The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1ß (IL-1ß), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

Contactomics of Microglia and Intercellular Communication.

Microglia represent the main immunocompetent cell type in the parenchyma of the brain and the spinal cord, with roles extending way beyond their immune functions. While emerging data show the pivotal role of microglia in brain development, brain health and brain diseases, the exact mechanisms through which microglia contribute to complex neuroimmune interactions are still largely unclear. Understanding the communication between microglia and other cells represents an important cornerstone of these interactions, which may provide novel opportunities for therapeutic interventions in neurological or psychiatric disorders. As such, in line with studying the effects of the numerous soluble mediators that influence neuroimmune processes, attention on physical interactions between microglia and other cells in the CNS has increased substantially in recent years. In this chapter, we briefly summarize the latest literature on "microglial contactomics" and its functional implications in health and disease.

Microglia contribute to neuronal synchrony despite endogenous ATP-related phenotypic transformation in acute mouse brain slices.

Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.

NMDA receptors in hippocampal GABAergic synapses and their role in nitric oxide signaling.

GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDA receptor (NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that--similar to glutamatergic synapses--the Ca(2+) transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 µm NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO-cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.

Nitric oxide signaling modulates synaptic transmission during early postnatal development.

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions.

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

Microglia monitor and protect neuronal function through specialized somatic purinergic junctions.

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.

Hippocampal GABAergic synapses possess the molecular machinery for retrograde nitric oxide signaling.

Nitric oxide (NO) plays an important role in synaptic plasticity as a retrograde messenger at glutamatergic synapses. Here we describe that, in hippocampal pyramidal cells, neuronal nitric oxide synthase (nNOS) is also associated with the postsynaptic active zones of GABAergic symmetrical synapses terminating on their somata, dendrites, and axon initial segments in both mice and rats. The NO receptor nitric oxide-sensitive guanylyl cyclase (NOsGC) is present in the brain in two functional subunit compositions: alpha1beta1 and alpha2beta1. The beta1 subunit is expressed in both pyramidal cells and interneurons in the hippocampus. Using immunohistochemistry and in situ hybridization methods, we describe that the alpha1 subunit is detectable only in interneurons, which are always positive for beta1 subunit as well; however, pyramidal cells are labeled only for beta1 and alpha2 subunits. With double-immunofluorescent staining, we also found that most cholecystokinin- and parvalbumin-positive and smaller proportion of the somatostatin- and nNOS-positive interneurons are alpha1 subunit positive. We also found that the alpha1 subunit is present in parvalbumin- and cholecystokinin-positive interneuron terminals that establish synapses on somata, dendrites, or axon initial segments. Our results demonstrate that NOsGC, composed of alpha1beta1 subunits, is selectively expressed in different types of interneurons and is present in their presynaptic GABAergic terminals, in which it may serve as a receptor for NO produced postsynaptically by nNOS in the very same synapse.

Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling.

A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type- and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ(9)-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ(9)-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

NMDA receptors in GABAergic synapses during postnatal development.

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6-7 and we observed a direct colocalization of GABA(A)- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca(2+)-influx through the NMDA receptors.

GABAB and CB1 cannabinoid receptor expression identifies two types of septal cholinergic neurons.

Septohippocampal cholinergic neurons play key roles in learning and memory processes, and in the generation of hippocampal theta rhythm. The range of receptors for endogenous modulators expressed on these neurons is unclear. Here we describe GABA(B) 1a/b receptor (GABA(B)R) and type 1 cannabinoid receptor (CB(1)R) expression in rat septal cholinergic [i.e. choline acetyltransferase (ChAT)-positive] cells. Using double immunofluorescent staining, we found that almost two-thirds of the cholinergic cells in the rat medial septum were GABA(B)R positive, and that these cells had significantly larger somata than did GABA(B)R-negative cholinergic neurons. We detected CB(1)R labelling in somata after axonal protein transport was blocked by colchicine. In these animals about one-third of the cholinergic cells were CB(1)R positive. These cells again had larger somata than CB(1)R-negative cholinergic neurons. The analyses confirmed that the size of GABA(B)R-positive and CB(1)R-positive cholinergic cells were alike, and all CB(1)R-positive cholinergic cells were GABA(B)R positive as well. CB(1)R-positive cells were invariably ChAT positive. All retrogradely labelled septohippocampal cholinergic cells were positive for GABA(B)R and at least half of them also for CB(1)R. These data shed light on the existence of at least two cholinergic cell types in the medial septum: one expresses GABA(B)R and CB(1)R, has large somata and projects to the hippocampus, whereas the other is negative for GABA(B)R and CB(1)R and has smaller somata. The results also suggest that cholinergic transmission in the hippocampus is fine-tuned by endocannabinoid signalling.