Noemí Santana

Antipsychotic drugs reverse the disruption in prefrontal cortex function produced by NMDA receptor blockade with phencyclidine

NMDA receptor (NMDA-R) antagonists are extensively used as schizophrenia models because of their ability to evoke positive and negative symptoms as well as cognitive deficits similar to those of the illness. Cognitive deficits in schizophrenia are associated with prefrontal cortex (PFC) abnormalities. These deficits are of particular interest because an early improvement in cognitive performance predicts a better long-term clinical outcome. Here, we examined the effect of the noncompetitive NMDA-R antagonist phencyclidine (PCP) on PFC function to understand the cellular and network elements involved in its schizomimetic actions. PCP induces a marked disruption of the activity of the PFC in the rat, increasing and decreasing the activity of 45% and 33% of the pyramidal neurons recorded, respectively (22% of the neurons were unaffected). Concurrently, PCP markedly reduced cortical synchrony in the delta frequency range (0.3–4 Hz) as assessed by recording local field potentials. The subsequent administration of the antipsychotic drugs haloperidol and clozapine reversed PCP effects on pyramidal cell firing and cortical synchronization. PCP increased c-fos expression in PFC pyramidal neurons, an effect prevented by the administration of clozapine. PCP also enhanced c-fos expression in the centromedial and mediodorsal (but not reticular) nuclei of the thalamus, suggesting the participation of enhanced thalamocortical excitatory inputs. These results shed light on the involvement of PFC in the schizomimetic action of NMDA-R antagonists and show that antipsychotic drugs may partly exert their therapeutic effect by normalizing a disrupted PFC activity, an effect that may add to subcortical dopamine receptor blockade.

Quantitative analysis of the expression of dopamine D1 and D2 receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex

Mesocortical dopamine (DA) is a key neurotransmitter in cognitive processes and is involved in schizophrenia and antipsychotic drug action. DA exerts a highly complex modulation of network activity in prefrontal cortex (PFC), possibly due to the recruitment of multiple signaling pathways and to specialized cellular localizations of DA receptors in cortical microcircuits. Using double in situ hybridization, we quantitatively assessed the expression of D(1) and D(2) receptor messenger RNAs (mRNAs) in pyramidal and gamma-aminobutyric acidergic (GABAergic) neurons of rat PFC. The proportion of pyramidal and GABA cells expressing these transcripts shows great regional variability in PFC, with little overlap (layer V). More pyramidal and GABA cells express D(1) than D(2) receptors. D(1) receptors are expressed by a greater proportion of GABA than pyramidal neurons, yet the number of D(1)-positive pyramidal cells outnumbers D(1)-positive interneurons due to the greater abundance of pyramidal neurons. Occasional PFC cells show high levels of mRNA, similar to those in striatal neurons. Finally, pyramidal and GABAergic cells expressing the same transcript were almost never found in close apposition, yet D(2)-containing pyramidal neurons were often found close to non-D(2) GABA neurons. Thus, cellular and network DA actions in PFC are region and layer specific and may depend on precise cellular interactions.

5-HT1A Receptor Agonists Enhance Pyramidal Cell Firing in Prefrontal Cortex Through a Preferential Action on GABA Interneurons

5-HT(1A) receptors (5-HT1AR) are expressed by pyramidal and γ-aminobutyric acidergic (GABAergic) neurons in medial prefrontal cortex (mPFC). Endogenous serotonin inhibits mPFC pyramidal neurons via 5-HT1AR while 5-HT1AR agonists, given systemically, paradoxically excite ventral tegmental area-projecting pyramidal neurons. This enhances mesocortical dopamine function, a process involved in the superior efficacy of atypical antipsychotic drugs on negative and cognitive symptoms of schizophrenia. Moreover, the 5-HT1AR-induced increase of pyramidal discharge may also contribute to the maintenance of activity patterns required for working memory, impaired in schizophrenia. Given the importance of these processes, we examined the neurobiological basis of pyramidal activation through 5-HT1AR using the prototypical agent 8-OH-DPAT. (±)8-OH-DPAT (7.5 μg/kg i.v.) increased discharge rate and c-fos expression in rat mPFC pyramidal neurons. Local blockade of GABA(A) inputs with gabazine (SR-95531) avoided (±)8-OH-DPAT-induced excitations of pyramidal neurons. Moreover, (±)8-OH-DPAT administration reduced the discharge rate of mPFC fast-spiking GABAergic interneurons at doses exciting pyramidal neurons. Activation of other 5-HT1AR subpopulations (raphe nuclei or hippocampus) does not appear to contribute to pyramidal excitations. Overall, the present data suggest a preferential action of (±)8-OH-DPAT on 5-HT1AR in GABAergic interneurons. This results in pyramidal disinhibition and subsequent downstream excitations of subcortical structures reciprocally connected with PFC, such as midbrain dopaminergic neurons.

