Laura Pozzi

Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine

Caffeine has been imbibed since ancient times in tea and coffee, and more recently in colas. Caffeine owes its psychostimulant action to a blockade of adenosine A2A receptors, but little is known about its intracellular mechanism of action. Here we show that the stimulatory effect of caffeine on motor activity in mice was greatly reduced following genetic deletion of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000). Results virtually identical to those seen with caffeine were obtained with the selective A2A antagonist SCH 58261. The depressant effect of the A2A receptor agonist, CGS 21680, on motor activity was also greatly attenuated in DARPP-32 knockout mice. In support of a role for DARPP-32 in the action of caffeine, we found that, in striata of intact mice, caffeine increased the state of phosphorylation of DARPP-32 at Thr 75. Caffeine increased Thr 75 phosphorylation through inhibition of PP-2A-catalysed dephosphorylation, rather than through stimulation of cyclin-dependent kinase 5 (Cdk5)-catalysed phosphorylation, of this residue. Together, these studies demonstrate the involvement of DARPP-32 and its phosphorylation/dephosphorylation in the stimulant action of caffeine.

A Whole-Brain Atlas of Inputs to Serotonergic Neurons of the Dorsal and Median Raphe Nuclei

The serotonin system is proposed to regulate physiology and behavior and to underlie mood disorders; nevertheless, the circuitry controlling serotonergic neurons remains uncharacterized. We therefore generated a comprehensive whole-brain atlas defining the monosynaptic inputs onto forebrain-projecting serotonergic neurons of dorsal versus median raphe based on a genetically restricted transsynaptic retrograde tracing strategy. We identified discrete inputs onto serotonergic neurons from forebrain and brainstem neurons, with specific inputs from hypothalamus, cortex, basal ganglia, and midbrain, displaying a greater than anticipated complexity and diversity in cell-type-specific connectivity. We identified and functionally confirmed monosynaptic glutamatergic inputs from prefrontal cortex and lateral habenula onto serotonergic neurons as well as a direct GABAergic input from striatal projection neurons. In summary, our findings emphasize the role of hyperdirect inputs to serotonergic neurons. Cell-type-specific classification of connectivity patterns will allow for further functional analysis of the diverse but specific inputs that control serotonergic neurons during behavior.

Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum

The two mitogen‐activated protein kinases (MAPKs), extracellular signal‐regulated protein kinase 1 and 2 (ERK1/2), are involved in the control of gene expression via phosphorylation and activation of the transcription factors cyclic AMP response element binding protein (CREB) and Elk‐1. Here, we have examined the effect of haloperidol and clozapine, two anti‐psychotic drugs, and eticlopride, a selective dopamine D2 receptor antagonist, on the state of phosphorylation of ERK1/2, CREB and Elk‐1, in the mouse dorsal striatum. Administration of the typical anti‐psychotic haloperidol stimulated the phosphorylation of ERK1/2, CREB and Elk‐1. Virtually identical results were obtained using eticlopride. In contrast, the atypical anti‐psychotic clozapine reduced ERK1/2, CREB and Elk‐1 phosphorylation. This opposite regulation was specifically exerted by haloperidol and clozapine on ERK, CREB, and Elk‐1 phosphorylation, as both anti‐psychotic drugs increased the phosphorylation of the dopamine‐ and cyclic AMP‐regulated phosphoprotein of 32 kDa (DARPP‐32) at the cyclic AMP‐dependent protein kinase (PKA) site. The activation of CREB and Elk‐1 induced by haloperidol appeared to be achieved via different signalling pathways, as inhibition of ERK1/2 activation abolished the stimulation of Elk‐1 phosphorylation without affecting CREB phosphorylation. This study shows that haloperidol and clozapine induce distinct patterns of phosphorylation in the dorsal striatum. The results provide a novel biochemical paradigm elucidating the molecular mechanisms underlying the distinct therapeutic actions of typical and atypical anti‐psychotic agents.

