaging studies. Despite the fact that most radiotracers for positron emission tomography studies are characterized in non‐human primates (NHPs), data on regional differences of the effect of AMPH in NHPs are very limited. This study examined the impact of AMPH on extracellular dopamine (DA) levels in the medial prefrontal cortex and the caudate of NHPs using microdialysis. In addition to differences in magnitude, we observed striking differences in the temporal profile of extracellular DA levels between these regions that can likely be attributed to differences in the regulation of dopamine uptake and biosynthesis. The present data suggest that cortical DA levels may remain elevated longer than in the caudate which may contribute to the clinical profile of the actions of AMPH.

     

Acute ischemic cardiac dysfunction is attenuated via gene transfer of a peptide inhibitor of the beta-adrenergic receptor kinase (betaARK1)

Acute myocardial ischemia is a critical adverse effect potentially occurring during cardiac procedures. A peptide inhibitor of the beta-adrenergic receptor kinase (betaARK1), betaARKct, has been successful in rescuing chronic myocardial ischemia. The present study focused on the effects of adenoviral-mediated betaARKct (Adv-betaARKct) delivery on left ventricle (LV) dysfunction induced by acute coronary occlusion. Rabbits received intracoronary delivery of phosphate-buffered saline (PBS) (n=9) or 5x10(11) viral particles of betaARKct (n=8). A loose prolene 5-0 Potz-loop suture was placed around the circumflex coronary artery (LCx) with both ends buried under the skin. Four days later, the suture was retrieved and pulled to occlude the LCx. Ischemia was confirmed by immediate ECG changes. LV function was continuously recorded for 45 min. Contractility (LVdP/dtmax), relaxation (LVdP/dtmin) and end diastolic pressure (EDP) were less impaired in the betaARKct group as compared to PBS (P<0.05, two-way ANOVA). betaAR density was higher in the ischemic area of the LV in the betaARKct group (betaARKct: 71.9+/-4.6 fmol/mg protein, PBS: 54.5+/-4.0 fmol/mg protein, P<0.05). Adenylyl cyclase activity was also improved basally and in response to betaAR stimulation. betaARK1 activation was less in the betaARKct group (P<0.05). Therefore, inhibition of myocardial betaARK1 may represent a new strategy to prevent LV dysfunction induced by acute coronary ischemia.     

Chelation of hippocampal zinc enhances long‐term potentiation and synaptic tagging/capture in CA1 pyramidal neurons of aged rats: implications to aging and memory

Aging is associated with decline in cognitive functions, prominently in the memory consolidation and association capabilities. Hippocampus plays a crucial role in the formation and maintenance of long‐term associative memories, and a significant body of evidence shows that impairments in hippocampal function correlate with aging‐related memory loss. A number of studies have implicated alterations in hippocampal synaptic plasticity, such as long‐term potentiation (LTP), in age‐related cognitive decline although exact mechanisms underlying are not completely clear. Zinc deficiency and the resultant adverse effects on cognition have been well studied. However, the role of excess of zinc in synaptic plasticity, especially in aging, is not addressed well. Here, we have investigated the hippocampal zinc levels and the impairments in synaptic plasticity, such as LTP and synaptic tagging and capture (STC), in the CA1 region of acute hippocampal slices from 82‐ to 84‐week‐old male Wistar rats. We report increased zinc levels in the hippocampus of aged rats and also deficits in the tetani‐induced and dopaminergic agonist‐induced late‐LTP and STC. The observed deficits in synaptic plasticity were restored upon chelation of zinc using a cell‐permeable chelator. These data suggest that functional plasticity and associativity can be successfully established in aged neural networks by chelating zinc with cell‐permeable chelating agents.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Differential Interactions of Desipramine with Amphetamine and Methamphetamine: Evidence that Amphetamine Releases Dopamine from Noradrenergic Neurons in the Medial Prefrontal Cortex

