GABAergic neurogliaform cells represent local sources of insulin in the cerebral cortex.

Concentrations of insulin in the brain are severalfold higher than blood plasma levels. Insulin in the brain regulates the metabolism, molecular composition, and cognitive performance of microcircuits and reduces food intake; cerebral insulin levels are altered in diabetes, aging, obesity, and Alzheimer's disease. Released by pancreatic ß cells, insulin passes the blood-brain barrier, but sources of locally released insulin still remain unclear. We find that insulin is strongly expressed in GABAergic neurogliaform cells in the cerebral cortex of the rat detected by single-cell digital PCR. Focal application of glucose or glibenclamide to neurogliaform cells mimics the excitation suppressing effect of external insulin on local microcircuits via insulin receptors. Thus, neurogliaform cells might link GABAergic and insulinergic action in cortical microcircuits.

Regulation of cortical microcircuits by unitary GABA-mediated volume transmission.

GABA (gamma-aminobutyric acid) is predominantly released by local interneurons in the cerebral cortex to particular subcellular domains of the target cells. This suggests that compartmentalized, synapse-specific action of GABA is required in cortical networks for phasic inhibition. However, GABA released at the synaptic cleft diffuses to receptors outside the postsynaptic density and thus tonically activates extrasynaptic GABA(A) and GABA(B) receptors, which include subtypes of both receptor families especially sensitive to low concentrations of GABA. The synaptic and extrasynaptic action of GABA corroborates the idea that neurons of the brain use synaptic (or wiring) transmission and non-synaptic (or volume) transmission for communication. However, re-uptake mechanisms restrict the spatial extent of extrasynaptic GABA-mediated effects, and it has been proposed that the concerted action of several presynaptic interneurons, the sustained firing of individual cells or an increase in release-site density is required to reach ambient GABA levels sufficient to activate extrasynaptic receptors. Here we show that individual neurogliaform cells release enough GABA for volume transmission within the axonal cloud and, thus, that neurogliaform cells do not require synapses to produce inhibitory responses in the overwhelming majority of nearby neurons. Neurogliaform cells suppress connections between other neurons acting on presynaptic terminals that do not receive synapses at all in the cerebral cortex. They also reach extrasynaptic, delta-subunit-containing GABA(A) (GABA(Adelta)) receptors responsible for tonic inhibition. We show that GABA(Adelta) receptors are localized to neurogliaform cells preferentially among cortical interneurons. Neurosteroids, which are modulators of GABA(Adelta) receptors, alter unitary GABA-mediated effects between neurogliaform cells. In contrast to the specifically placed synapses formed by other interneurons, the output of neurosteroid-sensitive neurogliaform cells represents the ultimate form of the lack of spatial specificity in GABA-mediated systems, leading to long-lasting network hyperpolarization combined with widespread suppression of communication in the local circuit.

Synaptic Organization-Function Relationships of Amygdala Interneurons Supporting Associative Learning.

Coordinated activity of basolateral amygdala (BLA) GABAergic interneurons (INs) and glutamatergic principal cells (PCs) is critical for associative learning, however the microcircuit organization-function relationships of distinct IN classes remain uncertain. Here, we show somatostatin (SOM) INs provide inhibition onto, and are excited by, local PCs, whereas vasoactive intestinal peptide (VIP) INs are driven by extrinsic afferents. Parvalbumin (PV) INs inhibit PCs and are activated by local and extrinsic inputs. Thus, SOM and VIP INs exhibit complementary roles in feedback and feedforward inhibition, respectively, while PV INs contribute to both microcircuit motifs. Functionally, each IN subtype reveals unique activity patterns across fear- and extinction learning with SOM and VIP INs showing most divergent characteristics, and PV INs display an intermediate phenotype parallelling synaptic data. Finally, SOM and PV INs dynamically track behavioral state transitions across learning. These data provide insight into the synaptic microcircuit organization-function relationships of distinct BLA IN classes.

A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction.

