A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Intracellular chloride concentration ([Cl⁻]_i) is a major determinant of neuronal excitability, as synaptic inhibition is primarily mediated by chloride-permeable receptors (1). In the mature brain, [Cl⁻]_i is maintained at low levels by chloride extrusion, which renders γ-aminobutyric acid (GABA) hyperpolarizing (2) and counteracts activity-dependent chloride loads (3). GABAergic inhibition in the adult is crucial not only for preventing runaway excitation of glutamatergic cells (4) but also for entraining neuronal assemblies into oscillations underlying cognitive processing (5). However, the capacity of chloride extrusion is low during early brain development (6, 7). Additionally, immature neurons are equipped with chloride uptake mechanisms, particularly with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (8–12). NKCC1 contributes to the maintenance of high [Cl⁻]_i in the developing brain (13), favoring depolarization through GABA_A receptor (GABA_AR) activation in vivo (14, 15).When GABA acts as a depolarizing neurotransmitter, neural circuits generate burst-like spontaneous activity (16–20), which is crucial for their developmental refinement (21–24). In vitro evidence indicates that GABAergic interneurons promote neuronal synchrony in an NKCC1-dependent manner (10, 12, 25–28). However, the in vivo developmental functions of NKCC1 are far from understood (29, 30). One fundamental question is to what extent NKCC1 and GABAergic depolarization supports correlated spontaneous activity in the neonatal brain. In the neocortex, GABA imposes spatiotemporal inhibition on network activity already in the neonatal period (14, 25, 31, 32). Whether a similar situation applies to other brain regions is unknown, as two recent chemo- and optogenetic studies in the hippocampus yielded opposing results (25, 33). Manipulations of the chloride driving force are potentially suited to resolve these divergent findings, but pharmacological (34–36) or conventional knockout (10, 11, 37) strategies suffer from unspecific effects that complicate interpretations.Here, we overcome this limitation by selectively deleting Slc12a2 (encoding NKCC1) from telencephalic glutamatergic neurons. We show that chloride uptake via NKCC1 promotes synchronized activity in acute hippocampal slices, but has weak and event type-dependent effects in CA1 in vivo. Long-term loss of NKCC1 leads to subtle changes of network dynamics in the adult, leaving synaptic development unperturbed and behavioral performance intact. Our data suggest that NKCC1-dependent chloride uptake is largely dispensable for several key aspects of hippocampal development in vivo.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Basal forebrain mediates prosocial behavior via disinhibition of midbrain dopamine neurons

Sociability is fundamental for our daily life and is compromised in major neuropsychiatric disorders. However, the neuronal circuit mechanisms underlying prosocial behavior are still elusive. Here we identify a causal role of the basal forebrain (BF) in the control of prosocial behavior via inhibitory projections that disinhibit the midbrain ventral tegmental area (VTA) dopamine (DA) neurons. Specifically, BF somatostatin-positive (SST) inhibitory neurons were robustly activated during social interaction. Optogenetic inhibition of these neurons in BF or their axon terminals in the VTA largely abolished social preference. Electrophysiological examinations further revealed that SST neurons predominantly targeted VTA GABA neurons rather than DA neurons. Consistently, optical inhibition of SST neuron axon terminals in the VTA decreased DA release in the nucleus accumbens during social interaction, confirming a disinhibitory action. These data reveal a previously unappreciated function of the BF in prosocial behavior through a disinhibitory circuitry connected to the brain’s reward system.

Prosocial behavior is of great importance to our daily life and represents the cornerstone of human society (1, 2). Conversely, impairments in social cognition and social interaction are commonly observed in a number of neuropsychiatric disorders, notably autism spectrum disorders (ASDs) (3, 4). However, in striking contrast to a rapid increase in the number of individuals afflicted, the effective treatment options for social deficits remain inadequate. Essentially, a deeper mechanistic understanding of social behavior and social impairment is currently needed.Since the concept of “the social brain” was coined in the late 20th century (5, 6), the study of social neuroscience has thrived. A growing body of studies from both human beings and animals of different species have been conducted to unravel the mystery behind social behavior at multiple levels varying from molecules to circuits (7–12). Not surprisingly, mirroring the complex nature of social behavior, a number of brain regions have been discovered to be involved in the process of social perception, social cognition, and social interaction over the past decades (13–22). Among many mechanistic understandings of social behavior, one outstanding advance in recent years is the recognition that the brain’s reward system plays a critical role in prosocial interaction behavior (14, 16, 17, 19). Particularly, the projection from the midbrain ventral tegmental area (VTA) dopamine (DA) neurons to the nucleus accumbens (NAc) is found to produce a rewarding effect and to mediate prosocial behavior in mice (14). However, the neural mechanism by which social information is relayed to the VTA, which leads to control of social behavioral manifestation, is still elusive.The basal forebrain (BF) is a collection of brain structures located in the rostroventral forebrain and was traditionally defined by the presence of cholinergic projection neurons (23). Accordingly, previous studies have been mostly focused on cholinergic neurons and have revealed essential roles of this neuronal population in the regulation of arousal, attention, learning, and memory (24–26). In addition to cholinergic neurons, the BF also comprises other major neuronal types including glutamatergic neurons and GABAergic neurons expressing either somatostatin (SST) or parvalbumin (PV) (27, 28). Recently, a diversity of brain functions has started to be unraveled for the noncholinergic neuronal types in the BF as well. For example, BF SST inhibitory neurons promote high-calorie food intake (29), yet glutamatergic neurons drive food avoidance through projections to the lateral hypothalamus (30). Also, cortically projecting BF PV inhibitory neurons regulate cortical gamma band rhythms (31). However, the functional role of the BF in social behavior and its neuronal substrate has not been investigated.Neuronal structure abnormalities in the BF have been identified in patients with ASDs, which are characterized by severe impairments in social cognition and interaction (32–34). Consistently, brain-imaging studies have also highlighted functional reduction of the BF in low-functioning autistic children (35). Does the BF play a causal role in social behavior? Also, what is the neuronal circuitry behind this mechanism? These are significant questions to be explored. Recent neural tracing studies with advanced viral genetic tools reveal dense anatomical connections between the BF and the VTA (36–38). Thus, we hypothesized that the BF projections to the VTA may carry information important for prosocial behavior.     

