Neuropeptide S Activates Paraventricular Oxytocin Neurons to Induce Anxiolysis.

Neuropeptides, such as neuropeptide S (NPS) and oxytocin (OXT), represent potential options for the treatment of anxiety disorders due to their potent anxiolytic profile. In this study, we aimed to reveal the mechanisms underlying the behavioral action of NPS, and present a chain of evidence that the effects of NPS within the hypothalamic paraventricular nucleus (PVN) are mediated via actions on local OXT neurons in male Wistar rats. First, retrograde studies identified NPS fibers originating in the brainstem locus coeruleus, and projecting to the PVN. FACS identified prominent NPS receptor expression in PVN-OXT neurons. Using genetically encoded calcium indicators, we further demonstrated that NPS reliably induces a transient increase in intracellular Ca2+ concentration in a subpopulation of OXT neurons, an effect mediated by NPS receptor. In addition, intracerebroventricular (i.c.v.) NPS evoked a significant somatodendritic release of OXT within the PVN as assessed by microdialysis in combination with a highly sensitive radioimmunoassay. Finally, we could show that the anxiolytic effect of NPS seen after i.c.v. or intra-PVN infusion requires responsive OXT neurons of the PVN and locally released OXT. Thus, pharmacological blockade of OXT receptors as well as chemogenetic silencing of OXT neurons within the PVN prevented the effect of synthetic NPS. In conclusion, our results indicate a significant role of the OXT system in mediating the effects of NPS on anxiety, and fill an important gap in our understanding of brain neuropeptide interactions in the context of regulation of emotional behavior within the hypothalamus.SIGNIFICANCE STATEMENT Given the rising scientific interest in neuropeptide research in the context of emotional and stress-related behaviors, our findings demonstrate a novel intrahypothalamic mechanism involving paraventricular oxytocin neurons that express the neuropeptide S receptor. These neurons respond with transient Ca2+ increase and somatodendritic oxytocin release following neuropeptide S stimulation. Thereby, oxytocin neurons seem essential for neuropeptide S-induced anxiolysis, as this effect was blocked by pharmacological and chemogenetic inhibition of the oxytocin system.

GluN2B-containing NMDA receptors promote wiring of adult-born neurons into olfactory bulb circuits.

In the developing telencephalon, NMDA receptors (NMDARs) are composed of GluN1 and GluN2B subunits. These "young" NMDARs set a brake on synapse recruitment in neurons of the neonatal cortex. The functional role of GluN2B for synapse maturation of adult-born granule cells (GCs) in the olfactory bulb has not been established and may differ from that of differentiating neurons in immature brain circuits with sparse activity. We genetically targeted GCs by sparse retroviral delivery in mouse subventricular zone that allows functional analysis of single genetically modified cells in an otherwise intact environment. GluN2B-deficient GCs did not exhibit impairment with respect to the first developmental milestones such as synaptogenesis, dendrite formation, and maturation of inhibitory synaptic inputs. However, GluN2B deletion prevented maturation of glutamatergic synaptic input. This severe impairment in synaptic development was associated with a decreased response to novel odors and eventually led to the demise of adult-born GCs. The effect of GluN2B on GC survival is subunit specific, since it cannot be rescued by GluN2A, the subunit dominating mature NMDAR function. Our observations indicate that, GluN2B-containing NMDARs promote synapse activation in adult-born GCs that integrate in circuits with high and correlated synaptic activity. The function of GluN2B-containing NMDARs on synapse maturation can thus be bidirectional depending on the environment.

Subventricular zone-derived neuroblasts use vasculature as a scaffold to migrate radially to the cortex in neonatal mice.

Neurons continue to be generated in the subventricular zone (SVZ) throughout postnatal development and adulthood in rodents. Whereas in adults, virtually all neuroblasts migrate tangentially to the olfactory bulb via the rostral migratory stream (RMS), in neonates, a substantial fraction migrate radially through the corpus callosum (CC) to the cortex. Mechanisms of radial cortical migration have remained unknown. We investigated this by taking recourse to enhanced green fluorescent protein (EGFP)-labeled neuroblasts in the CC and deep cortical layers of neonatal mice and found that they are frequently located adjacent to vasculature. Using time-lapse 2-photon microscopy in acute brain slices, we demonstrate that EGFP-labeled neuroblasts migrate along blood vessels. Although in close proximity to blood vessels, migrating neuroblasts are separated from endothelial cells by 1-2 layers of astrocytic processes, as revealed by electron microscopal studies of retrovirally labeled postnatally born cells. We propose that 2 factors could contribute to the decline of radial migration to the cortex during postnatal development, namely the establishment of a glial sheath delineating the RMS and a gradual decrease in the density of blood vessels in the CC. Together, our data provide evidence for a new mode of radial cortical migration of SVZ-generated neurons involving vasculature and astrocytes.

