Mihály Hajós

A Novel Positive Allosteric Modulator of the α7 Neuronal Nicotinic Acetylcholine Receptor: In Vitro and In Vivo Characterization

Several lines of evidence suggest a link between the α7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimers disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the α7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human α7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by α4β2, α3β4, and α9α10 nAChRs. PNU-120596 increased the channel mean open time of α7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of α7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances α7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Interaction between a selective 5-HT1A receptor antagonist and an SSRI in vivo: effects on 5-HT cell firing and extracellular 5-HT.

1 The acute inhibitory effect of selective 5‐hydroxytryptamine (serotonin) reuptake inhibitors (SSRIs) on 5‐HT neuronal activity may offset their ability to increase synaptic 5‐HT in the forebrain. 2Here, we determined the effects of the SSRI, paroxetine, and a novel selective 5‐HT1A receptor antagonist, WAY 100635, on 5‐HT cell firing in the dorsal raphe nucleus (DRN), and on extracellular 5‐HT in both the DRN and the frontal cortex (FCx). Extracellular electrophysiological recording and brain microdialysis were used in parallel experiments, in anaesthetized rats. 3 Paroxetine dose‐dependently inhibited the firing of 5‐HT neurones in the DRN, with a maximally effective dose of approximately 0.8 mg kg−1, i.v. WAY 100635 (0.1 mg kg−1, i.v.) both reversed the inhibitory effect of paroxetine and, when used as a pretreatment, caused a pronounced shift to the right of the paroxetine dose‐response curve. 4 Paroxetine (0.8 mg kg−1, i.v.), doubled extracellular 5‐HT in the DRN, but did not alter extracellular 5‐HT in the FCx. A higher dose of paroxetine (2.4 mg kg−1, i.v.) did increase extracellular 5‐HT in the FCx, but to a lesser extent than in the DRN. Whereas 0.8 mg kg−1, i.v. paroxetine alone had no effect on extracellular 5‐HT in the FCx, in rats pretreated with WAY 100635 (0.1 mg kg−1), paroxetine (0.8 mg kg−1, i.v.) markedly increased extracellular 5‐HT in the FCx. 5 In conclusion, pretreatment with the selective 5‐HT1A receptor antagonist, WAY 100635, blocked the inhibitory effect of paroxetine on 5‐HT neuronal activity in the DRN and, at the same time, markedly enhanced the effect of paroxetine on extracellular 5‐HT in the FCx. These results may be relevant to recent clinical observations that 5‐HT1A receptor antagonists in combination with SSRIs have a rapid onset of antidepressant effect.

An electrophysiological and neuroanatomical study of the medial prefrontal cortical projection to the midbrain raphe nuclei in the rat.

In this study we utilized electrophysiological and pathway tracing methods to investigate the projections from the medial prefrontal cortex to the midbrain raphe nuclei of the rat. Initial pathway tracing experiments using retrograde (horseradish peroxidase conjugates with wheatgerm agglutinin or choleratoxin B subunit) and anterograde (Phaseolus vulgaris-leucoagglutinin) markers demonstrated a direct, bilateral projection to the dorsal raphe nucleus and median raphe nucleus from the medial prefrontal cortex, and the origin of this projection was localized predominantly in the ventral medial prefrontal cortex (infralimbic/dorsal penduncular cortices). Using chloral hydrate-anaesthetized rats, extracellular recordings were made mostly from 5-hydroxytryptamine neurons in the dorsal raphe nucleus, but non-5-hydroxytryptamine dorsal raphe neurons were also studied, as was a small number of 5-hydroxytryptamine neurons in the median raphe nucleus. In an initial study, electrical stimulation of the ventral medial prefrontal cortex caused a post-stimulus inhibition in the majority (49/56) of dorsal raphe 5-hydroxytryptamine neurons tested (mean duration of inhibition, 200+/-17 ms); in some cases (8/56) the inhibition was preceded by short-latency (26 +/-3 ms) orthodromic activation, and a small number of cells was antidromically activated (6/56). Both single spiking and burst-firing 5-hydroxytryptamine neurons in the dorsal raphe nucleus responded in the same way, and median raphe 5-hydroxytryptamine neurons were also inhibited (5/5). In contrast, few (2/12) of the non-5-hydroxytryptamine dorsal raphe neurons tested were inhibited by ventral medial prefrontal cortex stimulation. The effects of stimulation of the dorsal and ventral medial prefrontal cortex were compared on the same raphe 5-hydroxytryptamine neurons (n=17): ventral medial prefrontal cortex stimulation inhibited 16/17 of these neurons while only 8/17 were inhibited by dorsal medial prefrontal cortex stimulation. Finally, the inhibitory effect of ventral medial prefrontal cortex stimulation on 5-hydroxytryptamine cell-firing was not altered by 5-hydroxytryptamine depletion with p-chlorophenylalanine or by systemic administration of the selective 5-hydroxytryptamine1A receptor antagonist WAY 100635. The latter findings indicate that the inhibition is not due to release of raphe 5-hydroxytryptamine which could theoretically arise from anti- or orthodromically activated 5-hydroxytryptamine neurons. Our results show that stimulation of the ventral medial prefrontal cortex causes a marked post-stimulus inhibition in the vast majority of midbrain raphe 5-hydroxytryptamine neurons tested. It seems likely that the projection from ventral medial prefrontal cortex to the midbrain raphe nuclei mediates the responses of 5-hydroxytryptamine neurons to cortical stimulation. These data are relevant to recent discoveries of functional and structural abnormalities in the medial prefrontal cortex of patients with major depressive illness.

Preclinical Characterization of Selective Phosphodiesterase 10A Inhibitors: A New Therapeutic Approach to the Treatment of Schizophrenia

We have recently proposed the hypothesis that inhibition of the cyclic nucleotide phosphodiesterase (PDE) 10A may represent a new pharmacological approach to the treatment of schizophrenia (Curr Opin Invest Drug 8:54–59, 2007). PDE10A is highly expressed in the medium spiny neurons of the mammalian striatum (Brain Res 985:113–126, 2003; J Histochem Cytochem 54:1205–1213, 2006; Neuroscience 139:597–607, 2006), where the enzyme is hypothesized to regulate both cAMP and cGMP signaling cascades to impact early signal processing in the corticostriatothalamic circuit (Neuropharmacology 51:374–385, 2006; Neuropharmacology 51:386–396, 2006). Our current understanding of the physiological role of PDE10A and the therapeutic utility of PDE10A inhibitors derives in part from studies with papaverine, the only pharmacological tool for this target extensively profiled to date. However, this agent has significant limitations in this regard, namely, relatively poor potency and selectivity and a very short exposure half-life after systemic administration. In the present report, we describe the discovery of a new class of PDE10A inhibitors exemplified by TP-10 (2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid), an agent with greatly improved potency, selectivity, and pharmaceutical properties. These new pharmacological tools enabled studies that provide further evidence that inhibition of PDE10A represents an important new target for the treatment of schizophrenia and related disorders of basal ganglia function.

Neurophysiological biomarkers for drug development in schizophrenia

Schizophrenia represents a pervasive deficit in brain function, leading to hallucinations and delusions, social withdrawal and a decline in cognitive performance. As the underlying genetic and neuronal abnormalities in schizophrenia are largely unknown, it is challenging to measure the severity of its symptoms objectively, or to design and evaluate psychotherapeutic interventions. Recent advances in neurophysiological techniques provide new opportunities to measure abnormal brain functions in patients with schizophrenia and to compare these with drug-induced alterations. Moreover, many of these neurophysiological processes are phylogenetically conserved and can be modelled in preclinical studies, offering unique opportunities for use as translational biomarkers in schizophrenia drug discovery.