Amphetamine is more effective than methamphetamine at raising dopamine levels in the prefrontal cortex. The current study tested the hypothesis that norepinephrine transporters are involved in this difference. Using microdialysis, dopamine, norepinephrine, and serotonin were measured in the rat prefrontal cortex after administration of methamphetamine or amphetamine, with and without perfusion of desipramine. Amphetamine raised norepinephrine levels more than methamphetamine did. Desipramine raised dopamine and serotonin levels but did not alter metabolite levels. Desipramine attenuated the increase in dopamine by amphetamine while increasing the dopamine released by methamphetamine. These data suggest that methamphetamine and amphetamine differ in altering prefrontal cortical dopamine levels and in interacting with norepinephrine transporters. It is proposed that amphetamine releases dopamine in the prefrontal cortex primarily through norepinephrine transporters, whereas methamphetamine interacts minimally with norepinephrine transporters.     

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.     

Rapid microelectrode measurements and the origin and regulation of extracellular glutamate in rat prefrontal cortex

Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal versus astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (sodium channel blocker), produced a significant (～ 40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally applied ω-conotoxin (MVIIC; ～ 50%; calcium channel blocker), and the mGluR(2/3) agonist, LY379268 (～ 20%), and a significant increase with the mGluR(2/3) antagonist LY341495 (～ 40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (glutamate transporter inhibitor) produced an ～ 120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of tetrodotoxin completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the microelectrode array technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically -evoked event is entirely neuronally derived.     

Reducing the harm of stress: medications to rescue the prefrontal cortex and overcome bad habits: the science of stress: focus on the brain, breaking bad habits, and chronic disease

Our brain is sensitive to stress. Both acute and chronic stress cause cognitive deficits and induce chronic disorders such as drug addiction. In a June 2011 conference at Yale entitled "The Science of Stress: Focus on the Brain, Breaking Bad Habits, and Chronic Disease," Drs. Amy Arnsten and Sherry Mckee discussed the roles of prefrontal cortex in the treatment of stress impairments and addiction. Medications to strengthen the prefrontal function, such as prazosin and guanfacine, may reduce the harm of stress and help overcome smoking and alcohol abuse.     

In vivo evidence that constitutive activity of serotonin2C receptors in the medial prefrontal cortex participates in the control of dopamine release in the rat nucleus accumbens: differential effects of inverse agonist versus antagonist

Control of the mesoaccumbens dopamine (DA) pathway by central serotonin_2C receptors (5-HT_2CRs) involves different 5-HT_2CR populations located within multiple brain areas. Here, using in vivo microdialysis in halothane-anesthetized rats, we assessed the role of medial prefrontal cortex (mPFC) 5-HT_2CRs in the control of basal and activated accumbal DA outflow, to identify the modalities of their recruitment and the role of 5-HT_2CR constitutive activity. Intra-mPFC injection of the 5-HT_2CR inverse agonist SB 206553 (0.5 μg/0.2 μL), without effect by itself, decreased accumbal DA outflow induced by morphine (2.5–10 mg/kg, s.c.), haloperidol (0.01 mg/kg, s.c.) or GBR 12909 (2.5 mg/kg, i.p.). Conversely, intra-mPFC injection of the 5-HT_2CR antagonist SB 242084 (0.5 μg/0.2 μL), without effect by itself, decreased the effect of 10 mg/kg morphine, the only drug enhancing basal 5-HT outflow in the mPFC. The inhibitory effect of SB 206553 on 2.5 mg/kg morphine-stimulated DA outflow was suppressed by the concomitant intra-mPFC injection of SB 242084. Finally, changes of basal DA outflow induced by the 5-HT_2CR agonist Ro 60-0175 (3 mg/kg, i.p.) or SB 206553 (5 mg/kg, i.p.) were unaffected by intra-mPFC injection of SB 242084. These results, showing that 5-HT_2CR antagonist and inverse agonist behave differently in vivo, demonstrate that mPFC 5-HT_2CRs facilitate activated accumbal DA outflow and that 5-HT_2CR constitutive activity participates in this interaction.