Cannabinoid Action Depends on Phosphorylation of Dopamine- and cAMP-Regulated Phosphoprotein of 32 kDa at the Protein Kinase A Site in Striatal Projection Neurons

Herbal cannabis, smoked in the form of marihuana or hashish, is the most common illicit drug consumed in the Western world. In the brain, cannabinoids interact with neuronal CB1 receptors, thereby producing a marked reduction of motor activity. Here, we report that the motor depressant effect produced by the cannabinoid receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) is attenuated by genetic inactivation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), which is abundantly expressed in the medium spiny neurons of the striatum. Point mutation of Thr34, the protein kinase A (PKA) phosphorylation site of DARPP-32, produces a similar reduction in the effect of the CB1 agonist. In contrast, point mutation of Thr75, a site on DARPP-32 specifically phosphorylated by cyclin-dependent kinase 5, does not affect the behavioral response to CP55,940. Activation of CB1 receptors, either by an agonist or by inhibition of reuptake of endogenous cannabinoids, stimulates phosphorylation at Thr34, thereby converting DARPP-32 into an inhibitor of protein phosphatase-1. Genetic inactivation either of dopamine D2 receptors or of adenosine A2A receptors reduces the phosphorylation of DARPP-32 at Thr34 and the motor depression produced by CP55,940. Our data indicate that a considerable proportion of the psychomotor effect of cannabinoids can be accounted for by a signaling cascade in striatal projection neurons involving PKA-dependent phosphorylation of DARPP-32, achieved via modulation of dopamine D2 and adenosine A2A transmission.

Evidence that Extracellular Concentrations of Dopamine Are Regulated by Noradrenergic Neurons in the Frontal Cortex of Rats

Abstract: Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1‐[2‐[bis(4‐fluorphenyl)methoxy]‐ethyl]‐4‐(3‐phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6‐hydroxydopamine (6 µg/µl) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6‐hydroxydopamine‐treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6‐hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10−5 mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.

Stimulation of 5-hydroxytryptamine (5-HT2C) receptors in the ventrotegmental area inhibits stress-induced but not basal dopamine release in the rat prefrontal cortex

The present study investigated whether 5‐HT2C receptors in the ventrotegmental area and prefrontal cortex regulate basal and stimulus‐evoked dopamine release in the prefrontal cortex. Using the in vivo microdialysis technique in conscious rats, we studied the effect of a selective 5‐HT2C receptor agonist, Ro60–0175, on basal and immobilization stress‐induced dopamine release in the prefrontal cortex. Ro60–0175 intraperitoneally (2.5 mg/kg) and into the ventrotegmental area (10 µg/0.5 µL) completely antagonized the effect of stress on extracellular dopamine without altering basal levels. Infusion of 10 µm Ro60–0175 through the cortical probe had no significant effect on basal and stress‐induced dopamine release. SB242084 (10 mg/kg), a selective antagonist of 5‐HT2C receptors, significantly increased basal extracellular dopamine and completely prevented the effect of intraperitoneal and intraventrotegmental Ro60–0175 on the stress‐induced rise of extracellular dopamine, but had no effect itself in stressed rats. The results show that Ro60–0175 suppresses cortical dopamine release induced by immobilization stress through the stimulation of 5‐HT2C receptors in the ventrotegmental area. While confirming that endogenous 5‐HT acting on 5‐HT2C receptors tonically inhibit basal dopamine release in the prefrontal cortex, the present findings suggest that the stimulation of 5‐HT2C receptors with an exogenous agonist preferentially inhibit stimulated release.

Fluoxetine increases extracellular dopamine in the prefrontal cortex by a mechanism not dependent on serotonin: a comparison with citalopram.