Selective siRNA-mediated suppression of 5-HT1A autoreceptors evokes strong anti-depressant-like effects.

Depression is a major health problem worldwide. Most prescribed anti-depressants, the selective serotonin reuptake inhibitors (SSRI) show limited efficacy and delayed onset of action, partly due to the activation of somatodendritic 5-HT1A-autoreceptors by the excess extracellular serotonin (5-HT) produced by SSRI in the raphe nuclei. Likewise, 5-HT1A receptor (5-HT1AR) gene polymorphisms leading to high 5-HT1A-autoreceptor expression increase depression susceptibility and decrease treatment response. In this study, we report on a new treatment strategy based on the administration of small-interfering RNA (siRNA) to acutely suppress 5-HT1A-autoreceptor-mediated negative feedback mechanisms. We developed a conjugated siRNA (C-1A-siRNA) by covalently binding siRNA targeting 5-HT1A receptor mRNA with the SSRI sertraline in order to concentrate it in serotonin axons, rich in serotonin transporter (SERT) sites. The intracerebroventricular (i.c.v.) infusion of C-1A-siRNA to mice resulted in its selective accumulation in serotonin neurons. This evoked marked anti-depressant-like effects in the forced swim and tail suspension tests, but did not affect anxiety-like behaviors in the elevated plus-maze. In parallel, C-1A-siRNA administration markedly decreased 5-HT1A-autoreceptor expression and suppressed 8-OH-DPAT-induced hypothermia (a pre-synaptic 5-HT1AR effect in mice) without affecting post-synaptic 5-HT1AR expression in hippocampus and prefrontal cortex. Moreover, i.c.v. C-1A-siRNA infusion augmented the increase in extracellular serotonin evoked by fluoxetine in prefrontal cortex to the level seen in 5-HT1AR knockout mice. Interestingly, intranasal C-1A-siRNA administration produced the same effects, thus opening the way to the therapeutic use of C-1A-siRNA. Hence, C-1A-siRNA represents a new approach to treat mood disorders as monotherapy or in combination with SSRI.

Activation of Thalamocortical Networks by the N-methyl-D-aspartate Receptor Antagonist Phencyclidine: Reversal by Clozapine

BACKGROUND Noncompetitive N-methyl-D-aspartate receptor antagonists are widely used as pharmacological models of schizophrenia. Their neurobiological actions are still poorly understood, although the prefrontal cortex (PFC) appears as a key target area. METHODS We examined the effect of phencyclidine (PCP) on neuronal activity of the mediodorsal (MD) and centromedial (CM) thalamic nuclei, reciprocally connected with the PFC, using extracellular recordings (n = 50 neurons from 35 Wistar rats) and c-fos expression. RESULTS Phencyclidine (.25 mg/kg intravenous [IV]) markedly disorganized the activity of MD/CM neurons, increasing (424%) and decreasing (41%) the activity of 57% and 20% of the recorded neurons, respectively (23% remained unaffected). Phencyclidine reduced delta oscillations (.15-4 Hz) as assessed by recording local field potentials. The subsequent clozapine administration (1 mg/kg IV) reversed PCP effects on neuronal discharge and delta oscillations. Double in situ hybridization experiments revealed that PCP (10 mg/kg intraperitoneal [IP]) markedly increased c-fos expression in glutamatergic neurons of several cortical areas (prefrontal, somatosensory, retrosplenial, entorhinal) and in thalamic nuclei, including MD/CM. Phencyclidine also increased c-fos expression in the amygdala; yet, it had a small effect in the hippocampus. Phencyclidine did not increase c-fos expression in gamma-aminobutyric acidergic cells except in hippocampus, amygdala, somatosensory, and retrosplenial cortices. Clozapine (5 mg/kg IP) had no effect by itself but significantly prevented PCP-induced c-fos expression. CONCLUSIONS Phencyclidine likely exerts its psychotomimetic action by increasing excitatory neurotransmission in thalamo-cortico-thalamic networks involving, among others, PFC, retrosplenial, and somatosensory cortices. The antipsychotic action of clozapine includes, among other actions, an attenuation of the neuronal hyperactivity in thalamocortical networks.

Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions

RationaleIdentifying the etiological factors in anxiety and depression is critical to develop more efficacious therapies. The inhibitory serotonin1A receptors (5-HT1AR) located on 5-HT neurons (autoreceptors) limit antidepressant responses and their expression may be increased in treatment-resistant depressed patients.ObjectivesRecently, we reported that intranasal administration of modified small interference RNA (siRNA) molecules targeting 5-HT1AR in serotonergic neurons evoked antidepressant-like effects. Here we extended this finding using marketed siRNAs against 5-HT1AR (1A-siRNA) to reduce directly the 5-HT1A autoreceptor expression and evaluate its biological consequences under basal conditions and in response to stressful situations.MethodsAdult mice were locally infused with vehicle, nonsense siRNA, and 1A-siRNA into dorsal raphe nucleus (DR). 5-HT1AR knockout mice (1A-KO) were also used. Histological approaches, in vivo microdialysis, and stress-related behaviors were performed to assess the effects of 5-HT1A autoreceptor knockdown.ResultsIntra-DR 1A-siRNA infusion selectively reduced 5-HT1AR mRNA and binding levels and canceled 8-OH-DPAT-induced hypothermia. Basal extracellular 5-HT in medial prefrontal cortex (mPFC) did not differ among treatments. However, 1A-siRNA-treated mice displayed less immobility in the tail suspension and forced swim tests, as did 1A-KO mice. This was accompanied by a greater increase in prefrontal 5-HT release during tail suspension test. Moreover, intra-DR 1A-siRNA infusion augmented the increase of extracellular 5-HT in mPFC evoked by fluoxetine, up to the level in 1A-KO mice.ConclusionTogether with our previous report, the present results indicate that acute suppression of 5-HT1A autoreceptor expression evokes robust antidepressant-like effects, likely mediated by an increased capacity of serotonergic neurons to release 5-HT in stressful conditions.

Phencyclidine Inhibits the Activity of Thalamic Reticular Gamma-Aminobutyric Acidergic Neurons in Rat Brain

BACKGROUND The neurobiological basis of action of noncompetitive N-methyl-D-aspartate acid receptor (NMDA-R) antagonists is poorly understood. Electrophysiological studies indicate that phencyclidine (PCP) markedly disrupts neuronal activity with an overall excitatory effect and reduces the power of low-frequency oscillations (LFO; <4 Hz) in thalamocortical networks. Because the reticular nucleus of the thalamus (RtN) provides tonic feed-forward inhibition to the rest of the thalamic nuclei, we examined the effect of PCP on RtN activity, under the working hypothesis that NMDA-R blockade in RtN would disinhibit thalamocortical networks. METHODS Drug effects (PCP followed by clozapine) on the activity of RtN (single unit and local field potential recordings) and prefrontal cortex (PFC; electrocorticogram) in anesthetized rats were assessed. RESULTS PCP (.25-.5 mg/kg, intravenous) reduced the discharge rate of 19 of 21 RtN neurons to 37% of baseline (p < .000001) and the power of LFO in RtN and PFC to ~20% of baseline (p < .001). PCP also reduced the coherence between PFC and RtN in the LFO range. A low clozapine dose (1 mg/kg intravenous) significantly countered the effect of PCP on LFO in PFC but not in RtN and further reduced the discharge rate of RtN neurons. However, clozapine administration partly antagonized the fall in coherence and phase-locking values produced by PCP. CONCLUSIONS PCP activates thalamocortical circuits in a bottom-up manner by reducing the activity of RtN neurons, which tonically inhibit thalamic relay neurons. However, clozapine reversal of PCP effects is not driven by restoring RtN activity and may involve a cortical action.