Foraging trade-offs,flagellar arrangements,and flow architecture of planktonic protists

Unicellular flagellated protists are a key element in aquatic microbial food webs. They all use flagella to swim and to generate feeding currents to encounter prey and enhance nutrient uptake. At the same time, the beating flagella create flow disturbances that attract flow-sensing predators. Protists have highly diverse flagellar arrangements in terms of number of flagella and their position, beat pattern, and kinematics, but it is unclear how the various arrangements optimize the fundamental trade-off between resource acquisition and predation risk. Here we describe the near-cell flow fields produced by 15 species and demonstrate consistent relationships between flagellar arrangement and swimming speed and between flagellar arrangement and flow architecture, and a trade-off between resource acquisition and predation risk. The flow fields fall in categories that are qualitatively described by simple point force models that include the drag force of the moving cell body and the propulsive forces of the flagella. The trade-off between resource acquisition and predation risk varies characteristically between flow architectures: Flagellates with multiple flagella have higher predation risk relative to their clearance rate compared to species with only one active flagellum, with the exception of the highly successful dinoflagellates that have simultaneously achieved high clearance rates and stealth behavior due to a unique flagellar arrangement. Microbial communities are shaped by trade-offs and environmental constraints, and a mechanistic explanation of foraging trade-offs is a vital part of understanding the eukaryotic communities that form the basis of pelagic food webs.

Unicellular flagellated protists play a key role in the biogeochemical cycles of the global ocean. Their photosynthetic activity and grazing on microbes are major processes in the microbial food web, and they may control the populations of bacteria and cyanobacteria (1). By being grazed, they transfer primary production to higher trophic levels (2–4). Thus, flagellates are both consumers and prey, but we do not understand how their resource acquisition trades off against predation mortality, or how this trade-off shapes their foraging behavior.In the low Reynolds number (Re) world of protists, viscosity impedes predator-prey contact. The physical mechanisms that nevertheless allow flagellates to daily clear a volume of water for prey that corresponds to approximately 10⁶ times their own cell volume (5, 6) are not well understood. Many marine flagellates are mixotrophic and can acquire resources both through photosynthesis and by eating other organisms (7). Their demand for inorganic mineral nutrients is also constrained by viscosity that retards the advective enhancement of diffusive uptake (8).To encounter prey and enhance advective transport of nutrients, protists may swim or create a feeding current through the beating of one or several flagella (9, 10). However, the beating of flagella produces fluid disturbances that exposes the flagellate to its rheotactic (flow-sensing) predators (11). Small flagellates are grazed by microzooplankton, many of which perceive their prey from the fluid disturbance that the prey generates (12, 13). Thus, there are fundamental foraging trade-offs. Such trade-offs are largely unexplored among the eukaryotic microbes that form the basis of aquatic food webs. This is crucial, because the diversity of microbial communities is determined by such trade-offs in concert with environmental constraints (14–17). Microbial diversity in turn governs the functionality and “services” of microbial communities, and hence also their role in ocean biogeochemistry (18, 19).Here we explore the trade-off between resource acquisition and predation risk in marine nanoflagellates and microflagellates by describing the flow fields produced by the action of their flagella. The quantification of near-cell feeding currents has been reported in only a few species of free-swimming protists (10, 20). The kinematics, wave patterns, and arrangement and number of flagella are highly diverse among flagellated protists (Fig. 1). Theoretical models suggest that the feeding currents and fluid signal generated by a swimming cell depends on the arrangement of the flagella (11, 13, 21, 22). We use microparticle image velocimetry (µPIV) to visualize and quantify the flow fields generated by free-swimming planktonic protists with diverse flagellar arrangements and beat patterns. We show how the different modes of swimming produce very different flow architectures and demonstrate a trade-off between resource acquisition and predation risk in flagellated protists.Open in a separate windowFig. 1.Schematic overview of the diverse flagellar arrangements and beat patterns represented in this study. Latin names below each taxonomic group indicate the species (or other taxonomic unit) examined. Flagellar hairs are drawn when feasible, but some flagellar morphologies (e.g., the dinoflagellates) are deliberately simplified (25, 63). Redrawn from several sources; not to scale.     

Cell type-specific lipid storage changes in Parkinson’s disease patient brains are recapitulated by experimental glycolipid disturbance

Neurons are dependent on proper trafficking of lipids to neighboring glia for lipid exchange and disposal of potentially lipotoxic metabolites, producing distinct lipid distribution profiles among various cell types of the central nervous system. Little is known of the cellular distribution of neutral lipids in the substantia nigra (SN) of Parkinson’s disease (PD) patients and its relationship to inflammatory signaling. This study aimed to determine human PD SN neutral lipid content and distribution in dopaminergic neurons, astrocytes, and microglia relative to age-matched healthy subject controls. The results show that while total neutral lipid content was unchanged relative to age-matched controls, the levels of whole SN triglycerides were correlated with inflammation-attenuating glycoprotein non-metastatic melanoma protein B (GPNMB) signaling in human PD SN. Histological localization of neutral lipids using a fluorescent probe (BODIPY) revealed that dopaminergic neurons and midbrain microglia significantly accumulated intracellular lipids in PD SN, while adjacent astrocytes had a reduced lipid load overall. This pattern was recapitulated by experimental in vivo inhibition of glucocerebrosidase activity in mice. Agents or therapies that restore lipid homeostasis among neurons, astrocytes, and microglia could potentially correct PD pathogenesis and disease progression.

Both neurons and glia depend on tight regulation and exchange of lipids for proper function. Typically through lipid transport mechanisms (1, 2), the continuous exchange of lipids is essential for maintaining physiological function as brain lipid content, transport, and distribution are complex and critical aspects of neuropathology. Recently, both clinical findings and experimental studies have implicated lipid storage and trafficking in the pathogenesis of Parkinson’s disease (PD) and related disorders (3, 4). Reduced function of lysosomal hydrolases that are associated with lysosomal storage disorders increases the risk for PD and results in brain pathology similar to that seen in most sporadic and genetic forms of the disease (5–11).One of the strongest genetic risk factors for PD is heterozygous loss-of-function mutations in GBA1, encoding the lysosomal hydrolase glucocerebrosidase (GCase) (12–14), a deficiency that causes systemic accumulation of its glycolipid substrate glucosylceramide (GlcCer). GCase activity is reduced with aging of both the human and murine brain (6, 15), and age is the overall greatest risk factor for developing PD (16). In contrast, the more severe loss of GCase activity in homozygous GBA1 mutant carriers causes the lysosomal storage disease Gaucher disease (GD) (17). Conduritol beta epoxide (CBE), an irreversible inhibitor of GCase, causes widespread accumulation of GlcCer and related glycosphingolipids in mice. In this model, there is marked increase of high molecular weight alpha-synuclein (aSYN) and deposition of proteinase K-resistant aSYN resembling that seen in PD (18–20). aSYN is a constituent of the classical Lewy bodies and Lewy neurites (21) found in surviving dopaminergic neurons in postmortem PD materials as a standard pathological criterion for PD (22). aSYN has a lipid-binding domain, and aSYN protein–lipid interactions are potentially perturbed in PD (reviewed in refs. 17, 23, 24). We recently demonstrated that excessive aSYN can deposit into lipid compartments, and that this process is reversible under increased lysosomal β-hexosaminidase expression (25).Several in vitro physiological studies have demonstrated that a reduction in neuronal neutral lipid storage or knockdown of fatty acid desaturases protects cultured neurons from degeneration (26–28). However, as neurons exhibit limited capacity to synthesize, metabolize, and transport lipid species under physiological conditions (29), other resident cells of the substantia nigra (SN), such as glial cells, are required to maintain lipid homeostasis in the brain. Astrocyte health and lipid exchange function—and microglial activation—are potentially central to PD pathogenesis and other age-dependent neurodegenerative diseases (30–33). Brain resident microglia can accumulate and generate lipids, which may propagate inflammatory processes through, for example, TREM2 binding of apolipoproteins (34–38). Proinflammatory cytokines present in chronic conditions are attenuated by the binding of glycoprotein nonmetastatic melanoma protein B (GPNMB) to the CD44 receptor on astrocytes (39). In GD patient serum, GPNMB level is significantly correlated with disease severity (40), and GPNMB is increased in human PD SN and following CBE-induced glycolipid accumulation in mice (41).There are surprisingly little data on lipid distribution patterns in PD-affected cell types given the relevance of lipid homeostasis, aSYN–lipid interactions, and lipidopathy-associated inflammatory signatures as they relate to PD (17, 41). In this study, we measured the differences in total lipid content and cellular distribution between PD and healthy subject (HS) SN and compared them with a lipid-associated neuroinflammatory signal, GPNMB. To quantify cell type-specific intracellular lipid content, colocalization analysis was performed on human postmortem SN sections that were costained for neutral lipids and markers of dopaminergic neurons, astrocytes, and microglia. Compared with HS SN, the lipid content of PD dopaminergic neurons and microglia was significantly higher, and that of astrocytes was significantly lower. To understand a possible mechanistic reason for this lipid distribution pattern, we used an in vivo mouse model of glycolipid dysregulation and attendant aSYN accumulation through GCase inhibition by CBE. This in vivo model recapitulated the lipid distribution pattern that we observed in human PD SN. Based on these data, we propose that PD is characterized by a unique neutral lipid distribution signature in neurons, astrocytes, and microglia that can be recapitulated experimentally by glycolipid dysregulation.     

Mechanisms and plasticity of chemogenically induced interneuronal suppression of principal cells

How do firing patterns in a cortical circuit change when inhibitory neurons are excited? We virally expressed an excitatory designer receptor exclusively activated by a designer drug (Gq-DREADD) in all inhibitory interneuron types of the CA1 region of the hippocampus in the rat. While clozapine N-oxide (CNO) activation of interneurons suppressed firing of pyramidal cells, unexpectedly the majority of interneurons also decreased their activity. CNO-induced inhibition decreased over repeated sessions, which we attribute to long-term synaptic plasticity between interneurons and pyramidal cells. Individual interneurons did not display sustained firing but instead transiently enhanced their activity, interleaved with suppression of others. The power of the local fields in the theta band was unaffected, while power at higher frequencies was attenuated, likely reflecting reduced pyramidal neuron spiking. The incidence of sharp wave ripples decreased but the surviving ripples were associated with stronger population firing compared with the control condition. These findings demonstrate that DREADD activation of interneurons brings about both short-term and long-term circuit reorganization, which should be taken into account in the interpretation of chemogenic effects on behavior.

The chemogenetic technology DREADD (designer receptors exclusively activated by designer drugs) is a widely used experimental method to control neuronal activity with an exogenous receptor that is engineered to respond selectively to an injectable agonist (1–4). In contrast to traditional pharmacology, chemogenetic techniques are both generalizable and specific because a receptor–agonist combination can be used for cell type-specific activation or inhibition of different neural populations in any brain region (4). The most prevalent DREADD platform exploits the human muscarinic receptors hM3Dq and hM4Di that are not activated by endogenous neurotransmitters but via the “designer drug” clozapine N-oxide (CNO). Subsequent experiments identified that the pharmacological actuator of CNO in the brain is the metabolically derived clozapine, arising from systemic CNO administration (4, 5). A recent improvement of the method introduced an ion channel-based platform for more potent neuronal activation and silencing that is controlled by pharmacologically selective actuator modules (6). Because of their easy use and assumed selective action, chemogenetic tools have become popular in animal research, and there is growing interest in developing chemogenetic techniques for clinical therapeutics (3).DREADD techniques have the advantage of activating or suppressing neurons over longer time periods, allowing for testing the contribution of specific neuron classes to behavior. However, it is unlikely that CNO brings about sustained activation or suppression in all target neurons uniformly and continuously because activation of interconnected inhibitory neuron populations often brings about unpredictable effects (7–11). Long-lasting excitation or inhibition of neurons typically induces synaptic plasticity and homeostatic regulations (10–13) but such potential circuit modifications have not yet been examined in connection with DREADD.The distal-less homeobox 5 and 6 (Dlx5/6) genes are specifically and transiently expressed by all forebrain GABAergic interneurons during embryonic development (14) and the recombinant adeno-associated virus (rAAV-hDLX) restricts gene expression to GABAergic interneurons in several species tested (15). Using rAAV-hDLX-Gq-DREADD in the hippocampus allowed us to investigate the mechanisms of chemogenetic modulation of interneuronal activity in behaving rats. CNO activation of all types of interneurons in the CA1 hippocampal region leads to a paradoxical decrease of the overall firing of many interneurons, coupled with a several-fold decrease of pyramidal cell firing. Interneurons did not display uniform sustained firing but, instead, enhanced activity of subgroups was interleaved with suppression of others. The sustained suppression of pyramidal cell activity was often interrupted by population bursts underlying sharp wave ripples. During such events, spiking of pyramidal cells was enhanced compared with control conditions. These findings demonstrate that DREADD activation of interneurons leads to dynamic circuit reorganization, which should be considered in the interpretation of chemogenic mechanisms in behavior (5, 16–21).     

Targeting presynaptic H3 heteroreceptor in nucleus accumbens to improve anxiety and obsessive-compulsive-like behaviors

Anxiety commonly co‐occurs with obsessive-compulsive disorder (OCD). Both of them are closely related to stress. However, the shared neurobiological substrates and therapeutic targets remain unclear. Here we report an amelioration of both anxiety and OCD via the histamine presynaptic H3 heteroreceptor on glutamatergic afferent terminals from the prelimbic prefrontal cortex (PrL) to the nucleus accumbens (NAc) core, a vital node in the limbic loop. The NAc core receives direct hypothalamic histaminergic projections, and optogenetic activation of hypothalamic NAc core histaminergic afferents selectively suppresses glutamatergic rather than GABAergic synaptic transmission in the NAc core via the H3 receptor and thus produces an anxiolytic effect and improves anxiety- and obsessive-compulsive-like behaviors induced by restraint stress. Although the H3 receptor is expressed in glutamatergic afferent terminals from the PrL, basolateral amygdala (BLA), and ventral hippocampus (vHipp), rather than the thalamus, only the PrL– and not BLA– and vHipp–NAc core glutamatergic pathways among the glutamatergic afferent inputs to the NAc core is responsible for co-occurrence of anxiety- and obsessive-compulsive-like behaviors. Furthermore, activation of the H3 receptor ameliorates anxiety and obsessive-compulsive-like behaviors induced by optogenetic excitation of the PrL–NAc glutamatergic afferents. These results demonstrate a common mechanism regulating anxiety- and obsessive-compulsive-like behaviors and provide insight into the clinical treatment strategy for OCD with comorbid anxiety by targeting the histamine H3 receptor in the NAc core.

Anxiety disorders and obsessive-compulsive disorder (OCD) are disabling psychiatric conditions and the major contributors to global burden of nonfatal illness (1). OCD is characterized by recurrent thoughts (obsessions) and/or repetitive behaviors (compulsions) that are aimed at reducing the anxiety caused by obsessions (2, 3), indicating a close correlation between anxiety and OCD. Indeed, anxiety disorders have been reported epidemiologically as the most frequent comorbid conditions with OCD (3, 4). Therefore, common pathologies may be present in anxiety disorders and OCD, and elucidation of the shared neural substrates will lead to greater insight into their pathophysiology and treatment.The nucleus accumbens (NAc) is a main component of the ventral striatum and a pivotal node in limbic basal ganglia loop, whose dysfunction may result in psychiatric diseases such as anxiety and OCD (5, 6). Accumulating experimental and clinical evidence indicates that the NAc, particularly the core compartment, holds a key position in motivation, emotion, and cognition and is strongly implicated in the psychopathology and treatment of anxiety and OCD. It has been reported that trait anxiety and OCD risk are positively correlated with the volume of NAc (7, 8). Functional neuroimaging reveals that the NAc activation correlates positively with the severity of human anxiety and obsessive-compulsive symptoms in OCD patients (9, 10). More importantly, deep brain stimulation (DBS) targeting the NAc core has been found to improve obsessive-compulsive symptoms and decrease ratings of anxiety in patients suffering from treatment-resistant OCD or depression (11, 12). Therefore, NAc core may be a potential common neural substrate for the clinical and neuropathological overlap between anxiety and OCD.The NAc core receives dense glutamatergic projections from the limbic system, including the prefrontal cortex, basolateral amygdala (BLA), and ventral hippocampus (vHipp), and integrates cognitive and affective information to instigate motivational approach behaviors (13, 14). In addition, the NAc core is regulated by various neuromodulators, such as orexin, serotonin, and histamine, from several brain regions (15–17). Among them, central histamine is synthesized and released by the histaminergic neurons restrictedly concentrated in the tuberomammillary nucleus (TMN) of the hypothalamus and serves as a general modulator for whole-brain activity via the mediation of histamine H1 to H4 receptors (18, 19). Accordingly, the aberrant histamine signaling is closely associated with sleep, motor, cognitive, and psychiatric conditions (18, 20, 21). In the clinic, drugs targeting the presynaptic H3 receptor have been used for prescribed treatment of various psychiatric and neurologic disorders (22). Interestingly, a high density of the H3 receptor has been found in NAc (23, 24). Therefore, in the present study, we create a transgenic rat strain expressing Cre recombinase in histidine decarboxylase (HDC, the histamine-synthesizing enzyme) neurons and employ anterograde axonal tract tracings, whole-cell patch clamp recordings, optogenetic and chemogenetic manipulation, and behavioral tests to explore the role of hypothalamic histaminergic afferents and the H3 receptor in the NAc core in regulation of anxiety and obsessive-compulsive-like behaviors. We find that optogenetic activation of hypothalamic TMN–NAc core histaminergic projections produces an anxiolytic effect and ameliorates obsessive-compulsive-like behaviors induced by restraint stress, which is due to H3 receptor–mediated suppression of glutamatergic transmission in a common prelimbic prefrontal cortex (PrL)–NAc core pathway.     

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABA_AR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.

Innate immunity, an evolutionally conserved behavior, constitutes the first defense line of multiple organisms to prevent microbial infections (1). The nematode Caenorhabditis elegans has been used as a model host for human opportunistic pathogen Pseudomonas aeruginosa infection (2) to identify evolutionarily conserved mechanisms of innate immunity. Typically, p38/PMK-1 mitogen-activated protein kinases (MAPKs) (3) and insulin/insulin-like signaling (IIS)/DAF-2 signaling cascades are recognized as two key components of the C. elegans intestinal innate immune response upon P. aeruginosa strain PA14 infection (4), as they are in mammals (3, 4). Moreover, increasing evidence has revealed several neural mechanisms as also being involved in the regulation of innate immunity. For example, G protein–coupled receptor (GPCR) NPR-1– and soluble guanylate cyclase GCY-35–expressing sensory neurons actively suppress the immune response of nonneuronal tissues (5). Additionally, a putative octopamine GPCR, OCTR-1, which is expressed and functions in the C. elegans sensory neurons ASH and ASI (6), down-regulates the unfolded protein response genes pqn/abu to further suppress the immune response of nonneuronal tissues (5, 6).Recent studies demonstrate that dopaminergic signaling inhibits innate immunity (7) whereas neuronal acetylcholine stimulates muscarinic signaling in the epithelium and activates the epithelial canonical Wnt pathway to promote the ability to defend against bacterial infection (8). Moreover, insulin-like peptide INS-7 secreted by the nervous system functions in a cell-nonautonomous manner to activate the IIS/DAF-2 pathway and modulate the intestinal innate immunity of C. elegans (9).GABAergic signaling constitutes a major inhibitory neurotransmission system that plays crucial roles in the central nervous system, especially for maintaining the balance between excitation and inhibition of neuronal networks (10). Disruption of this balance is not only linked to several neuropsychiatric disorders including schizophrenia, autism, and epilepsy (11) but also implicated in autoimmune disease (12). Up to date, multiple lines of evidence have shown that GABAergic signaling cell-autonomously modulates the immune response in immune cells (13–15). However, the roles of GABAergic synapses in innate immunity remain unknown.Here, we found that the nematode C. elegans harboring a deficiency in GABAergic neuromuscular junctions (NMJs) exhibits enhanced resistance to pathogens. P. aeruginosa PA14 infection increases synaptic expression of GABAergic synaptic components at the nerve cord of worms and enhances the strength of GABAergic transmission. Moreover, we identified an insulin-like peptide, INS-31, acting downstream of GABAergic NMJs and in body wall muscle (BWM) to control intestinal innate immunity in a cell-nonautonomous manner. This work reveals a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.     

Selective filtering of excitatory inputs to nucleus accumbens by dopamine and serotonin

The detailed mechanisms by which dopamine (DA) and serotonin (5-HT) act in the nucleus accumbens (NAc) to influence motivated behaviors in distinct ways remain largely unknown. Here, we examined whether DA and 5-HT selectively modulate excitatory synaptic transmission in NAc medium spiny neurons in an input-specific manner. DA reduced excitatory postsynaptic currents (EPSCs) generated by paraventricular thalamus (PVT) inputs but not by ventral hippocampus (vHip), basolateral amygdala (BLA), or medial prefrontal cortex (mPFC) inputs. In contrast, 5-HT reduced EPSCs generated by inputs from all areas except the mPFC. Release of endogenous DA and 5-HT by methamphetamine (METH) and (±)3,4-methylenedioxymethamphetamine (MDMA), respectively, recapitulated these input-specific synaptic effects. Optogenetic inhibition of PVT inputs enhanced cocaine-conditioned place preference, whereas mPFC input inhibition reduced the enhancement of sociability elicited by MDMA. These findings suggest that the distinct, input-specific filtering of excitatory inputs in the NAc by DA and 5-HT contribute to their discrete behavioral effects.

The nucleus accumbens (NAc), a major node of classic mesolimbic reward circuitry, plays a critical role in a variety of adaptive and pathological motivated behaviors by integrating information carried by inputs from a broad range of brain areas with distinct, yet overlapping functions (1–6). Output from the NAc is provided by medium spiny neurons (MSNs), the activity of which strongly depends on excitatory inputs from these brain areas, most prominently the ventral hippocampus (vHip), periventricular thalamus (PVT), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) (3, 7–11). The NAc is also a behaviorally important target for two of the brain’s major neuromodulatory systems, dopamine (DA) and serotonin (5-HT) (1, 5, 6, 12–14). DA release in the NAc, whether caused by drugs of abuse or optogenetic stimulation, is powerfully reinforcing and plays a critical role in shaping operant responses (1, 4–6, 15–17). In contrast, unlike DA release, release of 5-HT in the NAc, generated either pharmacologically or optogenetically, is not acutely reinforcing but can powerfully influence sociability (18, 19).The robust differences in the behavioral consequences of DA and 5-HT release in the NAc suggest that these neuromodulators must influence MSN activity in, perhaps profoundly, different ways. Yet little is known about the detailed mechanisms by which these neuromodulators accomplish this task. Because of the importance of excitatory input in controlling MSN activity and the fact that both DA and 5-HT are well established to modulate excitatory synaptic transmission in the NAc (18, 20–23), we hypothesized that an important mechanism by which these neuromodulators might distinctly influence MSN activity is by differentially filtering incoming information from major input structures. Specifically, we hypothesized that DA and 5-HT would depress excitatory synaptic transmission in distinct, input-specific manners. Because of methodological limitations prior to the advent of optogenetics, virtually all previous work examining DA and 5-HT modulation of excitatory transmission in the NAc used bulk electrical stimulation of unknown inputs.Consistent with our hypothesis, exogenously applied DA and 5-HT, as well as release of endogenous DA and 5-HT, depressed excitatory synaptic transmission in distinct, input-specific manners. Input-specific optogenetic inhibition of excitatory inputs to the NAc revealed input-specific effects on conditioned place preference and sociability assays, which are affected by NAc release of DA and 5-HT, respectively. Together, these results provide evidence that the input-specific filtering of excitatory input from distinct brain regions contributes to the behavioral effects of DA and 5-HT release in the NAc and provides a foundation for further work elucidating the neural mechanisms by which modulation of NAc activity influences motivated behaviors.     

Stac protein regulates release of neuropeptides

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca²⁺. However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca²⁺ channels (CaChs) and excitation–contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca²⁺ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Neuropeptides are required for a myriad of brain functions, such as regulation of complex social behaviors, including emotional behavior (1). The activities of many neuropeptides within the brain are now well-established, but, despite the important role neuropeptides play, our understanding of the mechanisms by which neuropeptides are released by neurons and how release is regulated is not as advanced as that of classical neurotransmitters. A greater understanding of neuropeptide release could help untangle the mechanisms of complex behaviors as well as reveal therapeutic targets that could modulate aberrant behaviors.Release of neuropeptides, which are packaged in dense core vesicles (DCVs) rather than in small synaptic vesicles that contain neurotransmitters, often involves activation of L-type Ca²⁺ channels (CaChs) and Ca²⁺-induced Ca²⁺ release (CICR) (2). Much of what is known about DCV exocytosis is from the study of nonneural cells. For example, in adrenal chromaffin cells, the exocytosis of DCVs containing catecholamines and/or peptide hormones involves CICR either via the ryanodine receptor (RyR) or inositol trisphosphate receptor (iP₃R) (3, 4) initiated by an influx of Ca²⁺ through voltage-gated CaChs, such as L-type CaChs. In neurons, the release of neuropeptides is more complex. Neuropeptides can be released from dendrites, cell bodies, axons, and presynaptic terminals (5–7), and release can involve a kiss-and-run mechanism (8). DCVs are not tightly clustered as are small synaptic vesicles, and DCVs are generally not associated with presynaptic specializations for release of neurotransmitters, such as active zones (9). Furthermore, the DCVs within the central nervous system (CNS) are not as numerous nor as large as they are in chromaffin cells and neurohypophyseal terminals. In many neurons, the release of neuropeptides requires bursts of action potentials (10, 11). Based primarily upon pharmacological experiments, it appears that an influx of Ca²⁺ via L-type CaChs is necessary for release of neuropeptides from some neurons (8, 12–18), with some cases also involving Ca²⁺ release from internal stores while others not. This suggests the possibility that, in neurons, neuropeptide release may be initiated by an influx of Ca²⁺ via L-type CaCh.Drosophila provide an ideal system to study neuropeptide release due to the vast array of molecular and genetic tools available for manipulating them. Numerous neuropeptides are expressed by the Drosophila nervous system, including proctolin by motor neurons (19, 20). Furthermore, larval motor neurons fire bursts of high frequency action potentials (21, 22) so motor boutons are likely to have the long-lasting increases in Ca²⁺ transients in motor boutons that are thought to be required for the release of neuropeptides. Motor boutons contain a network of endoplasmic reticulum (ER) (23), and there is a RyR-dependent release of Ca²⁺ from the ER presumably via CICR, which is required for sustained release of neuropeptides at the neuromuscular junction (NMJ) of third instar larvae (24). Elegant dynamic examination of DCVs in boutons with fluorescence recovery after photobleaching showed that they are immobile in the resting state, but they move randomly and release neuropeptides for several minutes following activity-dependent Ca²⁺ increases (25). Furthermore, simultaneous photobleaching and imaging of DCVs suggest that DCVs release neuropeptides via multiple rounds of kiss-and-run events (26). Thus, in Drosophila, neuropeptide release at the NMJ appears to involve CICR from the ER.One way to regulate the release of neuropeptides is to control changes in cytoplasmic Ca²⁺ levels. In mammals, stac3 is a member of a small family of genes, along with stac1 and stac2, which are expressed by subsets of neurons (27–29). Stac3 was identified as a regulator of L-type CaChs and excitation–contraction (EC) coupling in zebrafish skeletal muscles (30, 31). Stac3 also regulates EC coupling in murine skeletal muscles (32) and is causal for the congenital Native American myopathy (30). EC coupling is the process that transduces changes in muscle membrane voltage to initiate release of Ca²⁺ from the sarcoplasmic reticulum (SR) and contraction. In vertebrate skeletal muscles, EC coupling is mediated by the L-type CaCh DHPR, which is in the transverse tubule membrane (t-tubules) and is the voltage sensor for EC coupling, and the RyR, which is the Ca²⁺ release channel in the SR (33–36). In zebrafish, Stac3 regulates EC coupling by colocalizing with DHPR and RyR, and by regulating DHPR stability and functionality, including the response to voltage of DHPRs, but not trafficking of DHPRs (31, 37).The in vivo function of the stac1 and stac2 genes expressed by neurons is, however, unknown. Recently, a stac-like gene, Dstac, was identified in Drosophila (38). There is a single stac gene in Drosophila, and it is expressed both by muscles and a subset of neurons, including in the lateral ventral neurons (LN_Vs) that express the neuropeptide, pigment-dispersing factor (PDF), in the brain. Previously, genetic manipulation of PDF demonstrated the necessity of PDF for circadian rhythm (39). Interestingly, knocking down Dstac selectively in the PDF neurons disrupted circadian rhythm, suggesting the hypothesis that Dstac regulates the release of neuropeptides such as PDF. Since Stac3 regulates the L-type CaCh in vertebrate skeletal muscle, Dstac might control neuropeptide release via regulation of the single L-type CaCh in Drosophila neurons (Dmca1D) (40). We tested this hypothesis by examining the role of Dstac for neuropeptide release by the more accessible presynaptic boutons of motor neurons at the NMJs of third instar larvae.     

Thirst recruits phasic dopamine signaling through subfornical organ neurons

Thirst is a highly potent drive that motivates organisms to seek out and consume balance-restoring stimuli. The detection of dehydration is well understood and involves signals of peripheral origin and the sampling of internal milieu by first order homeostatic neurons within the lamina terminalis—particularly glutamatergic neurons of the subfornical organ expressing CaMKIIa (SFO^CaMKIIa). However, it remains unknown whether mesolimbic dopamine pathways that are critical for motivation and reinforcement integrate information from these “early” dehydration signals. We used in vivo fiber photometry in the ventral tegmental area and measured phasic dopamine responses to a water-predictive cue. Thirst, but not hunger, potentiated the phasic dopamine response to the water cue. In euvolemic rats, the dipsogenic hormone angiotensin II, but not the orexigenic hormone ghrelin, potentiated the dopamine response similarly to that observed in water-deprived rats. Chemogenetic manipulations of SFO^CaMKIIa revealed bidirectional control of phasic dopamine signaling during cued water reward. Taking advantage of within-subject designs, we found predictive relationships between changes in cue-evoked dopamine response and changes in behavioral responses—supporting a role for dopamine in motivation induced by homeostatic need. Collectively, we reveal a putative mechanism for the invigoration of goal-directed behavior: internal milieu communicates to first order, need state-selective circuits to potentiate the mesolimbic dopamine system’s response to cues predictive of restorative stimuli.

The invigoration of goal-directed behaviors is fundamentally grounded in homeostatic need. The maintenance of body fluid is a robust demonstration of the homeostasis-to-action arc, where minute changes can alter an animal’s motivation to seek and consume previously neutral (e.g., water) or even aversive (e.g., salt) stimuli (see refs. 1 and 2 for review). Plasma volume and osmolality are monitored through multiple mechanisms. For example, specialized cells of the kidney sense decreases in plasma perfusion and initiate the renin–angiotensin cascade resulting in the elevation of the hormone angiotensin II (AngII)—which acts in the central nervous system to increase water and sodium consumption (3–5). Circumventricular organs, particularly the lamina terminalis (i.e., subfornical organ [SFO], organum vasculosum [OVLT], and median preoptic nucleus [MnPO]), are thought to be central first order detectors of changes in body fluid composition (2, 6) and respond to AngII (7, 8). A series of elegant studies have shown that activation of specific populations of SFO neurons are sufficient to drive water consumption even in euvolemia (i.e., normal body fluid balance) (9, 10). These same SFO neurons increase their activity in response to dehydration and their activity is reduced when thirsty animals begin drinking (11).While the SFO detects body fluid imbalance, it must relay this information to marshal motive circuits for seeking and consuming fluid in response to need. Indeed, thirst recruits widespread networks across the brain, including those involved in motivation (12). Phasic activity of midbrain dopamine neurons in the ventral tegmental area (VTA) and dopamine release in the nucleus accumbens (NAc) play critical roles in motivation. Phasic dopamine responses are linked to stimulus valence (13–17), salience (18–22), reinforcement (23), and goal-directed action (24, 25) with recent work suggesting that their roles in these psychological constructs are not mutually exclusive (26). Perturbations in homeostasis tune dopamine responses. For example, hunger powerfully augments phasic dopamine responses to cues that predict food (27–29)—an effect recapitulated by central delivery of gut hormones that regulate hunger and satiety (28, 30). Changes in body fluid homeostasis [dehydration (31) or sodium depletion (32)] generate state-specific phasic dopamine responses to the intraoral delivery of fluid balance-restoring stimuli (water or hypertonic saline) or their predictive cues. How information about fluid balance reaches dopamine neurons remains unclear.Circumventricular organs, with their fenestrated blood–brain barrier, represent a potentially efficient way of communicating signals of peripheral origin that relate physiological need to central motive circuits. However, it remains unknown if and how central first order homeostatic neurons can modulate phasic dopamine signaling in the service of motivation. Understanding how homeostasis is communicated to the mesolimbic system is crucial for understanding the development of the enhanced cue reactivity that can underlie excessive ingestive behaviors. We trained rats to expect brief access to water in response to a cue and recorded either activity from VTA dopamine neurons or NAc dopamine release using fiber photometry. Thirst, but not hunger, potentiated dopamine responses to the water-predictive cue. Central delivery of AngII and modulation of excitatory SFO neurons using designer receptors exclusively activated by designer drug (DREADDs) recapitulated the effects of thirst. Collectively, the data support an intimate relationship between first order detectors of homeostatic imbalance and a system critical for converting motivation to action.     

Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP₂), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP₂ via the canonical pathway guanylyl cyclase/3′,5′-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.

Gonadotropin-releasing hormone (GnRH)-secreting neurons integrate multiple physiological and environmental cues and translate them into GnRH secretory patterns, to drive gonadotrophs, which in turn control the gonads. In both sexes, tonic GnRH pulses regulate gametogenesis and steroidogenesis via luteinizing hormone (LH) and follicle-stimulating pulses. Females require a GnRH surge to trigger LH surge and ovulation [reviewed in (1, 2)]. However, disruption of the normal pulsatile GnRH secretion impairs fertility in both sexes (3). Although insufficient for optimal reproductive health (4), the direct action of kisspeptins (KP) on GnRH neurons, via the KP receptor Kiss1r, is required for fertility (4–6). KP neurons play a critical regulatory role in the hypothalamic–pituitary–gonadal axis, including puberty onset (7–9), the preovulatory GnRH surge (5, 9, 10), and tonic GnRH pulses (6, 11). In rodents, two KP neuronal subpopulations exist with distinct functions: the anteroventral periventricular nucleus (AVPV) subpopulation, linked to puberty onset [reviewed in (12)] and preovulatory surge [reviewed in (13)], and the arcuate nucleus (ARC) subpopulation, also known as Kisspeptin-Neurokinin B-Dynorphin (KNDy) neurons, linked to tonic pulses (reviewed in ref. 14).Exogenous KP at the GnRH cell body evokes a long-lasting increase in intracellular calcium levels ([Ca²⁺]_i) (15), often leading to the summation of individual oscillations into [Ca²⁺]_i plateaus (15, 16). This observation is in agreement with an increase in electrical activity where GnRH neurons in acute slices go from burst firing to tonic firing after KP application (17–19). One could argue that this prolonged response is an artifact of the exogenously applied KP. However, endogenously released KP by AVPV stimulation also evokes a long-lasting increase in firing rate (20). Under normal conditions, [Ca²⁺]_i oscillations are driven by bursts of action potentials (AP) (21, 22). Yet, AP are not necessary for the KP-evoked [Ca²⁺]_i response to occur, as it is driven by multiple effectors including transient receptor potential-canonical channels (TRPC), voltage-gated calcium channels (VGCC), and inositol 1,4,5-trisphosphate receptors (InsP3R) (15, 16, 19, 23). Thus, the versatility of Kiss1r signaling pathway underlies the functionality of KP projections along GnRH neuron processes (24), with KP locally applied on nerve terminals also evoking a long-lasting increase in [Ca²⁺]_i (16).While the long-lasting KP response is suitable to trigger the preovulatory surge, it seems incompatible with the KNDy model for tonic pulses. GnRH and LH pulses occur every ∼20 min in ovariectomized mice (25). Indeed, the KNDy model provides on-/off- signals for KNDy neurons, neurokinin B and dynorphin respectively, and an on-signal for GnRH neurons, KP. However, this model lacks an off-signal for GnRH neurons. KNDy neurons trigger GnRH/LH pulses via Kiss1r (26), but the “extinction” of KNDy neurons by dynorphin is not obligatory for the termination of GnRH/LH pulses in rodents (27, 28), and dynorphin is lacking in human KP-neurokinin neurons (29).In fact, how the response in firing and [Ca²⁺]_i to KP relate to GnRH secretion is unknown. In acute coronal brain slices, the KP-evoked increase in firing at the cell body cannot be repeated (19, 30). In contrast, in acute sagittal brain slices, using fast scan cyclic voltammetry, the KP-evoked GnRH secretion from cells in the preoptic area (POA) and fibers in the median eminence (ME) can be triggered repeatedly (31). The difference in repeatability at the cell body is puzzling. One explanation could be a technical consequence of brain slicing or conventional patch clamp. Another explanation could be a dissociation between firing and [Ca²⁺]_i during the second KP application. In fact, the common feature of the KP-evoked increase in [Ca²⁺]_i and GnRH secretion, at the GnRH neuron cell body and nerve terminal, is that it is AP-independent (15, 16, 31). The current study uses calcium imaging and electrophysiology to address the mechanisms that allow 1) [Ca²⁺]_i in GnRH neurons to return to baseline after KP stimulation, and 2) GnRH neurons to respond to a second KP stimulation (i.e., repeatability) and shows nitric oxide as an important component for tonic GnRH neuronal pulses.     

