Deletion of the α2δ-1 calcium channel subunit increases excitability of mouse chromaffin cells.

High voltage-gated Ca2+ channels (HVCCs) shape the electrical activity and control hormone release in most endocrine cells. HVCCs are multi-subunit protein complexes formed by the pore-forming α1 and the auxiliary ß, α2δ and γ subunits. Four genes code for the α2δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α2δ-1 isoform. Here we show that α2δ-1 deletion led to ∼60% reduced HVCC Ca2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α2δ-1-/- MCCs compared to wild-type (WT), demonstrating that α2δ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca2+ influx, α2δ-1-/- MCCs showed reduced spontaneous electrical activity with action potentials (APs) having a shorter half-maximal duration caused by faster rising and decay slopes. However, the induced electrical activity showed opposite effects with α2δ-1-/- MCCs displaying significantly higher AP frequency in the tonic firing mode as well as an increase in the number of cells firing AP bursts compared to WT. This gain-of-function phenotype was caused by reduced functional activation of Ca2+-dependent K+ currents. Additionally, despite the reduced HVCC Ca2+ influx, the intracellular Ca2+ transients and vesicle exocytosis or endocytosis were unaltered in α2δ-1-/- MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α2δ-1 genetic deletion reduces Ca2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the α2δ-1 high voltage-gated Ca2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca2+ influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca2+ transients are unaffected by the reduced HVCC Ca2+ influx. Deletion of α2δ-1 reduces the immediately releasable pool vesicle exocytosis but has no effect on catecholamine (CA) release in response to sustained stimuli. The increased electrical activity and CA release from MCCs might contribute to the previously reported cardiovascular phenotype of patients carrying α2δ-1 loss-of-function mutations.

Reinstatement of synaptic plasticity in the aging brain through specific dopamine transporter inhibition.

Aging-related neurological deficits negatively impact mental health, productivity, and social interactions leading to a pronounced socioeconomic burden. Since declining brain dopamine signaling during aging is associated with the onset of neurological impairments, we produced a selective dopamine transporter (DAT) inhibitor to restore endogenous dopamine levels and improve cognitive function. We describe the synthesis and pharmacological profile of (S,S)-CE-158, a highly specific DAT inhibitor, which increases dopamine levels in brain regions associated with cognition. We find both a potentiation of neurotransmission and coincident restoration of dendritic spines in the dorsal hippocampus, indicative of reinstatement of dopamine-induced synaptic plasticity in aging rodents. Treatment with (S,S)-CE-158 significantly improved behavioral flexibility in scopolamine-compromised animals and increased the number of spontaneously active prefrontal cortical neurons, both in young and aging rodents. In addition, (S,S)-CE-158 restored learning and memory recall in aging rats comparable to their young performance in a hippocampus-dependent hole board test. In sum, we present a well-tolerated, highly selective DAT inhibitor that normalizes the age-related decline in cognitive function at a synaptic level through increased dopamine signaling.

Fear extinction rescuing effects of dopamine and L-DOPA in the ventromedial prefrontal cortex.

The ventromedial prefrontal cortex (vmPFC; rodent infralimbic cortex (IL)), is posited to be an important locus of fear extinction-facilitating effects of the dopamine (DA) bio-precursor, L-DOPA, but this hypothesis remains to be formally tested. Here, in a model of impaired fear extinction (the 129S1/SvImJ inbred mouse strain; S1), we monitored extracellular DA dynamics via in vivo microdialysis in IL during fear extinction and following L-DOPA administration. Systemic L-DOPA caused sustained elevation of extracellular DA levels in IL and increased neuronal activation in a subpopulation of IL neurons. Systemic L-DOPA enabled extinction learning and promoted extinction retention at one but not ten days after training. Conversely, direct microinfusion of DA into IL produced long-term fear extinction (an effect that was insensitive to ɑ-/ß-adrenoreceptor antagonism). However, intra-IL delivery of a D1-like or D2 receptor agonist did not facilitate extinction. Using ex vivo multi-electrode array IL neuronal recordings, along with ex vivo quantification of immediate early genes and DA receptor signalling markers in mPFC, we found evidence of reduced DA-evoked mPFC network responses in S1 as compared with extinction-competent C57BL/6J mice that were partially driven by D1 receptor activation. Together, our data demonstrate that locally increasing DA in IL is sufficient to produce lasting rescue of impaired extinction. The finding that systemic L-DOPA increased IL DA levels, but had only transient effects on extinction, suggests L-DOPA failed to reach a threshold level of IL DA or produced opposing behavioural effects in other brain regions. Collectively, our findings provide further insight into the neural basis of the extinction-promoting effects of DA and L-DOPA in a clinically relevant animal model, with possible implications for therapeutically targeting the DA system in anxiety and trauma-related disorders.