Preclinical and clinical studies implicate endocannabinoids (eCBs) in fear extinction, but the underlying neural circuit basis of these actions is unclear. Here, we employed in vivo optogenetics, eCB biosensor imaging, ex vivo electrophysiology, and CRISPR-Cas9 gene editing in mice to examine whether basolateral amygdala (BLA)-projecting medial prefrontal cortex (mPFC) neurons represent a neural substrate for the effects of eCBs on extinction. We found that photoexcitation of mPFC axons in BLA during extinction mobilizes BLA eCBs. eCB biosensor imaging showed that eCBs exhibit a dynamic stimulus-specific pattern of activity at mPFC→BLA neurons that tracks extinction learning. Furthermore, using CRISPR-Cas9-mediated gene editing, we demonstrated that extinction memory formation involves eCB activity at cannabinoid CB1 receptors expressed at vmPFC→BLA synapses. Our findings reveal the temporal characteristics and a neural circuit basis of eCBs' effects on fear extinction and inform efforts to target the eCB system as a therapeutic approach in extinction-deficient neuropsychiatric disorders.

Differential distribution of KCC2 along the axo-somato-dendritic axis of hippocampal principal cells.

The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role in adjusting intracellular Cl(-) concentrations. The lack of KCC2 in the plasma membrane of the axon initial segment (AIS) of pyramidal cells contributes to variable reversal potentials for perisomatic γ-aminobutyric acid (GABA)(A) receptor-mediated postsynaptic potentials, but the distribution of KCC2 in pyramidal dendrites remains to be established. We applied high-resolution pre-embedding immunolocalization to quantify KCC2 concentrations along dendritic, somatic and axonal regions of rat hippocampal principal cells. Confirming our results on neocortical pyramidal cells, membranes of AIS of CA1 pyramidal cells and dentate granule cells contained 6.4 ± 11.9% and 6.6 ± 14.1% of somatic KCC2 concentrations, respectively. Concentrations of KCC2 in basal dendritic shafts of stratum (str.) oriens were similar to somatic levels (109.2 ± 48.8%). Along apical dendritic shafts of CA1 pyramidal cells, the concentration of KCC2 showed a complex profile: normalized to somatic levels, the density of KCC2 was 124.5 ± 15.7%, 79 ± 12.4% and 98.2 ± 33.5% in the proximal and distal part of str. radiatum and in str. lacunosum moleculare, respectively. Dendritic spines of CA1 receiving excitatory inputs contained 39.9 ± 8.5% of KCC2 concentration measured in shafts of the same dendritic segments targeted by GABAergic inputs. Dendrites of dentate granule cells showed higher KCC2 concentration compared with the soma (148.9 ± 54%), but no concentration gradient was detected between proximal and distal dendrites. In conclusion, the density of KCC2 in hippocampal principal cells increases along the axo-somato-dendritic axis with cell type-specific distribution profiles within the dendritic tree.

An endocannabinoid-regulated basolateral amygdala-nucleus accumbens circuit modulates sociability.

Deficits in social interaction (SI) are a core symptom of autism spectrum disorders (ASDs); however, treatments for social deficits are notably lacking. Elucidating brain circuits and neuromodulatory signaling systems that regulate sociability could facilitate a deeper understanding of ASD pathophysiology and reveal novel treatments for ASDs. Here we found that in vivo optogenetic activation of the basolateral amygdala-nucleus accumbens (BLA-NAc) glutamatergic circuit reduced SI and increased social avoidance in mice. Furthermore, we found that 2-arachidonoylglycerol (2-AG) endocannabinoid signaling reduced BLA-NAc glutamatergic activity and that pharmacological 2-AG augmentation via administration of JZL184, a monoacylglycerol lipase inhibitor, blocked SI deficits associated with in vivo BLA-NAc stimulation. Additionally, optogenetic inhibition of the BLA-NAc circuit markedly increased SI in the Shank3B-/- mouse, an ASD model with substantial SI impairment, without affecting SI in WT mice. Finally, we demonstrated that JZL184 delivered systemically or directly to the NAc also normalized SI deficits in Shank3B-/- mice, while ex vivo JZL184 application corrected aberrant NAc excitatory and inhibitory neurotransmission and reduced BLA-NAc-elicited feed-forward inhibition of NAc neurons in Shank3B-/- mice. These data reveal circuit-level and neuromodulatory mechanisms regulating social function relevant to ASDs and suggest 2-AG augmentation could reduce social deficits via modulation of excitatory and inhibitory neurotransmission in the NAc.

Cyclooxygenase-2 inhibition reduces anxiety-like behavior and normalizes enhanced amygdala glutamatergic transmission following chronic oral corticosterone treatment.