Role of the medial prefrontal cortex in 5‐HT1A receptor‐induced inhibition of 5‐HT neuronal activity in the rat

We examined the involvement of the frontal cortex in the 5‐HT1A receptor‐induced inhibition of 5‐HT neurones in the dorsal raphe nucleus (DRN) of the anaesthetized rat using single‐unit recordings complemented by Fos‐immunocytochemistry. Both transection of the frontal cortex as well as ablation of the medial region of the prefrontal cortex (mPFC) significantly attenuated the inhibition of 5‐HT neurones induced by systemic administration of the 5‐HT1A receptor agonist, 8‐OH‐DPAT (0.5–16 μg kg−1, i.v.). In comparison, the response to 8‐OH‐DPAT was not altered by ablation of the parietal cortex. The inhibitory effect of 8‐OH‐DPAT was reversed by the 5‐HT1A receptor antagonist, WAY 100635 (0.1 mg kg−1, i.v.) in all neurones tested. In contrast, cortical transection did not alter the sensitivity of 5‐HT neurones to iontophoretic application of 8‐OH‐DPAT into the DRN. Similarly, cortical transection did not alter the sensitivity of 5‐HT neurones to systemic administration of the selective 5‐HT reuptake inhibitor, paroxetine (0.1–0.8 mg kg−1, i.v.). 8‐OH‐DPAT evoked excitation of mPFC neurones at doses (0.5–32 μg kg−1, i.v.) in the range of those which inhibited 5‐HT cell firing. At higher doses (32–512 μg kg−1, i.v.) 8‐OH‐DPAT inhibited mPFC neurones. 8‐OH‐DPAT (0.1 mg kg−1, s.c.) also induced Fos expression in the mPFC. The neuronal excitation and inhibition, as well as the Fos expression, were antagonized by WAY 100635. These data add further support to the view that the inhibitory effect of 5‐HT1A receptor agonists on the firing activity of DRN 5‐HT neurones involves, in part, activation of a 5‐HT1A receptor‐mediated postsynaptic feedback loop centred on the mPFC.

Evidence for a role of GABA interneurones in the cortical modulation of midbrain 5-hydroxytryptamine neurones

Recent electrophysiological studies demonstrate that the ventral medial prefrontal cortex has a powerful inhibitory influence on 5-hydroxytryptamine (5-HT) neurones in the dorsal raphe nucleus. Here we utilised a combination of anatomical and electrophysiological methods to characterise the cellular substrate underlying this effect.Anterograde tracing (Phaseolus vulgaris leucoagglutinin) using electron microscopy demonstrated a pathway from the ventral medial prefrontal cortex that makes neuronal contacts throughout the dorsal raphe nucleus. These contacts were predominantly asymmetrical synapses adjoining GABA immunoreactive dendrites and spines. In vivo extracellular recordings were made in the dorsal raphe nucleus of the anaesthetised rat from a subpopulation of non-5-HT neurones. These neurones were fast-firing, irregular and with short spike width, properties strongly reminiscent of immunochemically identified GABA interneurones in other brain regions. Recordings of classical 5-HT neurones were also included. Electrical stimulation of the ventral medial prefrontal cortex elicited a rapid onset (16 ms latency), orthodromic excitation of the non-5-HT neurones (13/25 neurones). This stimulation also caused a pronounced inhibition of most 5-HT neurones tested, with a longer latency (30 ms), and this was partially blocked by locally applied bicuculline. These data provide the first evidence that the ventral medial prefrontal cortex influences the activity of large numbers of raphe 5-HT neurones by targeting a local network of GABA neurones. This circuitry predicts that physiological and pathological changes in the ventral medial prefrontal cortex will impact on significant parts of the forebrain 5-HT system.

Activation of Cannabinoid-1 Receptors Disrupts Sensory Gating and Neuronal Oscillation: Relevance to Schizophrenia