The human channel gating-modifying A749G CACNA1D (Cav1.3) variant induces a neurodevelopmental syndrome-like phenotype in mice.

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum-projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.

Chirality Matters: Fine-Tuning of Novel Monoamine Reuptake Inhibitors Selectivity through Manipulation of Stereochemistry.

The high structural similarity, especially in transmembrane regions, of dopamine, norepinephrine, and serotonin transporters, as well as the lack of all crystal structures of human isoforms, make the specific targeting of individual transporters rather challenging. Ligand design itself is also rather limited, as many chemists, fully aware of the synthetic and analytical challenges, tend to modify lead compounds in a way that reduces the number of chiral centers and hence limits the potential chemical space of synthetic ligands. We have previously shown that increasing molecular complexity by introducing additional chiral centers ultimately leads to more selective and potent dopamine reuptake inhibitors. Herein, we significantly extend our structure-activity relationship of dopamine transporter-selective ligands and further demonstrate how stereoisomers of defined absolute configuration may fine-tune and direct the activity towards distinct targets. From the pool of active compounds, using the examples of stereoisomers 7h and 8h, we further showcase how in vitro activity significantly differs in in vivo drug efficacy experiments, calling for proper validation of individual stereoisomers in animal studies. Furthermore, by generating a large library of compounds with defined absolute configurations, we lay the groundwork for computational chemists to further optimize and rationally design specific monoamine transporter reuptake inhibitors.

The Novel Analogue of Modafinil CE-158 Protects Social Memory against Interference and Triggers the Release of Dopamine in the Nucleus Accumbens of Mice.

Previous studies have shown that atypical dopamine-transporter-inhibitors such as modafinil and its analogues modify behavioral and cognitive functions in rodents. Here, we tested potential promnestic effects of the novel, more dopamine-transporter selective modafinil analogue CE-158 in the social discrimination memory task in male mice. Systemic administration of CE-158 1 h before the social learning event prevented the impairment of social-recognition memory following retroactive interference 3 h after the learning session of a juvenile conspecific. This effect was dose-dependent, as mice treated with 10 mg/kg, but not with 1 mg/kg CE-158, were able to discriminate between the novel and familiar conspecific despite the presentation of an interference stimulus, both 3 h and 6 h post learning. However, when 10 mg/kg of the drug was administered after learning, CE-158 failed to prevent social memory from interference. Paralleling these behavioral effects, the systemic administration of 10 mg/kg CE-158 caused a rapid and sustained elevation of extracellular dopamine in the nucleus accumbens, a brain area where dopaminergic signaling plays a key role in learning and memory function, of freely moving mice, while 1 mg/kg was not sufficient for altering dopamine levels. Taken together, our findings suggest promnestic effects of the novel dopamine-transporter-inhibitor CE-158 in a social recognition memory test that may be in part mediated via increased dopamine-neurotransmission in the nucleus accumbens. Thus, selective-dopamine-transporter-inhibitors such as CE-158 may represent interesting drug candidates for the treatment of memory complaints observed in humans with cognitive impairments and dementia.

Brain-derived neurotrophic factor expression in serotonergic neurons improves stress resilience and promotes adult hippocampal neurogenesis.

The neurotrophin brain-derived neurotrophic factor (BDNF) stimulates adult neurogenesis, but also influences structural plasticity and function of serotonergic neurons. Both, BDNF/TrkB signaling and the serotonergic system modulate behavioral responses to stress and can lead to pathological states when dysregulated. The two systems have been shown to mediate the therapeutic effect of antidepressant drugs and to regulate hippocampal neurogenesis. To elucidate the interplay of both systems at cellular and behavioral levels, we generated a transgenic mouse line that overexpresses BDNF in serotonergic neurons in an inducible manner. Besides displaying enhanced hippocampus-dependent contextual learning, transgenic mice were less affected by chronic social defeat stress (CSDS) compared to wild-type animals. In parallel, we observed enhanced serotonergic axonal sprouting in the dentate gyrus and increased neural stem/progenitor cell proliferation, which was uniformly distributed along the dorsoventral axis of the hippocampus. In the forced swim test, BDNF-overexpressing mice behaved similarly as wild-type mice treated with the antidepressant fluoxetine. Our data suggest that BDNF released from serotonergic projections exerts this effect partly by enhancing adult neurogenesis. Furthermore, independently of the genotype, enhanced neurogenesis positively correlated with the social interaction time after the CSDS, a measure for stress resilience.

Induction of deltaFosB in the periaqueductal gray by stress promotes active coping responses.

We analyzed the influence of the transcription factor DeltaFosB on learned helplessness, an animal model of affective disorder wherein a subset of mice exposed to inescapable stress (IS) develop a deficit in escape behavior. Repeated IS induces DeltaFosB in the ventrolateral periaqueductal gray (vlPAG), and levels of the protein are highly predictive of an individual's subsequent behavorial deficit-with the strongest DeltaFosB induction observed in the most resilient animals. Induction of DeltaFosB by IS predominates in substance P-positive neurons in the vlPAG, and the substance P gene, a direct target for DeltaFosB, is downregulated upon DeltaFosB induction. Local overexpression of DeltaFosB in the vlPAG using viral-mediated gene transfer dramatically reduces depression-like behaviors and inhibits stress-induced release of substance P. These results indicate that IS-induced accumulation of DeltaFosB in the vlPAG desensitizes substance P neurons enriched in this area and opposes behavioral despair by promoting active defense responses.

Increased in vivo release of neuropeptide S in the amygdala of freely moving rats after local depolarisation and emotional stress.

Intracerebral microdialysis in conjunction with a highly sensitive radioimmunoassay was used to study the in vivo release of neuropeptide S (NPS) within the amygdala of freely moving rats. NPS was consistently detected in basolateral amygdala dialysates and the release considerably enhanced in response to local depolarisation as well as exposure to forced swim stress. Thus, our data demonstrate for the first time emotional stress-induced release of NPS in the amygdala supporting a functional role of endogenous NPS in stress/anxiety-related phenomena.

Structure-Activity Relationships of Novel Thiazole-Based Modafinil Analogues Acting at Monoamine Transporters.

Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping disorders and investigated as potential therapeutics against cocaine addiction and for cognitive enhancement. Our continuous effort to find modafinil analogues with higher inhibitory activity on and selectivity toward the dopamine transporter (DAT) has previously led to the promising thiazole-containing derivatives CE-103, CE-111, CE-123, and CE-125. Here, we describe the synthesis and activity of a series of compounds based on these scaffolds, which resulted in several new selective DAT inhibitors and gave valuable insights into the structure-activity relationships. Introduction of the second chiral center and subsequent chiral separations provided all four stereoisomers, whereby the S-configuration on both generally exerted the highest activity and selectivity on DAT. The representative compound of this series was further characterized by in silico, in vitro, and in vivo studies that have demonstrated both safety and efficacy profile of this compound class.

Differential Effects of Novel Dopamine Reuptake Inhibitors on Interference With Long-Term Social Memory in Mice.

In the laboratory, long-term social recognition memory (SRM) in mice is highly susceptible to proactive and retroactive interference. Here, we investigate the ability of novel designed dopamine (DA) re-uptake inhibitors (rac-CE-123 and S-CE-123) to block retroactive and proactive interference, respectively. Our data show that administration of rac-CE-123 30 min before learning blocks retroactive interference that has been experimentally induced at 3 h, but not at 6 h, post-learning. In contrast, S-CE-123 treatment 30 min before learning blocked the induction of retroactive interference at 6 h, but not 3 h, post-learning. Administration of S-CE-123 failed to interfere with proactive interference at both 3 h and 6 h. Analysis of additional behavioral parameters collected during the memory task implies that the effects of the new DA re-uptake inhibitors on retroactive and proactive interference cannot easily be explained by non-specific effects on the animals' general social behavior. Furthermore, we assessed the mechanisms of action of drugs using intracerebral in vivo-microdialysis technique. The results revealed that administration of rac-CE-123 and S-CE-123 dose-dependently increased DA release within the nucleus accumbens of freely behaving mice. Thus, the data from the present study suggests that the DA re-uptake inhibitors tested protect the consolidation of long-term social memory against interference for defined durations after learning. In addition, the data implies that DA signaling in distinct brain areas including the nucleus accumbens is involved in the consolidation of SRM in laboratory mice.

Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429.

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Neurokinin 1 receptor antagonism promotes active stress coping via enhanced septal 5-HT transmission.

Antagonists of the substance P (SP) preferring neurokinin 1 receptor (NK1R) represent a promising novel class of drugs for the treatment of stress-related disorders such as depression and anxiety disorders; however, the involved neuronal pathways releasing SP in response to stressors are ill defined. By using in vivo microdialysis in combination with a highly sensitive and selective radioimmunoassay we found that exposure to forced swim stress increased SP release in the rat lateral septum (LS), a key area in processing emotions and stress responses. Acute administration of the selective NK1R antagonist L-822429 injected either systemically or locally into the LS reduced passive and facilitated active stress-coping strategies in the forced swim test. This effect seems to be mediated by enhanced intraseptal serotonergic transmission via serotonin (5-HT)1A receptors since NK1R blockade reversed the swim stress-induced decrease to an increase in extracellular 5-HT efflux, and furthermore the behavioral effects of L-822429 were blocked by intraseptal 5-HT1A receptor antagonism. A direct heterosynaptic regulation by NK1R on 5-HT release from serotonergic fibers was ruled out by immunocytochemistry at the light and electron microscopic level indicating involvement of GABAergic interneuron(s) in this interaction. Taken together, our data identify the LS as a critical brain area for the involvement of SP transmission in the modulation of stress responses and demonstrate that NK1R blockade can elicit a functionally significant facilitatory effect on 5-HT transmission, which does not necessarily involve the previously proposed interaction with neuronal firing at the cell body level of raphe neurons.

Stress-induced release of substance P in the locus coeruleus modulates cortical noradrenaline release.

Several lines of evidence implicate the neuropeptide substance P (SP) in the modulation of emotional behavior. Interaction between SP and noradrenergic systems has been proposed to be important in the regulation of stress, depression, and anxiety mechanisms; however, most evidence so far is based on studies in unchallenged and/or anesthetized animals. Thus, by using a dual-probe microdialysis approach in freely moving animals, the aim of the present study was to investigate whether a relevant stressor can trigger the release of SP in the locus coeruleus (LC) and whether and how this response modulates noradrenaline (NA) transmission both in the LC and in the medial prefrontal cortex (mPFC), an important LC terminal region involved in emotional processing. While confirming previous reports that neurokinin 1 receptor (NK1R) antagonists activate cortical noradrenergic transmission under resting conditions, we present evidence that this interaction is opposite during stress challenge. Our results show that exposure to forced swimming considerably enhanced the release of SP and NA in the LC. Administration of a selective NK1R antagonist into the LC potentiated this NA response within the LC but abolished the stress-induced increase in NA release within the mPFC. These findings demonstrate stress-induced increase in endogenous extracellular SP levels within the LC exerting a facilitatory effect on the noradrenergic pathway to the mPFC. The attenuation of stress-induced hyperactivation of this pathway by NK1R antagonists, presumably via enhancing NA and autoinhibition in the LC, may contribute to the therapeutic efficacy of these drugs known to ameliorate symptoms of stress-related disorders.

Exploring the role of neuropeptide S in the regulation of arousal: a functional anatomical study.

Neuropeptide S (NPS) is a regulatory peptide expressed by limited number of neurons in the brainstem. The simultaneous anxiolytic and arousal-promoting effect of NPS suggests an involvement in mood control and vigilance, making the NPS-NPS receptor system an interesting potential drug target. Here we examined, in detail, the distribution of NPS-immunoreactive (IR) fiber arborizations in brain regions of rat known to be involved in the regulation of sleep and arousal. Such nerve terminals were frequently apposed to GABAergic/galaninergic neurons in the ventro-lateral preoptic area (VLPO) and to tyrosine hydroxylase-IR neurons in all hypothalamic/thalamic dopamine cell groups. Then we applied the single platform-on-water (mainly REM) sleep deprivation method to study the functional role of NPS in the regulation of arousal. Of the three pontine NPS cell clusters, the NPS transcript levels were increased only in the peri-coerulear group in sleep-deprived animals, but not in stress controls. The density of NPS-IR fibers was significantly decreased in the median preoptic nucleus-VLPO region after the sleep deprivation, while radioimmunoassay and mass spectrometry measurements showed a parallel increase of NPS in the anterior hypothalamus. The expression of the NPS receptor was, however, not altered in the VLPO-region. The present results suggest a selective activation of one of the three NPS-expressing neuron clusters as well as release of NPS in distinct forebrain regions after sleep deprivation. Taken together, our results emphasize a role of the peri-coerulear cluster in the modulation of arousal, and the importance of preoptic area for the action of NPS on arousal and sleep.

Release of oxytocin in the rat central amygdala modulates stress-coping behavior and the release of excitatory amino acids.

Previous experiments have indicated that the release of oxytocin (OXT) occurs in various hypothalamic and extrahypothalamic brain areas. In the present study, we investigated in male rats whether swim stress triggers the release of OXT in the central amygdala (CeA), a key area in processing emotions and stress responses. Further, we examined the physiological significance of OXT released within the CeA for behavioral responses during forced swimming as well as effects on the local release of selected amino acids including glutamate, aspartate, arginine, taurine, and GABA, which are thought to modulate processing of emotions. Exposure to a 10-min forced swimming session caused a significant increase in OXT release (200%, p<0.01) within, but not outside, the CeA as monitored by microdialysis. Administration of the OXT receptor antagonist des-Gly-NH2d(CH2)5(Tyr(Me)2Thr4)OVT via inverse microdialysis into the amygdala before and during exposure to swimming reduced the floating time by 55% (p<0.05) and increased the swimming time by 29% (p<0.05) indicative of a more active stress-coping strategy. Simultaneously, local administration of the OXT receptor antagonist caused a significant increase in the stress-induced release of the excitatory amino acids glutamate and aspartate, whereas the basal release of these amino acids remained unchanged. Taken together, these findings demonstrate a significant activation of the oxytocinergic system in the CeA in response to swim stress. Furthermore, our data indicate that OXT receptor-mediated mechanisms within the amygdala are involved in the generation of passive stress-coping strategies, which might be mediated at least in part via its inhibitory influence on the local release of excitatory amino acids during stress.

Alterations in central neuropeptide expression, release, and receptor binding in rats bred for high anxiety: critical role of vasopressin.

To model aspects of trait anxiety/depression, Wistar rats were bred for extremes in either hyper (HAB)- or hypo(LAB)-anxiety as measured on the elevated plus-maze and in a variety of additional behavioral tests. Similar to psychiatric patients, HAB rats prefer passive stress-coping strategies, indicative of depression-like behavior, show hyper-reactivity of the hypothalamo-pituitary-adrenal axis, and a pathological response to the dexamethasone/corticotropin-releasing hormone (CRH) challenge test. Here we tested central mRNA expression, release patterns, and receptor binding of neuropeptides critically involved in the regulation of both anxiety-related behavior and the HPA axis. Thus, CRH, arginine-8-vasopressin (AVP), and oxytocin (OXT) were studied in brains of HAB and LAB males both under basal conditions and after exposure to a mild emotional stressor. In HAB rats, CRH mRNA was decreased in the bed nucleus of the stria terminalis only. While no significant difference in CRH1-receptor binding was found in any brain area, CRH2-receptor binding was elevated in the hypothalamic paraventricular nucleus (PVN), the ventromedial hypothalamus, and the central amygdala of HABs compared to LABs. AVP, but not OXT, mRNA expression as well as release of the neuropeptide, were higher in the PVN of HABs, whereas AVP V1a-receptor binding failed to show significant differences in any brain region studied. Remarkably, intra-PVN treatment of HABs with the AVP V1-receptor antagonist d (CH(2))(5) Tyr (Me) AVP resulted in a decrease in anxiety/depression-related behavior. The elevated expression and release of AVP within the PVN of HAB rats together with the behavioral effects of the AVP V1-receptor antagonist suggest a critical involvement of this neuropeptide in neuroendocrine and behavioral phenomena associated with trait anxiety/depression.