Chronic stress increases the probability of receiving an anxiety, depression, or chronic illness diagnosis. Pharmacological interventions that reduce the behavioral and physiological effects of chronic stress in animal models may represent novel approaches for the treatment of stress-related psychiatric disorders. Here, we examined the effects of cyclooxygenase-2 (COX-2) inhibition on anxiety-like behaviors and amygdala glutamatergic signaling after chronic non-invasive oral corticosterone (CORT) administration in mice. Treatment with the highly selective COX-2 inhibitor Lumiracoxib (LMX) reversed anxiety-like behavior induced by chronic CORT. Specifically, acute and repeated administration of LMX 5 mg kg-1 reduced chronic CORT-induced anxiety-like behavior measured using the elevated-plus maze, elevated-zero maze, and light-dark box tests. In contrast, LMX did not affect anxiety-like behaviors in naïve mice. Ex vivo electrophysiology studies revealed that repeated LMX treatment normalized chronic CORT-induced increases in spontaneous excitatory glutamatergic currents recorded from anterior, but not posterior, basolateral amygdala neurons. These data indicate COX-2 inhibition can reverse chronic CORT-induced increases in anxiety-like behaviors and amygdala glutamatergic signaling, and support further clinical investigation of selective COX-2 inhibitors for the treatment of affective and stress-related psychiatric disorders.

Dynamic remodeling of a basolateral-to-central amygdala glutamatergic circuit across fear states.

Acquisition and extinction of learned fear responses utilize conserved but flexible neural circuits. Here we show that acquisition of conditioned freezing behavior is associated with dynamic remodeling of relative excitatory drive from the basolateral amygdala (BLA) away from corticotropin releasing factor-expressing (CRF+) centrolateral amygdala neurons, and toward non-CRF+ (CRF-) and somatostatin-expressing (SOM+) neurons, while fear extinction training remodels this circuit back toward favoring CRF+ neurons. Importantly, BLA activity is required for this experience-dependent remodeling, while directed inhibition of the BLA-centrolateral amygdala circuit impairs both fear memory acquisition and extinction memory retrieval. Additionally, ectopic excitation of CRF+ neurons impairs fear memory acquisition and facilities extinction, whereas CRF+ neuron inhibition impairs extinction memory retrieval, supporting the notion that CRF+ neurons serve to inhibit learned freezing behavior. These data suggest that afferent-specific dynamic remodeling of relative excitatory drive to functionally distinct subcortical neuronal output populations represents an important mechanism underlying experience-dependent modification of behavioral selection.

Role of Striatal Direct Pathway 2-Arachidonoylglycerol Signaling in Sociability and Repetitive Behavior.

BACKGROUND: Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal 2-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored. METHODS: Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of 2-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor-expressing or adenosine A2a receptor-expressing medium spiny neurons (MSNs) to determine the role of 2-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of 2-AG deficiency in the ventral and dorsal striatum. RESULTS: Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of 2-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming. CONCLUSIONS: These data suggest a role for 2-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for 2-AG in regulating striatal direct-pathway MSNs.

Endocannabinoid signalling modulates susceptibility to traumatic stress exposure.

Stress is a ubiquitous risk factor for the exacerbation and development of affective disorders including major depression and posttraumatic stress disorder. Understanding the neurobiological mechanisms conferring resilience to the adverse consequences of stress could have broad implications for the treatment and prevention of mood and anxiety disorders. We utilize laboratory mice and their innate inter-individual differences in stress-susceptibility to demonstrate a critical role for the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in stress-resilience. Specifically, systemic 2-AG augmentation is associated with a stress-resilient phenotype and enhances resilience in previously susceptible mice, while systemic 2-AG depletion or CB1 receptor blockade increases susceptibility in previously resilient mice. Moreover, stress-resilience is associated with increased phasic 2-AG-mediated synaptic suppression at ventral hippocampal-amygdala glutamatergic synapses and amygdala-specific 2-AG depletion impairs successful adaptation to repeated stress. These data indicate amygdala 2-AG signalling mechanisms promote resilience to adverse effects of acute traumatic stress and facilitate adaptation to repeated stress exposure.

Electrophysiological Measurement of Cannabinoid-Mediated Synaptic Modulation in Acute Mouse Brain Slices.

Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when applied to ex vivo systems such as electrophysiological analysis in acute brain slices. This unit provides protocols for measuring cannabinoid and eCB-mediated synaptic signaling in mouse brain slices, including analysis of short-term, long-term, and tonic eCB signaling modes, and the unique considerations for working with eCBs and TRPV1/cannabinoid ligands in acute brain slices.

Fluoxetine Facilitates Fear Extinction Through Amygdala Endocannabinoids.