5-HT(3A) receptor-bearing white matter interstitial GABAergic interneurons are functionally integrated into cortical and subcortical networks.

In addition to axons and surrounding glial cells, the corpus callosum also contains interstitial neurons that constitute a heterogeneous cell population. There is growing anatomical evidence that white matter interstitial cells (WMICs) comprise GABAergic interneurons, but so far there is little functional evidence regarding their connectivity. The scarcity of these cells has hampered electrophysiological studies. We overcame this hindrance by taking recourse to transgenic mice in which distinct WMICs expressed enhanced green fluorescence protein (EGFP). The neuronal phenotype of the EGFP-labeled WMICs was confirmed by their NeuN positivity. The GABAergic phenotype could be established based on vasoactive intestinal peptide and calretinin expression and was further supported by a firing pattern typical for interneurons. Axons and dendrites of many EGFP-labeled WMICs extended to the cortex, hippocampus, and striatum. Patch-clamp recordings in acute slices showed that they receive excitatory and inhibitory input from cortical and subcortical structures. Moreover, paired recordings revealed that EGFP-labeled WMICs inhibit principal cells of the adjacent cortex, thus providing unequivocal functional evidence for their GABAergic phenotype and demonstrating that they are functionally integrated into neuronal networks.

Fear, love, and the origins of canid domestication: An oxytocin hypothesis.

The process of dog domestication likely involved at least two functional stages. The initial stage occurred when subpopulations of wolves became synanthropes, benefiting from life nearby or in human environments. The second phase was characterized by the evolution of novel forms of interspecific cooperation and social relationships between humans and dogs. Here, we discuss possible roles of the oxytocin system across these functional stages of domestication. We hypothesize that in early domestication, oxytocin played important roles in attenuating fear and stress associated with human contact. In later domestication, we hypothesize that oxytocin's most critical functions were those associated with affiliative social behavior, social engagement, and cooperation with humans. We outline possible neurobiological changes associated with these processes and present a Siberian fox model of canid domestication in which these predictions can be tested. Lastly, we identify limitations of current studies on the neuroendocrinology of domestication and discuss challenges and opportunities for future research.

Altered PVN-to-CA2 hippocampal oxytocin pathway and reduced number of oxytocin-receptor expressing astrocytes in heart failure rats.

Oxytocinergic actions within the hippocampal CA2 are important for neuromodulation, memory processing and social recognition. However, the source of the OTergic innervation, the cellular targets expressing the OT receptors (OTRs) and whether the PVN-to-CA2 OTergic system is altered during heart failure (HF), a condition recently associated with cognitive and mood decline, remains unknown. Using immunohistochemistry along with retrograde monosynaptic tracing, RNAscope and a novel OTR-Cre rat line, we show that the PVN (but not the supraoptic nucleus) is an important source of OTergic innervation to the CA2. These OTergic fibers were found in many instances in close apposition to OTR expressing cells within the CA2. Interestingly, while only a small proportion of neurons were found to express OTRs (~15%), this expression was much more abundant in CA2 astrocytes (~40%), an even higher proportion that was recently reported for astrocytes in the central amygdala. Using an established ischemic rat heart failure (HF) model, we found that HF resulted in robust changes in the PVN-to-CA2 OTergic system, both at the source and target levels. Within the PVN, we found an increased OT immunoreactivity, along with a diminished OTR expression in PVN neurons. Within the CA2 of HF rats, we observed a blunted OTergic innervation, along with a diminished OTR expression, which appeared to be restricted to CA2 astrocytes. Taken together, our studies highlight astrocytes as key cellular targets mediating OTergic PVN inputs to the CA2 hippocampal region. Moreover, they provide the first evidence for an altered PVN-to-CA2 OTergic system in HF rats, which could potentially contribute to previously reported cognitive and mood impairments in this animal model.

In vivo evidence for the involvement of the carboxy terminal domain in assembling connexin 36 at the electrical synapse.

Connexin 36 (Cx36)-containing electrical synapses contribute to the timing and amplitude of neural responses in many brain regions. A Cx36-EGFP transgenic was previously generated to facilitate their identification and study. In this study we demonstrate that electrical coupling is normal in transgenic mice expressing Cx36 from the genomic locus and suggest that fluorescent puncta present in brain tissue represent distributed electrical synapses. These qualities emphasize the usefulness of the Cx36-EGFP reporter as a tool for the detailed anatomical characterization of electrical synapses in fixed and living tissue. However, though the fusion protein is able to form gap junctions between Xenopus laevis oocytes it is unable to restore electrical coupling to interneurons in the Cx36-deficient mouse. Further experiments in transgenic tissue and non-neural cell lines reveal impaired transport to the plasma membrane as the possible cause. By analyzing the functional deficits exhibited by the fusion protein in vivo and in vitro, we identify a motif within Cx36 that may interact with other trafficking or scaffold proteins and thereby be responsible for its incorporation into electrical synapses.

Central and peripheral release of oxytocin: Relevance of neuroendocrine and neurotransmitter actions for physiology and behavior.

The hypothalamic neuropeptide oxytocin (OT) is critically involved in the modulation of socio-emotional behavior, sexual competence, and pain perception and anticipation. While intracellular signaling of OT and its receptor (OTR), as well as the functional connectivity of hypothalamic and extra-hypothalamic OT projections, have been recently explored, it remains elusive how one single molecule has pleotropic effects from cell proliferation all the way to modulation of complex cognitive processes. Moreover, there are astonishing species-dependent differences in the way OT regulates various sensory modalities such as touch, olfaction, and vision, which can be explained by differences in OTR expression in brain regions processing sensory information. Recent research highlights a small subpopulation of OT-synthesizing cells, namely, parvocellular cells, which merely constitute 1% of the total number of OT cells but act as "master cells' that regulate the activity of the entire OT system. In this chapter, we summarize the latest advances in the field of OT research with a particular focus on differences between rodents, monkeys and humans and highlight the main differences between OT and its "sister" peptide arginine-vasopressin, which often exerts opposite effects on physiology and behavior.

Territorial blueprint in the hippocampal system.

As we skillfully navigate through familiar places, neural computations of distances and coordinates escape our attention. However, we perceive clearly the division of space into socially meaningful territories. 'My space' versus 'your space' is a distinction familiar to all of us. Spatial frontiers are social in nature since they regulate individuals' access to utilities in space depending on hierarchy and affiliation. How does the brain integrate spatial geometry with social territory? We propose that the action of oxytocin (OT) in the entorhinal-hippocampal regions supports this process. Grounded on the functional role of the hypothalamic neuropeptide in the hippocampal system, we show how OT-induced plasticity may bias the geometrical coding of place and grid cells to represent social territories.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.

A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System.

Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram.

Functional characterization of intrinsic cholinergic interneurons in the cortex.

Acetylcholine is a major neurotransmitter that modulates cortical functions. In addition to basal forebrain neurons that give rise to the principal cholinergic input into the cortex, a second source constituted by intrinsic cholinergic interneurons has been identified. Although these cells have been characterized anatomically, little is known about their functional role in cortical microcircuits. The paucity of this cell population has been a major hindrance for detailed electrophysiological investigations. To facilitate functional studies, we generated transgenic mice that express enhanced green fluorescent protein (EGFP) in choline acetyltransferase (ChAT)-positive neurons. Aided by the transgene expression, the characterization of distinct cholinergic interneurons was possible. These cells were located in layer 2-3, had a bipolar morphology, were calretinin- and vasoactive intestinal peptide positive, but had a non-GABAergic phenotype. Paired recordings showed that EGFP/ChAT-positive neurons receive excitatory and inhibitory input from adjacent principal cells and various types of interneurons. However, EGFP/ChAT-positive neurons do not exert direct postsynaptic responses in neighboring neurons. Interestingly, prolonged activation of EGFP-labeled cholinergic neurons induces an increase in spontaneous EPSCs in adjacent pyramidal neurons. This indirect effect is mediated by nicotinic receptors that are presumably presynaptically localized. Thus, intrinsic bipolar cholinergic neurons can modulate cortical function locally.

Oxytocin Signaling in the Lateral Septum Prevents Social Fear during Lactation.

Oxytocin (OXT)-mediated behavioral responses to social and stressful cues have extensively been studied in male rodents. Here, we investigated the capacity of brain OXT receptor (OXTR) signaling in the lateral septum (LS) to prevent social fear expression in female mice using the social-fear-conditioning paradigm. Utilizing the activated OXT system during lactation, we show that lactating mice did not express fear 24 hr after social fear conditioning. Supporting the role of OXTR signaling in the LS in attenuation of social fear, synthetic OXT infusion or overexpression of OXTR in the LS diminished social fear expression, whereas constitutive OXTR knockout severely impaired social fear extinction in virgin mice. Subsequently, both pharmacological blockade of local OXTRs in the LS and chemogenetic silencing of supraoptic nucleus OXTergic afferents to the LS increased social fear expression in lactating mice. Hence, LS-projecting OXT neurons suppress social fear in female mice.

Assembling the Puzzle: Pathways of Oxytocin Signaling in the Brain.

Oxytocin (OT) is a neuropeptide, which can be seen to be one of the molecules of the decade due to its profound prosocial effects in nonvertebrate and vertebrate species, including humans. Although OT can be detected in various physiological fluids (blood, saliva, urine, cerebrospinal fluid) and brain tissue, it is unclear whether peripheral and central OT releases match and synergize. Moreover, the pathways of OT delivery to brain regions involved in specific behaviors are far from clear. Here, we discuss the evolutionarily and ontogenetically determined pathways of OT delivery and OT signaling, which orchestrate activity of the mesolimbic social decision-making network. Furthermore, we speculate that both the alteration in OT delivery and OT receptor expression may cause behavioral abnormalities in patients afflicted with psychosocial diseases.

Density of acetylcholine esterase (AchE) and tyrosine hydroxylase (TH) containing fibers in the amygdala of roman high- and low-avoidance rats.

The cholinergic and dopaminergic innervation of the amygdala plays an important role in attention, emotional arousal, aversive forms of associative learning, conditioned responses, and stress responsivity. Roman High- (RHA) and Low-Avoidance (RLA) rats are an ideal model to study the potential impact of this innervation on behavioral responses, because they were selected bidirectionally for differences in their two-way active avoidance performance. RHA rats are known to quickly acquire two-way active avoidance and show indications of enhanced impulsive behavior, novelty seeking, and vulnerability to substance abuse, whereas RLA rats exhibit a passive coping style with high levels of immobility and enhanced stress responsivity. In the present study, the density of acetylcholine esterase (AchE)-positive cholinergic fibers and tyrosine hydroxylase immunoreactive (TH-ir) fibers were analyzed in various amygdala nuclei. In comparison to RLA rats, RHA rats displayed a significantly higher density of AchE-positive fibers in the lateral nucleus (La), the major sensory input area of the amygdala. In contrast, RLA rats showed a higher density of TH-ir fibers in the lateral division of the central nucleus (CeL), which modulates amygdala output and is known to contain more corticotropin-releasing hormone (CRH) positive neurons in RLA than in RHA rats. The findings suggest that a higher density of AchE-positive fibers in the La of RHA rats may facilitate attentional mechanisms and aversive forms of associative learning in RHA rats, whereas the increased density of TH-ir fibers in the CeL of RLA rats may be involved in the regulation of enhanced CRH expression and stress responsivity.

A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing.

Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

Changes in the physiology of CA1 hippocampal pyramidal neurons in preplaque CRND8 mice.

Amyloid-ß protein (Aß) is thought to play a central pathogenic role in Alzheimer's disease. Aß can impair synaptic transmission, but little is known about the effects of Aß on intrinsic cellular properties. Here we compared the cellular properties of CA1 hippocampal pyramidal neurons in acute slices from preplaque transgenic (Tg+) CRND8 mice and wild-type (Tg-) littermates. CA1 pyramidal neurons from Tg+ mice had narrower action potentials with faster decays than neurons from Tg- littermates. Action potential-evoked intracellular Ca(2+) transients in the apical dendrite were smaller in Tg+ than in Tg- neurons. Resting calcium concentration was higher in Tg+ than in Tg- neurons. The difference in action potential waveform was eliminated by low concentrations of tetraethylammonium ions and of 4-aminopyridine, implicating a fast delayed-rectifier potassium current. Consistent with this suggestion, there was a small increase in immunoreactivity for Kv3.1b in stratum radiatum in Tg+ mice. These changes in intrinsic properties may affect information flow through the hippocampus and contribute to the behavioral deficits observed in mouse models and patients with early-stage Alzheimer's disease.

Long-range-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex.

The hippocampus and entorhinal cortex play a pivotal role in spatial learning and memory. The two forebrain regions are highly interconnected via excitatory pathways. Using optogenetic tools, we identified and characterized long-range γ-aminobutyric acid-releasing (GABAergic) neurons that provide a bidirectional hippocampal-entorhinal inhibitory connectivity and preferentially target GABAergic interneurons. Activation of long-range GABAergic axons enhances sub- and suprathreshold rhythmic theta activity of postsynaptic neurons in the target areas.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.