Shlomo Wagner

GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity

Mammals manifest circadian behaviour timed by an endogenous clock in the hypothalamic suprachiasmatic nucleus (SCN). Considerable progress has been made in identifying the molecular basis of the circadian clock, but the mechanisms by which it is translated into cyclic firing activity, high during the day and low at night, are still poorly understood. GABA (γ-aminobutyric acid), a common inhibitory neurotransmitter in the central nervous system, is particularly densely distributed within the SCN, where it is located in the majority of neuronal somata and synaptic terminals. Using an in vitro brain-slice technique, we have now studied the effect of bath-applied GABA on adult SCN neurons at various times of the day. We find that GABA acts as an inhibitory neurotransmitter at night, decreasing the firing frequency; but during the day GABA acts as an excitatory neurotransmitter, increasing the firing frequency. We show that this dual effect, which is mediated by GABAA receptors, may be attributed to an oscillation in intracellular chloride concentration. A likely explanation is that the amplitude of the oscillation in firing rate, displayed by individual neurons, is amplified by the dual effect of GABA in the SCNs GABAergic network.

A Multireceptor Genetic Approach Uncovers an Ordered Integration of VNO Sensory Inputs in the Accessory Olfactory Bulb

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.

Long-Term Social Recognition Memory Is Mediated by Oxytocin-Dependent Synaptic Plasticity in the Medial Amygdala

BACKGROUND Recognition of specific individuals is fundamental to mammalian social behavior and is mediated in most mammals by the main and accessory olfactory systems. Both these systems innervate the medial amygdala (MeA), where activity of the neuropeptide oxytocin is thought to mediate social recognition memory (SRM). The specific contribution of the MeA to SRM formation and the specific actions of oxytocin in the MeA are unknown. METHODS We used the social discrimination test to evaluate short-term and long-term SRM in adult Sprague-Dawley male rats (n = 38). The role of protein synthesis in the MeA was investigated by local application of the protein synthesis blocker anisomycin (n = 11). Synaptic plasticity was assessed in vivo by recording the MeA evoked field potential responses to stimulation of the main (n = 21) and accessory (n = 56) olfactory bulbs before and after theta burst stimulation. Intracerebroventricular administration of saline, oxytocin, or oxytocin receptor antagonist was used to measure the effect of oxytocin on synaptic plasticity. RESULTS Anisomycin application to the MeA prevented the formation of long-term SRM. In addition, the responses of MeA neurons underwent long-term depression (LTD) after theta burst stimulation of the accessory olfactory bulb, but not the main accessory bulb, in an oxytocin-dependent manner. No LTD was found in socially isolated rats, which are known to lack long-term SRM. Finally, accessory olfactory bulb stimulation before SRM acquisition blocked long-term SRM, supporting the involvement of LTD in the MeA in formation of long-term SRM. CONCLUSIONS Our results indicate that long-term SRM in rats involves protein synthesis and oxytocin-dependent LTD in the MeA.

GABA‐induced current and circadian regulation of chloride in neurones of the rat suprachiasmatic nucleus

1 We have shown previously that GABA, the main neurotransmitter in the suprachiasmatic nucleus (SCN), has dual effects on SCN neurones, excitatory during the day and inhibitory at night. This duality has been attributed to changes in [Cl−]i during the circadian cycle. To unravel the processes underlying these changes we investigated the biophysical properties of the GABAergic receptors and the regulation of [Cl−]i in SCN neurones. 2 We used voltage‐clamp methodology in conjunction with local application of GABA to characterise the current induced by GABA in SCN neurones within acute brain slices. This current, mediated via GABAA receptors, shows moderate voltage dependence, does not desensitise and can significantly alter [Cl−]i. 3 Loading or depletion of intracellular Cl− was induced by a train of GABA pulses. The recovery of intracellular Cl− was deduced from the change in [Cl−]i calculated from the response to a test GABA pulse presented at different intervals after the conditioning train of GABA application. The time course of recovery was described by an exponential curve. Recovery following Cl− depletion was slower than recovery from Cl− loading and was further delayed during the subjective night. 4 We concluded that: (a) SCN neurones express a large number of somatic GABAA receptors, which give rise to a modifiable, tonic Cl− conductance that modulates cell excitability; (b) two Cl− transport mechanisms operate in SCN neurones, one that replenishes the cell with Cl− following Cl− depletion and another that removes Cl− after Cl− loading; (c) the efficiency of the replenishing mechanism is reduced during the subjective night; and (d) this reduction explains a lower [Cl−]i during the night phase of the circadian cycle.

The Contribution of Oxytocin and Vasopressin to Mammalian Social Behavior: Potential Role in Autism Spectrum Disorder

Oxytocin (OT) and arginine-vasopressin (AVP) are 2 peptides that are produced in the brain and released via the pituitary gland to the peripheral blood, where they have diverse physiological functions. In the last 2 decades it has become clear that these peptides also play a central role in the modulation of mammalian social behavior by their actions within the brain. Several lines of evidence suggest their involvement in autism spectrum disorder (ASD), which is known to be associated with impaired social cognition and behavior. Recent clinical trials using OT administration to autistic patients have reported promising results. Here, we aim to describe the main data that suggest a connection between these peptides and ASD. Following a short illustration of several major topics in ASD biology we will (a) briefly describe the oxytocinergic and vasopressinergic systems in the brain, (b) discuss a few compelling cases manifesting the involvement of OT and AVP in mammalian social behavior, (c) describe data supporting the role of these peptides in human social cognition and behavior, and (d) discuss the possibility of the involvement of OT and AVP in ASD etiology, as well as the prospect of using these peptides as a treatment for ASD patients.

DNA Methylation of Specific CpG Sites in the Promoter Region Regulates the Transcription of the Mouse Oxytocin Receptor

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

The suprachiasmatic nucleus in stationary organotypic culture

Suprachiasmatic nuclei, derived from neonate rats, were maintained for several weeks in stationary organotypic culture. Hypothalamic slice explants, supported by Millicell filters and incubated in Petri dishes containing serum-based medium, flattened appreciably, yet preserved the organization of the suprachiasmatic nucleus and the surrounding hypothalamic tissue. After two to three weeks, cultures were fixed, and three neuronal sub-populations were identified as vasopressinergic, vasoactive intestinal peptide-containing, or GABA-containing. The GABAergic component of the cultured suprachiasmatic nucleus was particularly profuse, projecting extensively into the hypothalamic slice. Unilateral ablation of the nucleus in the explant dramatically reduced ipsilateral GABA-immunoreactivity in the slice. Explants in which an incision separated the bilateral suprachiasmatic nucleus from the paraventricular nucleus, deprived the latter of its fine-caliber GABA-immunoreactive input. Extra- or intra-cellular electrophysiological recordings from the suprachiasmatic nucleus were obtained in 51 of 58 cultures. The electrical properties of the long-term cultured suprachiasmatic nucleus were similar to those recorded in acute slices from adult rats. In six cultures recordings were extended for up to 10-24 h. Within long-term stationary organotypic cultures of the suprachiasmatic nucleus, sub-populations of neurons, intrinsic to the nucleus in vivo, were identified immunocytochemically. Lesion studies supported the observation that the main source of the GABAergic innervation within the entire hypothalamic slice explant appeared to be the suprachiasmatic nucleus. Electrophysiological studies confirmed the viability of the long-term cultured nucleus and revealed changes in spontaneous electrical activity that may indicate circadian fluctuation.

Brain region-specific methylation in the promoter of the murine oxytocin receptor gene is involved in its expression regulation

Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor α (ERα) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERα and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter.

Calcium-activated sustained firing responses distinguish accessory from main olfactory bulb mitral cells.

Many mammals rely on pheromones for mediating social interactions. Recent studies indicate that both the main olfactory system (MOS) and accessory olfactory system (AOS) detect and process pheromonal stimuli, yet the functional difference between these two chemosensory systems remains unclear. We hypothesized that the main functional distinction between the MOS and AOS is the type of sensory information processing performed by each system. Here we compared the electrophysiological responses of mitral cells recorded from the accessory olfactory bulb (AOB) and main olfactory bulb (MOB) in acute mouse brain slices to various stimuli and found them markedly different. The response of MOB mitral cells to brief (0.1 ms, 1–100 V) stimulation of their sensory afferents remained transient regardless of stimulus strength, whereas sufficiently strong stimuli evoked sustained firing in AOB mitral cells lasting up to several minutes. Using EPSC-like current injections (10–100 pA, 10 ms rise time constant, 5 s decay time constant) in the presence of various synaptic blockers (picrotoxin, CGP55845, APV, DNQX, E4CPG, and MSPG), we demonstrated that this difference is attributable to distinct intrinsic properties of the two neuronal populations. The AOB sustained responses were found to be mediated by calcium-activated nonselective cationic current induced by transient intense firing. This current was found to be at least partially mediated by TRPM4 channels activated by calcium influx. We hypothesize that the sustained activity of the AOS induces a new sensory state in the animal, reflecting its social context.

Oxytocin and Memory of Emotional Stimuli: Some Dance to Remember, Some Dance to Forget

An ever-growing body of evidence suggests that the hypothalamic neuropeptide oxytocin plays a central role in the regulation of mammalian social behavior and relationships. Yet, mammalian social interactions are extremely complex, involving both approach and avoidance behaviors toward specific individuals. While in the past oxytocin was conceived merely as a prosocial molecule that nonselectively facilitated affiliative emotions and behavior, it is now recognized that oxytocin plays a role in a wide range of social relationships, some of which involve negative emotions such as fear, aggression, and envy and lead to avoidance behavior. However, the way by which a single molecule such as oxytocin contributes to contrasting emotions and opposite behaviors is yet to be discovered. Here, we discuss the role of oxytocin in the modulation of emotional memories in rodents, focusing on two paradigms: social recognition and fear conditioning, representing approach and avoidance behaviors, respectively. We review recent pioneering studies that address the complex effects of oxytocin in a mechanistic approach, using genetic animal models and brain region-specific manipulations of oxytocin activity. These studies suggest that the multiple roles of oxytocin in social and fear behavior are due to its local effects in various brain areas, most notably distinct regions of the amygdala. Finally, we propose a model explaining some of the contradictory effects of oxytocin as products of the balance between two networks in the amygdala that are controlled by the medial prefrontal cortex.