Simone B. Sartori

Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms.

The L-type calcium channel (LTCC) isoforms Cav1.2 and Cav1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Cav1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Cav1.1 and Cav1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Cav1.2 and Cav1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Cav-DM) from mice expressing mutant Cav1.2 channels [Cav1.2DHP(-/-)] lacking high affinity for DHPs and from Cav1.3 knockouts [Cav1.3(-/-)]. (+)-[3H]isradipine binding to Cav1.2DHP(-/-) and Cav-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Cav1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Cav1.1 and Cav1.4 α1 subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold Kd increase) to the mutated Cav1.2 channels. Inhibition of (+)-[3H]isradipine binding to Cav1.2DHP(-/-) (predominantly Cav1.3) and wild-type (predominantly Cav1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Cav1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Cav1.1 and Cav1.4 are unlikely to serve as drug targets to treat CNS diseases.

The Central and Basolateral Amygdala Are Critical Sites of Neuropeptide Y/Y2 Receptor-Mediated Regulation of Anxiety and Depression

Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.

Tachykinin Receptors as Therapeutic Targets in Stress-Related Disorders

The first report demonstrating the therapeutic efficacy of an orally applied neurokinin-1 (NK1) receptor antagonist in depression was published 10 years ago. Although there were difficulties to reproduce this particular finding, a huge amount of data has been published since this time, supporting the potential therapeutic value of various tachykinin ligands as promising novel tools for the management of stress-related disorders including anxiety disorders, schizophrenia and depression. The present review summarizes evidence derived from anatomical, neurochemical, pharmacological and behavioral studies demonstrating the localization of tachykinin neuropeptides including substance P (SP), neurokinin A, neurokinin B and their receptors (NK1, NK2, NK3) in brain areas known to be implicated in stress-mechanisms, mood/anxiety regulation and emotion-processing; their role as neurotransmitters and/or neuromodulators within these structures and their interactions with other neurotransmitter systems including dopamine, noradrenaline and serotonin (5-hydroxytryptamine, 5-HT). Finally, there is clear functional evidence from animal and human studies that interference with tachykinin transmission can modulate emotional behavior. Based on these findings and on evidence of upregulated tachykinin transmission in individuals suffering from stress-related disorders, several diverse tachykinin receptor antagonists, as well as compounds with combined antagonist profile have been developed and are currently under clinical investigation revealing evidence for anxiolytic, antidepressant and antipsychotic efficacy, seemingly characterized by a low side effect profile. However, substantial work remains to be done to clarify the precise mechanism of action of these compounds, as well as the potential of combining them with established and experimental therapies in order to boost efficacy.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Fetal Down Syndrome Brains Exhibit Aberrant Levels of Neurotransmitters Critical for Normal Brain Development

BACKGROUND. In the immature developing fetal brain, amino acids (such as γ-aminobutyric acid, and taurine) and monoamines (serotonin, noradrenaline, and dopamine) act as developmental signals or regulators. In subjects with Down syndrome, dysfunctional brain development is evident from birth as reduction in brain weight, as well as volume reductions in specific brain regions, and an altered number of neurons, dendrites, and dendritic branching is observed. However, mechanisms that underlie the observed dysfunctional brain development in Down syndrome are not clear. OBJECTIVES. Because diverse amino acids and monoamines are critical for normal brain development, we wanted to determine whether dysfunctional brain development observed in subjects with Down syndrome is associated with altered brain amino acid and/or monoamine levels. DESIGN/METHODS. We quantified tissue concentrations of diverse amino acids, including γ-aminobutyric acid and taurine, and the monoamines serotonin, noradrenaline, and dopamine in the frontal cortex of fetal Down syndrome tissue at a gestational age of ∼20 weeks versus age-matched control aborted fetuses. RESULTS. Fetal Down syndrome brains showed reductions in the levels of serotonin, γ-aminobutyric acid, taurine, and dopamine in the frontal cortex. No alteration in the levels of arginine, aspartate, glutamine, glutamate, glycine, histidine, serine, or noradrenaline was observed. CONCLUSIONS. Serotonin, γ-aminobutyric acid, taurine, and dopamine are critical for the acquisition of brain morphologic features, neuronal and glia proliferation, and synapse formation. The detected reductions in the levels of these neurotransmitters may indicate potential mechanisms for the observed dysfunctional neuronal development in the Down syndrome fetal brain.

New paradigms for treatment-resistant depression

Clinical depression is a serious mental disorder characterized by low mood, anhedonia, loss of interest in daily activities, and other symptoms, and is associated with severe consequences including suicide and increased risk of cardiovascular events. Depression affects nearly 15% of the population. The standard of care for the last 50 years has focused on monoamine neurotransmitters, including such treatments as selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). However, these treatments have significant limitations: they can take weeks before showing mood‐altering effects, and only one to two out of ten patients shows clinical effects beyond those associated with placebo. A major paradigm shift in research into the treatment of depression is underway, based on promising results with the glutamatergic NMDA receptor antagonist ketamine. Further research has demonstrated the significance of glutamatergic pathways in depression and the association of this system with the stress pathway and magnesium homeostasis. Treatment with NMDA receptor antagonists and magnesium have shown the ability to sprout new synaptic connections and reverse stress‐induced neural changes, opening up promising new territory for the development of drugs to meet the unmet need in patients with clinical depression.

Magnesium deficiency induces anxiety and HPA axis dysregulation: modulation by therapeutic drug treatment.

Preclinical and some clinical studies suggest a relationship between perturbation in magnesium (Mg2+) homeostasis and pathological anxiety, although the underlying mechanisms remain largely unknown. Since there is evidence that Mg2+ modulates the hypothalamic-pituitary adrenal (HPA) axis, we tested whether enhanced anxiety-like behaviour can be reliably elicited by dietary Mg2+ deficiency and whether Mg2+ deficiency is associated with altered HPA axis function. Compared with controls, Mg2+ deficient mice did indeed display enhanced anxiety-related behaviour in a battery of established anxiety tests. The enhanced anxiety-related behaviour of Mg2+ deficient mice was sensitive to chronic desipramine treatment in the hyponeophagia test and to acute diazepam treatment in the open arm exposure test. Mg2+ deficiency caused an increase in the transcription of the corticotropin releasing hormone in the paraventricular hypothalamic nucleus (PVN), and elevated ACTH plasma levels, pointing to an enhanced set-point of the HPA axis. Chronic treatment with desipramine reversed the identified abnormalities of the stress axis. Functional mapping of neuronal activity using c-Fos revealed hyper-excitability in the PVN of anxious Mg2+ deficient mice and its normalisation through diazepam treatment. Overall, the present findings demonstrate the robustness and validity of the Mg2+ deficiency model as a mouse model of enhanced anxiety, showing sensitivity to treatment with anxiolytics and antidepressants. It is further suggested that dysregulations in the HPA axis may contribute to the hyper-emotionality in response to dietary induced hypomagnesaemia. This article is part of a Special Issue entitled ‘Anxiety and Depression’.

Altered GABA transmission in a mouse model of increased trait anxiety.

Anxiety disorders are the most prevalent central nervous system diseases imposing a high social burden to our society. Emotional processing is particularly controlled by GABA-ergic transmission in the amygdala. Using in situ hybridization and immunohistochemistry we now investigated changes in the expression of GABA synthesizing enzymes (GAD65 and GAD67), GABAA (α1–5, β1–3, γ1–2) and GABAB receptor subunits (GBBR1, GBBR2) in amygdaloid nuclei of high anxiety-related behavior (HAB) mice in comparison to mice selected for normal anxiety-related behavior (NAB). Levels of GAD65 and GAD67 mRNAs and protein, as well as those of GABA were increased in the amygdala of HAB mice. Relative to NAB controls, mRNA expression of the GABAA receptor subunits β1, β2 and γ2 was specifically increased in the basolateral amygdala of HAB mice while transcription of α5 and γ1 subunits was reduced in the central and medial amygdala. On the protein level, increases in β2 and γ2 subunit immunoreactivities were evident in the basolateral amygdala of HAB mice. No change in GABAB receptor expression was observed. These findings point towards an imbalanced GABA-ergic neurotransmission in the amygdala of HAB mice. On the other hand, FosB, a marker for neuronal activity, was increased in principal neurons of the basolateral amygdala in HAB mice, reflecting activation of excitatory neurons, possibly as a consequence of reduced GABA-ergic tonic inhibition through α5 and γ1 containing receptors. Ultimately these mechanisms may lead to the compensatory activation of GABA transmission, as indicated by the increased expression of GAD65/67 in HAB mice.

Behavioral and Neurobiological Effects of Deep Brain Stimulation in a Mouse Model of High Anxiety- and Depression-Like Behavior

Increasing evidence suggests that high-frequency deep brain stimulation of the nucleus accumbens (NAcb-DBS) may represent a novel therapeutic strategy for individuals suffering from treatment-resistant depression, although the underlying mechanisms of action remain largely unknown. In this study, using a unique mouse model of enhanced depression- and anxiety-like behavior (HAB), we investigated behavioral and neurobiological effects of NAcb-DBS. HAB mice either underwent chronic treatment with one of three different selective serotonin reuptake inhibitors (SSRIs) or received NAcb-DBS for 1 h per day for 7 consecutive days. Animals were tested in established paradigms revealing depression- and anxiety-related behaviors. The enhanced depression-like behavior of HAB mice was not influenced by chronic SSRI treatment. In contrast, repeated, but not single, NAcb-DBS induced robust antidepressant and anxiolytic responses in HAB animals, while these behaviors remained unaffected in normal depression/anxiety animals (NAB), suggesting a preferential effect of NAcb-DBS on pathophysiologically deranged systems. NAcb-DBS caused a modulation of challenge-induced activity in various stress- and depression-related brain regions, including an increase in c-Fos expression in the dentate gyrus of the hippocampus and enhanced hippocampal neurogenesis in HABs. Taken together, these findings show that the normalization of the pathophysiologically enhanced, SSRI-insensitive depression-like behavior by repeated NAcb-DBS was associated with the reversal of reported aberrant brain activity and impaired adult neurogenesis in HAB mice, indicating that NAcb-DBS affects neuronal activity as well as plasticity in a defined, mood-associated network. Thus, HAB mice may represent a clinically relevant model for elucidating the neurobiological correlates of NAcb-DBS.

Anxiety- rather than depression-like behavior is associated with adult neurogenesis in a female mouse model of higher trait anxiety- and comorbid depression-like behavior

Adult neurogenesis has been implicated in affective disorders and the action of antidepressants (ADs) although the functional significance of this association is still unclear. The use of animal models closely mimicking human comorbid affective and anxiety disorders seen in the majority of patients should provide relevant novel information. Here, we used a unique genetic mouse model displaying higher trait anxiety (HAB) and comorbid depression-like behavior. We demonstrate that HABs have a lower rate of hippocampal neurogenesis and impaired functional integration of newly born neurons as compared with their normal anxiety/depression-like behavior (NAB) controls. In HABs, chronic treatment with the AD fluoxetine alleviated their higher depression-like behavior and protected them from relapse for 3 but not 7 weeks after discontinuation of the treatment without affecting neurogenesis. Similar to what has been observed in depressed patients, fluoxetine treatment induced anxiogenic-like effects during the early treatment phase in NABs along with a reduction in neurogenesis. On the other hand, treatment with AD drugs with a particularly strong anxiolytic component, namely the neurokinin-1-receptor-antagonist L-822 429 or tianeptine, increased the reduced rate of neurogenesis in HABs up to NAB levels. In addition, challenge-induced hypoactivation of dentate gyrus (DG) neurons in HABs was normalized by all three drugs. Overall, these data suggest that AD-like effects in a psychopathological mouse model are commonly associated with modulation of DG hypoactivity but not neurogenesis, suggesting normalization of hippocampal hypoactivity as a neurobiological marker indicating successful remission. Finally, rather than to higher depression-related behavior, neurogenesis seems to be linked to pathological anxiety.