Pharmacologically elevating brain endocannabinoids (eCBs) share anxiolytic and fear extinction-facilitating properties with classical therapeutics, including the selective serotonin reuptake inhibitor, fluoxetine. There are also known functional interactions between the eCB and serotonin systems and preliminary evidence that antidepressants cause alterations in brain eCBs. However, the potential role of eCBs in mediating the facilitatory effects of fluoxetine on fear extinction has not been established. Here, to test for a possible mechanistic contribution of eCBs to fluoxetine's proextinction effects, we integrated biochemical, electrophysiological, pharmacological, and behavioral techniques, using the extinction-impaired 129S1/Sv1mJ mouse strain. Chronic fluoxetine treatment produced a significant and selective increase in levels of anandamide in the BLA, and an associated decrease in activity of the anandamide-catabolizing enzyme, fatty acid amide hydrolase. Slice electrophysiological recordings showed that fluoxetine-induced increases in anandamide were associated with the amplification of eCB-mediated tonic constraint of inhibitory, but not excitatory, transmission in the BLA. Behaviorally, chronic fluoxetine facilitated extinction retrieval in a manner that was prevented by systemic or BLA-specific blockade of CB1 receptors. In contrast to fluoxetine, citalopram treatment did not increase BLA eCBs or facilitate extinction. Taken together, these findings reveal a novel, obligatory role for amygdala eCBs in the proextinction effects of a major pharmacotherapy for trauma- and stressor-related disorders and anxiety disorders.

Cyclooxygenase-2 inhibition reduces stress-induced affective pathology.

Mood and anxiety disorders are the most prevalent psychiatric conditions and are exacerbated by stress. Recent studies have suggested cyclooxygenase-2 (COX-2) inhibition could represent a novel treatment approach or augmentation strategy for affective disorders including anxiety disorders and major depression. We show that traditional COX-2 inhibitors and a newly developed substrate-selective COX-2 inhibitor (SSCI) reduce a variety of stress-induced behavioral pathologies in mice. We found that these behavioral effects were associated with a dampening of neuronal excitability in the basolateral amygdala (BLA) ex vivo and in vivo, and were mediated by small-conductance calcium-activated potassium (SK) channel and CB1 cannabinoid receptor activation. Taken together, these data provide further support for the potential utility of SSCIs, as well as traditional COX-2 inhibitors, as novel treatment approaches for stress-related psychiatric disorders.

Dietary habits and counseling focused on phosphate intake in hemodialysis patients with hyperphosphatemia.

OBJECTIVE: To evaluate the dietary habits of hemodialysis patients with hyperphosphatemia and the effects of a dietetic intervention focused on limiting dietary phosphate load. DESIGN: Cross-sectional dietary evaluation and prospective intervention study. SETTING: Hospital hemodialysis units of Pisa and Pistoia, Italy. Subjects Forty-three stable adult hemodialysis patients, 20 of whom had phosphorus serum levels >5.5 mg/dL. INTERVENTION: Analysis of dietary composition and of the effects of individual dietetic counseling in an attempt to reduce phosphorus intake while preserving the same protein intake. MAIN OUTCOME MEASURES: Differences in nutrient intake between normophosphatemic and hyperphosphatemic patients, and changes in dietary phosphorus and phosphorus-protein ratio, serum phosphate, and calcium-phosphate product after dietetic intervention. RESULTS: No major differences in nutrient intake were detected between hyperphosphatemia and normophosphatemia patients, apart from a lower phosphorus-protein ratio (13.1 +/- 1.7 versus 14.1 +/- 2.1 mg/g, P < .05) in the former. After dietetic intervention in the hyperphosphatemia patients, phosphate and calcium intake decreased significantly (by 100 mg on average), whereas dietary protein did not change. A further decrease of the dietary phosphate-protein ratio (12.5 +/- 1.8 mg/g, P < .05) also occurred. Serum phosphate showed a trend to decrease in the intervention group, whereas the serum calcium-phosphate product decreased significantly (from 66.8 +/- 13.1 to 61.0 +/- 13.8 mg2 /dL2 , P < .05). CONCLUSIONS: In compliant and motivated patients, individual dietetic counseling may be useful in reducing phosphate load and in limiting the phosphate burden related to an adequate protein intake, with a potentially favorable impact on calcium-phosphate retention. A phosphate-controlled diet has a role in an integrated therapeutic approach to hyperphosphatemia and positive calcium-phosphorus balance in hemodialysis patients.

Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation.

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders.