Alfred Hetzenauer

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

Role of voltage-gated L-type Ca2+ channel isoforms for brain function.

Voltage-gated LTCCs (L-type Ca2+ channels) are established drug targets for the treatment of cardiovascular diseases. LTCCs are also expressed outside the cardiovascular system. In the brain, LTCCs control synaptic plasticity in neurons, and DHP (dihydropyridine) LTCC blockers such as nifedipine modulate brain function (such as fear memory extinction and depression-like behaviour). Voltage-sensitive Ca2+ channels Cav1 .2 and Cav1.3 are the predominant brain LTCCs. As DHPs and other classes of organic LTCC blockers inhibit both isoforms, their pharmacological distinction is impossible and their individual contributions to defined brain functions remain largely unknown. Here, we summarize our recent experiments with two genetically modified mouse strains, which we generated to explore the individual biophysical features of Cav1.2 and Cav1.3 LTCCs and to determine their relative contributions to various physiological peripheral and neuronal functions. The results described here also allow predictions about the pharmacotherapeutic potential of isoform-selective LTCC modulators.

Impaired extinction of learned fear in rats selectively bred for high anxiety – evidence of altered neuronal processing in prefrontal-amygdala pathways

The impaired extinction of acquired fear is a core symptom of anxiety disorders, such as post‐traumatic stress disorder, phobias or panic disorder, and is known to be particularly resistant to existing pharmacotherapy. We provide here evidence that a similar relationship between trait anxiety and resistance to extinction of fear memory can be mimicked in a psychopathologic animal model. Wistar rat lines selectively bred for high (HAB) or low (LAB) anxiety‐related behaviour were tested in a classical cued fear conditioning task utilizing freezing responses as a measure of fear. Fear acquisition was similar in both lines. In the extinction trial, however, HAB rats showed a marked deficit in the attenuation of freezing responses to repeated auditory conditioned stimulus presentations as compared with LAB rats, which exhibited rapid extinction. To gain information concerning the putatively altered neuronal processing associated with the differential behavioural response between HAB and LAB rats, c‐Fos expression was investigated in the main prefrontal‐amygdala pathways important for cued fear extinction. HAB compared to LAB rats showed an attenuated c‐Fos response to repeated conditioned stimulus presentations in infralimbic and cingulate cortices, as well as in the lateral amygdala, but facilitated the c‐Fos response in the medial part of the central amygdala. In conclusion, the present results support the notion that impaired extinction in high anxiety rats is accompanied by an aberrant activation profile in extinction‐relevant prefrontal‐amygdala circuits. Thus, HAB rats may represent a clinically relevant model to study the mechanisms and potential targets to accelerate delayed extinction processes in subjects with enhanced trait anxiety.

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’.

INCREASED NOVELTY-INDUCED MOTOR ACTIVITY AND REDUCED DEPRESSION-LIKE BEHAVIOR IN NEUROPEPTIDE Y (NPY)–Y4 RECEPTOR KNOCKOUT MICE

There is growing evidence that neuropeptide Y (NPY) acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y-receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble burying test and in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared with Y4 and Y2 knockout mice, respectively. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of pancreatic polypeptide (PP) induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion.

Brain activation pattern induced by stimulation of L-type Ca2+-channels: Contribution of CaV1.3 and CaV1.2 isoforms

Ca(V)1.2 and Ca(V)1.3, are the main dihydropyridine-sensitive L-type calcium channel isoforms in the brain. To reveal the contribution of each isoform to the neuronal activation pattern elicited by the dihydropyridine L-type calcium channel activator BayK 8644, we utilized Fos expression as a marker of neuronal activation in mutant mice (Ca(V)1.2(DHP-/-) mice) expressing dihydropyridine-insensitive Ca(V)1.2 L-type calcium channels. BayK 8644-treated wildtype mice displayed intense and widespread Fos expression throughout the neuroaxis in 77 of 80 brain regions quantified. The Fos response in Ca(V)1.2(DHP-/-) mice was greatly attenuated or absent in most of these areas, suggesting that a major part of the widespread Fos induction including most cortical areas was mediated by Ca(V)1.2 L-type calcium channels. BayK 8644-induced Fos expression in Ca(V)1.2(DHP-/-) mice indicating predominantly Ca(V)1.3 L-type calcium channel-mediated activation was noted in more restricted neuronal populations (20 of 80), in particular in the central amygdala, the bed nucleus of the stria terminalis, paraventricular hypothalamic nucleus, lateral preoptic area, locus coeruleus, lateral parabrachial nucleus, central nucleus of the inferior colliculus, and nucleus of the solitary tract. Our data indicate that selective stimulation of other than Ca(V)1.2 L-type calcium channels, mostly Ca(V)1.3, causes neuronal activation in a specific set of mainly limbic, hypothalamic and brainstem areas, which are associated with functions including integration of emotion-related behavior. Hence, selective modulation of Ca(V)1.3 L-type calcium channels could represent a novel (pharmacotherapeutic) tool to influence these CNS functions.

Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear

Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.

Individual contribution of metabotropic glutamate receptor (mGlu) 2 and 3 to c-Fos expression pattern evoked by mGlu2/3 antagonism.

Objectives and materials and methods The aims of the present study were (1) to determine the neuronal activation pattern elicited by the group II mGlu antagonist LY341495 and (2) to evaluate the contribution of each group II mGlu subtype by using wild-type (WT) and knockout (KO) mice lacking either mGlu2 or mGlu3. c-Fos expression was used as a marker of neuronal activation.Results and discussion In WT mice, LY341495 induced widespread c-Fos expression in 68 out of 92 brain areas, including limbic areas such as the amygdala, septum, prefrontal cortex, and hippocampus. LY341495-induced c-Fos response was markedly decreased in the medial part of the central amygdala (CeM) and lateral septum (LS) in mGlu3-KO mice, as well as in the lateral parabrachial nucleus (LPB) in both KO strains. In the majority of investigated areas, LY341495-induced c-Fos expression was similar in KO and WT mice. Analysis of the cellular and subcellular distribution of mGlu2 and mGlu3 revealed a prevailing presence of mGlu3-immunoreactivity in the CeM in glial processes and in postsynapstic neuronal elements, whereas only rare presynaptic axon terminals were found immunoreactive for mGlu2.ConclusionIn conclusion, our data indicate that group II mGlu blockade increases neuronal activation in a variety of brain areas, including many stress- and anxiety-related areas. The activation of two key brain areas, the CeM and LS, is mediated via mGlu3, while activation in the LPB involves both subtypes. Moreover, in the majority of investigated areas, LY341495-mediated neuronal activation appears to require a complex cross talk between group II mGlu subtypes or the action of LY341495 on additional receptors.

Modulation of magnesium deficiency-induced anxiety and HPA axis dysregulation by therapeutic drug treatment

Background Preclinical and some clinical studies suggest a relationship between perturbation in magnesium homeostasis and pathological anxiety, although the underlying mechanisms remain largely unknown. Since there is evidence that Mg modulates the hypothalamic-pituitary-adrenal (HPA) axis, we tested whether enhanced anxiety-like behaviour can be reliably elicited by dietary Mg restriction and whether Mg deficiency is associated with altered HPA axis function.

Increased novelty-induced motor activity and reduced depression-like behavior in NPY Y4 receptor knockout mice

There is growing evidence that neuropeptide Y acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble-burying test and motor-activity independent in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared to Y4 and Y2 knockout mice, respectively. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of PP induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion. Lack of central Y4 receptors appears to be responsible for the alterations in behavior seen in Y4 and Y2/Y4 knockout mice suggesting a potential new target to treat anxiety-related disorders.