BACKGROUND Impaired auditory gating and abnormal neuronal synchrony are indicators of dysfunctional information processing in schizophrenia patients and possible underlying mechanisms of their impaired sensory and cognitive functions. Because cannabinoid receptors and endocannabinoids have been linked to psychiatric disorders, including schizophrenia, the aim of this study was to evaluate the effects of cannabinoid-1 (CB1) receptor activation on sensory gating and neuronal oscillations in rats. METHODS Auditory sensory gating has been recorded from the hippocampus and entorhinal cortex (EC) in anesthetized rats. Neuronal network oscillations were recorded from the hippocampus, medial septum, EC, and medial prefrontal cortex in anesthetized and freely moving rats. Effects of systemic administration of CB1 receptor agonist CP-55940 were evaluated on these parameters. RESULTS CP-55940 significantly disrupted auditory gating both in the hippocampus and EC in anesthetized rats. Theta field potential oscillations were disrupted in the hippocampus and EC, with simultaneous interruption of theta-band oscillations of septal neurons. Administration of the CB1 receptor antagonist AM-251 reversed both the agonist-induced gating deficit and the diminished oscillations. In freely moving rats, CP-55940 significantly reduced theta and gamma power in the hippocampus, whereas in the EC, only gamma power was attenuated. However, novelty-induced theta and gamma activities were significantly diminished by CP-55940 in both the hippocampus and EC. CONCLUSIONS Our data indicate that activation of CB1 receptors interferes with neuronal network oscillations and impairs sensory gating function in the limbic circuitry, further supporting the connection between cannabis abuse and increased susceptibility of developing schizophrenia spectrum disorders.

Inhibition of median and dorsal raphe neurones following administration of the selective serotonin reuptake inhibitor paroxetine

Acute systemic injection of selective serotonin reuptake inhibitors (SSRIs) decreases 5-HT neuronal firing in the dorsal raphe nucleus (DRN). Recent data, however, question whether these drugs also inhibit the firing of 5-HT neurones in the median raphe nucleus (MRN). Using in vivo extracellular electrophysiological recording techniques in the chloral hydrate anaesthetised rat, we have tested the effect of acute administration of the SSRI, paroxetine, on 5-HT neuronal activity in the MRN and DRN. Presumed 5-HT neurones in the MRN displayed the same electrophysiological characteristics as those in the DRN, the only detectable difference being that MRN neurones showed a significantly (p < 0.001) slower mean ( ± SEM(n)) spontaneous firing rate (MRN, 5.6 ± 0.9 (14) spikes/10 s; DRN, 13.5 ± 1.6 (24) spikes/10 s). Paroxetine caused a dose-related (0.1–0.8 mg/kg i.v.) inhibition of all MRN neurones tested (n = 8), producing a complete cessation of cell-firing at the highest doses. DRN neurones (n = 9) responded in a similar fashion. Furthermore, paroxetine inhibited MRN and DRN neurones with almost identical potency (MRN ED50 259 ± 57 μg/kg i.v.: DRN ED50 243 ± 49 μg/kg i.v.). In the majority of cells tested, the effect of paroxetine was reversed by the 5-HT1A receptor antagonists spiperone or (+)WAY100135, implicating the involvement of the 5-HT1A autoreceptor. The selective 5-HT1A receptor agonist 8-OH-DPAT also inhibited the firing of MRN (n = 5) and DRN (n = 12) neurones and with equal potency (MRN ED50, 1.32 ± 0.40 μg/kg i.v.: DRN ED50, 1.19 ± 0.23 μg/kg i.v.). Our data indicate that paroxetine not only inhibits the firing of 5-HT neurones in the MRN but does so with equal potency to those in the DRN.

Preclinical pharmacology of the α4β2 nAChR partial agonist varenicline related to effects on reward, mood and cognition

The pharmacological properties and pharmacokinetic profile of the alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline provide an advantageous combination of free brain levels and functional potencies at the target receptor that for a large part explain its efficacy as a smoking cessation aid. Since alpha4beta2 and other nAChR subtypes play important roles in mediating central processes that control reward, mood, cognition and attention, there is interest in examining the effects of selective nAChR ligands such as varenicline in preclinical animal models that assess these behaviors. Here we describe results from studies on vareniclines effects in animal models of addiction, depression, cognition and attention and discuss these in the context of recently published preclinical and preliminary clinical studies that collected data on vareniclines effects on mood, cognition and alcohol abuse disorder. Taken together, the preclinical and the limited clinical data show beneficial effects of varenicline, but further clinical studies are needed to evaluate whether the preclinical effects observed in animal models are translatable to the clinic.