Abstract : Fluoxetine at 10 and 25 mg/kg increased (167 and 205%, respectively) the extracellular dopamine concentration in the prefrontal cortex, whereas 25 (but not 10) mg/kg citalopram raised (216%) dialysate dopamine. No compound modified dialysate dopamine in the nucleus accumbens. The effect of 25 mg/kg of both compounds on cortical extracellular dopamine was not significantly affected by 300 mg/kg p‐chlorophenylalanine (PCPA) (fluoxetine, saline, 235% ; PCPA, 230% ; citalopram, saline, 179% ; PCPA, 181%). PCPA depleted tissue and dialysate serotonin by ~90 and 50%, respectively, and prevented the effect of fluoxetine and citalopram on dialysate serotonin (fluoxetine, saline, 246% ; PCPA, 110% ; citalopram, saline, 155% ; PCPA, 96%). Citalopram significantly raised extracellular serotonin from 0.1 to 100 μM (251‐520%), whereas only 10 and 100 μM increased dialysate dopamine (143‐231%). Fluoxetine similarly increased extracellular serotonin (98‐336%) and dopamine (117‐318%). PCPA significnatly reduced basal serotonin and the effects of 100 μM fluoxetine (saline, 272% ; PCPA, 203%) and citalopram (saline, 345% ; PCPA 258%) on dialysate serotonin but did not modify their effect on dopamine (fluoxetine, saline, 220% ; PCPA, 202% ; citalopram, saline, 191% ; PCPA, 211%). The results clearly show that the effects of fluoxetine and of high concentrations of citalopram on extracellular dopamine do not depend on their effects on serotonin.

Regulation of striatal tyrosine hydroxylase phosphorylation by acute and chronic haloperidol.

The typical neuroleptic haloperidol increases the state of phosphorylation and activity of tyrosine hydroxylase (TH), the rate‐limiting enzyme in the synthesis of catecholamines. Here we show that the increases in TH phosphorylation produced by haloperidol at Ser31 and Ser40, two sites critically involved in the regulation of enzymatic activity, are abolished in dopamine D2 receptor‐null mice and mimicked by the selective dopamine D2 receptor antagonist, eticlopride. Moreover, the ability of haloperidol and eticlopride to stimulate phosphorylation at both seryl residues is prevented by treatment with SL327, a compound that blocks activation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2). We also show that chronic administration of haloperidol reduces the basal levels of phosphoSer31‐TH and decreases the ability of the drug to stimulate Ser40 phosphorylation. These results provide a model accounting for the stimulation exerted by haloperidol on dopamine synthesis. According to this model, haloperidol increases TH activity via blockade of dopamine D2 receptors, disinhibition of dopaminergic projection neurons and ERK1/2‐dependent phosphorylation of TH at Ser31 and Ser40. These studies also show that lower levels of phosphorylated TH are associated with chronic neuroleptic treatment and may be related to depressed dopaminergic transmission in nigrostriatal neurons.

Mice lacking NMDA receptors in parvalbumin neurons display normal depression-related behavior and response to antidepressant action of NMDAR antagonists.

The underlying circuit imbalance in major depression remains unknown and current therapies remain inadequate for a large group of patients. Discovery of the rapid antidepressant effects of ketamine - an NMDA receptor (NMDAR) antagonist – has linked the glutamatergic system to depression. Interestingly, dysfunction in the inhibitory GABAergic system has also been proposed to underlie depression and deficits linked to GABAergic neurons have been found with human imaging and in post-mortem material from depressed patients. Parvalbumin-expressing (PV) GABAergic interneurons regulate local circuit function through perisomatic inhibition and their activity is NMDAR-dependent, providing a possible link between NMDAR and the inhibitory system in the antidepressant effect of ketamine. We have therefore investigated the role of the NMDAR-dependent activity of PV interneurons for the development of depression-like behavior as well as for the response to rapid antidepressant effects of NMDAR antagonists. We used mutant mice lacking NMDA neurotransmission specifically in PV neurons (PV-Cre+/NR1f/f) and analyzed depression-like behavior and anhedonia. To study the acute and sustained effects of a single NMDAR antagonist administration, we established a behavioral paradigm of repeated exposure to forced swimming test (FST). We did not observe altered behavioral responses in the repeated FST or in a sucrose preference test in mutant mice. In addition, the behavioral response to administration of NMDAR antagonists was not significantly altered in mutant PV-Cre+/NR1f/f mice. Our results show that NMDA-dependent neurotransmission in PV neurons is not necessary to regulate depression-like behaviors, and in addition that NMDARs on PV neurons are not a direct target for the NMDAR-induced antidepressant effects of ketamine and MK801.