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin β-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent adverse effect of treatment in cancer patients and survivors (1). CIPN significantly impacts quality of life as damage to sensory nerves may be permanent, and is often a dose-limiting factor during cancer treatment (2–4). Patients with CIPN report pain-related symptoms, including allodynia, hyper- or hypoalgesia, or pain that can be more severe than the pain associated with the original cancer (4). Despite increasing data on agents that protect sensory nerves, our limited understanding of the mechanisms of CIPN impedes effective treatment (5). Studies from model systems may be helpful in identifying molecules that protect sensory neuron morphology and function from the effects of chemotherapeutics.In the present study, we explored the mechanisms of CIPN induced by paclitaxel using two established models: Drosophila larval nociceptive neurons (6, 7) and primary dorsal root ganglia (DRG) neurons isolated from adult mouse (8). Similar to other peripheral neuropathies, CIPN models using paclitaxel, bortezomib, oxaliplatin, and vincristine report changes in unmyelinated intraepidermal nerve fibers (IENFs) that detect painful or noxious stimuli (9–14). These small fibers are embedded in the epidermis, and continuously turn over coincident with the turnover of skin (9, 15). Drosophila class IV nociceptive neurons are a favored model for genetic studies of nociceptive neuron development and signaling mechanisms (16). Prior studies showed that class IV neuron morphology is sensitive to paclitaxel and demonstrated morphological changes of nociceptive neurons at the onset and the end stage of paclitaxel-induced pathology (6, 7). Specifically, chronic treatment of high doses (30 μM) induce fragmentation and simplification of branching of sensory terminals (6). Additionally, acute treatments of moderate doses (10 to 20 μM) induced hyperbranching of sensory arbors without changing the branch patterns or degeneration (7). Nociceptive neurons in Drosophila larvae detect multiple qualities of noxious stimuli (17, 18), and project naked nerve terminals that are partially embedded in the epidermis (19, 20). Larvae have a stereotyped behavioral response toward noxious stimuli that can serve as a readout of nociceptive neuron function (17, 21). Nociceptive neurons in Drosophila larvae may therefore serve as a good in vivo model to study morphological and functional changes to sensory neurons induced by chemotherapeutics.Paclitaxel binds to tubulin and prevents microtubule disassembly. It is a commonly used chemotherapeutic drug for treatment of solid cancers, such as breast, ovarian, and lung cancers, by virtue of its ability to inhibit cell division. Paclitaxel causes chronic sensory neuropathy in patients and animal models (22–24). Several CIPN animal and in vitro models have also revealed acute effects of paclitaxel (7, 8, 24–26). While the mechanisms of acute and chronic neurodegeneration are likely to be distinct (27), how long-term treatment of paclitaxel can affect sensory neuron morphology and function, and how neuronal arbors can be protected against long-term toxicity is not understood.Several studies have shown that nociceptive sensory terminals share a close relationship with specific extracellular structures, most notably epidermal cells and the extracellular matrix (ECM). Thus, in addition to direct effects on neurons, paclitaxel could conceivably destabilize terminals by disrupting relationships with the extracellular environment. Indeed, a study in zebrafish indicates that epidermal cells are directly affected by paclitaxel and that epidermal changes precede neuronal degradation, indicating that degradation of neuronal substrates contributes to degeneration of adjacent arbors (25). For the most part, however, extracellular contributions to neuropathy induced by chemotherapeutics are still poorly characterized. It is therefore important to determine how sensory terminals are maintained in the context of a dynamic extracellular environment that itself may be sensitive to chemotherapeutics. Integrins are a key mediator of the interaction between cells and the ECM, and impact dendrite stabilization and maintenance in both vertebrate and invertebrate systems (20, 28, 29). Prior studies in other systems indicate that integrin levels at the surface are maintained by continuous recycling via tight regulation of the endosomal pathway rather than degradation and de novo synthesis (30). Decreased recycling or increased degradation could lead to depletion of the surface receptors (31, 32) responsible for arbor maintenance and, in turn, degeneration of nociceptive terminals. We therefore explored whether integrin–ECM interactions may impact sensory neuron maintenance upon paclitaxel-induced toxicity and how the endosomal–lysosomal pathway may be linked to the maintenance of sensory neurons.Here, we have used Drosophila and isolated mouse DRG neurons to investigate the pathological effect of paclitaxel in sensory neurons. Morphological changes in Drosophila neurons occurred at paclitaxel doses that also caused changes in thermal nociceptive behaviors. Cell-specific overexpression of integrins protected nociceptive neurons from morphological alterations and prevented the thermal nociceptive behavior deficits caused by paclitaxel in Drosophila. Transduction of integrins also protected adult mouse DRG sensory neurons from paclitaxel-induced toxicity in vitro, indicating that integrin-mediated protection is conserved in a vertebrate model of CIPN. We provide evidence that paclitaxel alters intracellular trafficking in both Drosophila and mouse models of CIPN. Furthermore, our biochemical analysis indicates a reduction of integrin surface availability, suggesting paclitaxel-induced recycling defects in mouse DRG neurons in vitro. Our study suggests that altered interactions between sensory neurons and their extracellular environment are an important contributor to paclitaxel-induced neuronal pathology, and that preventing these changes may offer a therapeutic approach.     

Discrete TrkB-expressing neurons of the dorsomedial hypothalamus regulate feeding and thermogenesis

Mutations in the TrkB neurotrophin receptor lead to profound obesity in humans, and expression of TrkB in the dorsomedial hypothalamus (DMH) is critical for maintaining energy homeostasis. However, the functional implications of TrkB-fexpressing neurons in the DMH (DMH^TrkB) on energy expenditure are unclear. Additionally, the neurocircuitry underlying the effect of DMH^TrkB neurons on energy homeostasis has not been explored. In this study, we show that activation of DMH^TrkB neurons leads to a robust increase in adaptive thermogenesis and energy expenditure without altering heart rate or blood pressure, while silencing DMH^TrkB neurons impairs thermogenesis. Furthermore, we reveal neuroanatomically and functionally distinct populations of DMH^TrkB neurons that regulate food intake or thermogenesis. Activation of DMH^TrkB neurons projecting to the raphe pallidus (RPa) stimulates thermogenesis and increased energy expenditure, whereas DMH^TrkB neurons that send collaterals to the paraventricular hypothalamus (PVH) and preoptic area (POA) inhibit feeding. Together, our findings provide evidence that DMH^TrkB neuronal activity plays an important role in regulating energy expenditure and delineate distinct neurocircuits that underly the separate effects of DMH^TrkB neuronal activity on food intake and thermogenesis.

Impairments in energy homeostasis resulting from the compound effects of overeating and sedentary lifestyles have led to a profound increase in the rate of obesity around the world (1). Therapeutic strategies aimed at combating obesity by increasing energy expenditure or decreasing appetite have commonly failed due to counterregulatory mechanisms (2) and adverse side effects on cardiovascular physiology (3–5). To achieve safe and sustained weight loss, it will be essential to understand the mechanisms that govern and coordinate discrete physiological processes that contribute to energy homeostasis.Adaptive thermogenesis is the process by which energy is converted into heat and occurs primarily in brown adipose tissue (BAT) in response to environmental cues (6). BAT has a particularly high capacity for dissipating energy from fat and thus represents an important component of energy homeostasis. The dorsomedial hypothalamus (DMH) in the brain is centrally positioned in an established thermoregulatory neurocircuit, receiving inputs from the preoptic area (POA) (7–9) and sending excitatory projections to preautonomic neurons in the raphe pallidus (RPa) (10–13) that promote sympathetic activity in BAT, leading to increased thermogenesis. Direct chemical stimulation of the DMH (14) or activation of select populations of thermogenic DMH neurons (9, 11, 12, 15) leads to increased body temperature and energy expenditure but also significantly increases heart rate and blood pressure (12, 13, 15, 16). An inability to target increased sympathetic tone specifically in BAT without affecting other target tissues has greatly hampered strategies to treat obesity by targeting thermogenesis (4, 5).In addition to its influence on energy expenditure, the DMH also represents an important brain region in the regulation of feeding (17–19). Lesioning studies support an orexigenic role for the DMH (17), which can promote food intake through inhibitory projections to either the paraventricular hypothalamus (PVH) (18) or the arcuate nucleus (ARC) (20). Despite these early findings, evidence has also emerged that demonstrates the importance of anorexigenic populations of DMH neurons (19, 21, 22). We previously established that the activity of DMH neurons expressing the neurotrophin receptor TrkB (DMH^TrkB) is important for regulating feeding, showing that activation of DMH^TrkB neurons suppresses feeding and that deletion of the TrkB-encoding Ntrk2 gene in the DMH results in hyperphagia and obesity (21). Furthermore, humans with mutations in the TrkB-encoding NTRK2 gene exhibit severe obesity and impaired thermoregulation (23). However, it is unclear whether activation of DMH^TrkB neurons has a direct influence on adaptive thermogenesis. Additionally, the neurocircuitry through which DMH^TrkB neurons govern feeding or energy expenditure is unknown.Here, we demonstrate that DMH^TrkB neuronal activity potently promotes energy expenditure by elevating thermogenesis and physical activity with a notable lack of influence on heart rate and blood pressure. We further reveal that DMH^TrkB neurons send diverging projections to the RPa or the POA and PVH to differentially regulate energy expenditure and food intake, respectively.