The modulatory role of the lateral septum on neuroendocrine and behavioral stress responses.

The lateral septum (LS) has been shown to have a key role in emotional processes and stress responses. However, the exact role of the LS on stress modulation is not clear, as previous lesion studies mostly used electrolytic lesions, thereby destroying the whole septal area, including medial components and/or fibers of passage. The aim of the present study was therefore, to investigate the effects of selective excitotoxic ablation of the LS on neuroendocrine and behavioral stress responses in rats. Bilateral ibotenic acid lesions of the LS increased hypothalamo-pituitary-adrenocortical (HPA) axis responses to forced swim stress indicated by enhanced plasma ACTH and corticosterone responses and higher stress-induced c-Fos-like immunoreactivity in the paraventricular hypothalamic nucleus. Moreover, LS-lesioned animals showed a more passive coping style in the forced swim test indicated by increased floating and reduced struggling/swimming behavior compared with sham-lesioned controls. Interestingly, intraseptal corticosteroid receptor blockade modulated behavioral stress coping but failed to change HPA axis stress responses. Further experiments aimed at elucidating underlying neurochemical mechanisms revealed that intraseptal administration of the selective 5-HT(1A) receptor antagonist WAY-100635 increased and prolonged stress-induced ACTH and corticosterone levels mimicking lesion effects, while the agonist 8-OH-DPAT suppressed HPA axis activity facilitating the inhibitory role of the LS. In addition, 8-OH-DPAT-injected animals showed increased active and decreased passive coping strategies during forced swimming suggesting antidepressant efficacy. Taken together, our data suggest that the LS promotes active stress coping behavior and is involved in a HPA-inhibitory mechanism that is at least in part mediated by septal 5-HT(1A) receptors and does not involve a glucocorticoid mediated feedback mechanism.

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.

Substance P in stress and anxiety: NK-1 receptor antagonism interacts with key brain areas of the stress circuitry.

In a previous work it was demonstrated that emotional stressors trigger the in vivo release of the neuropeptide substance P (SP) in brain areas known to be implicated in stress and anxiety mechanisms, such as the amygdala, lateral septum, nucleus accumbens, and locus coeruleus. However, the specific role of SP within the hypothalamic paraventricular nucleus (PVN), the critical site of the neuroendocrine stress axis, is unclear. Studies performed in neurokinin-1-receptor (NK-1R) knockout mice have provided conflicting results. Therefore, the aim of the present study was to use a pharmacological approach and examine whether intracerebroventricular NK-1R-antagonist treatment modulates stress-induced neuronal activity in key brain areas of the stress circuitry, including the PVN. The elevated plus maze test was used as a mild stressor known to stimulate stress hormone secretion and c-Fos-expression in the PVN and simultaneously to obtain behavioral readout for anxiety-like behavior. Results demonstrate an anxiolytic-like effect of intracerebral NK-1R antagonism that is associated with an attenuation of the stress-induced c-Fos expression in the PVN and lateral septum. In the amygdala and the bed nucleus of stria terminalis, c-Fos induction by elevated plus maze exposure was much lower and was not influenced by NK-1R-antagonist treatment. Thus, our findings provide clear evidence that central NK-1R-blockade reduces neuronal activity in key brain areas of the stress circuitry, which is thought to be associated with attenuation of the neuroendocrine stress response. These findings support the idea that a stress-sensitive subset of the human psychiatric patients may particularly benefit from a pharmacological approach that interferes with SP transmission.