Expression of α1-adrenergic receptors in rat prefrontal cortex: cellular co-localization with 5-HT2A receptors

The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellular expression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dorsoventral gradient, with a lower percentage of α(1)AR-positive neurons in infralimbic cortex compared to anterior cingulate and prelimbic cortex. The expression of α(1A), α(1B) and α(1D) adrenergic receptors was segregated in different layers and subdivisions. In all them there is a high co-expression with 5-HT(2A)Rs (∼80%). These observations indicate that NE controls the activity of most PFC pyramidal neurons via α(1)ARs, either directly or indirectly, via GABAergic interneurons. Antipsychotic drugs can thus modulate the activity of PFC via α(1)AR blockade. The high co-expression with 5-HT(2A)Rs indicates a convergence of excitatory serotonergic and noradrenergic inputs onto the same neuronal populations. Moreover, atypical antipsychotics may exert a more powerful control of PFC function through the simultaneous blockade of α(1)ARs and 5-HT(2A)Rs.

Noradrenergic antidepressants increase cortical dopamine: Potential use in augmentation strategies

Most antidepressant treatments, based on serotonin (5-HT) and/or norepinephrine (NE) transporter blockade, show limited efficacy and slow onset of action, requiring the use of augmentation strategies. Here we report on a novel antidepressant strategy to selectively increase DA function in prefrontal cortex (PFC) without the potential tolerance problems associated to DA transporter blockade. This approach is based on previous observations indicating that extracellular DA in rat medial PFC (mPFC) - but not in nucleus accumbens (NAc) - arises from noradrenergic terminals and is sensitive to noradrenergic drugs. A low dose of reboxetine (3 mg/kg i.p.; NE reuptake inhibitor) non-significantly increased extracellular DA in mPFC. Interestingly, its combined administration with 5 mg/kg s.c. mirtazapine (non-selective α₂-adrenoceptor antagonist) increased extracellular DA in mPFC (264 ± 28%), but not in NAc. Extracellular NE (but not 5-HT) in mPFC was also enhanced by the combined treatment (472 ± 70%). Repeated (×3) reboxetine + mirtazapine administration produced a moderate additional increase in mPFC DA and markedly reduced the immobility time (-51%) in the forced-swim test. Neurochemical and behavioral effects of the reboxetine + mirtazapine combination persisted in rats pretreated with citalopram (3 mg/kg, s.c.), suggesting its potential usefulness to augment SSRI effects. In situ hybridization c-fos studies were performed to examine the brain areas involved in the above antidepressant-like effects, showing changes in c-fos expression in hippocampal and cortical areas. BDNF expression was also increased in the hippocampal formation. Overall, these results indicate a synergistic effect of the reboxetine + mirtazapine combination to increase DA and NE function in mPFC and to evoke robust antidepressant-like responses.

NMDA antagonist and antipsychotic actions in cortico-subcortical circuits

Cognitive deficits in schizophrenia are associated with prefrontal cortex (PFC) abnormalities. Schizophrenic patients show a reduced performance in tasks engaging the PFC and a reduction of markers of cellular integrity and function. Non-competitive N-methyl-Daspartate (NMDA) receptor antagonists are widely used as pharmacological models of schizophrenia due to their ability to exacerbate schizophrenia symptoms in patients and to elicit psychotomimetic actions in healthy volunteers. Also, these drugs evoke behavioral alterations in experimental animals that resemble schizophrenia symptoms. The PFC seems to be a key target area for these agents. However, the cellular and network elements involved are poorly known. Cognitive deficits are of particular interest since an early antipsychotic-induced improvement in cognitive performance predicts a better long-term clinical outcome.Here we report that the non-competitive NMDA receptor antagonist phencyclidine (PCP) induces a marked disruption of the activity of PFC. PCP administration increased the activity of a substantial proportion of pyramidal neurons, as evidenced by an increase in discharge rate and inc- fos expression. Examination of the effects of PCP on other brain areas revealed an increasedc- fos expression in a number of cortical and subcortical areas, but notably in thalamic nuclei projecting to the PFC. The administration of classical (haloperidol) and/or atypical (clozapine) antipsychotic drugs reversed PCP effects. These results indicate that PCP induces a marked disruption of the network activity in PFC and that antipsychotic drugs may partly exert their therapeutic effect by normalizing hyperactive cortico-thalamocortical circuits.