Perisomatic innervation and neurochemical features of giant pyramidal neurons in both hemispheres of the human primary motor cortex.

Betz cells-the gigantopyramidal neurons found in high amount in the primary motor cortex-are among of the most characteristic neuronal cells. A part of them contains the calcium-binding protein parvalbumin (PV) in primates. However, less is known about these cells in the human motor cortex despite their important role in different neurological disorders. Therefore, the aim of our study was to investigate the neurochemical features and perisomatic input properties of Betz cells in control human samples with short post-mortem interval. We used different microscopic techniques to investigate the primary motor cortex of both hemispheres. The soma size and density, and expression of PV of the Betz cells were investigated. Furthermore, we used confocal fluorescent and electron microscopy to examine their perisomatic input. The soma size and density showed moderate variability among samples and hemispheres. Post-mortem interval and hemispherical localization did not influence these features. Around 70% of Betz cells expressed PV, but in less intensity than the cortical interneurons. Betz neurons receive dense perisomatic input, which are mostly VIAAT- (vesicular inhibitory amino acid transporter) and PV immunopositive. In the electron microscope, we found PV-immunolabelled terminals with asymmetric-like synaptic structure, too. Terminals with morphologically similar synaptic specialisation were also found among vGluT2- (vesicular glutamate transporter type 2) immunostained terminals contacting Betz cells. Our data suggest that Betz cells' morphological properties showed less variability among subjects and hemispheres than the density of them. Their neurochemical and perisomatic input characteristics support their role in execution of fast and precise movements.

Thyrotropin-releasing hormone axonal varicosities appear to innervate dopaminergic neurons in the human hypothalamus.

Thyrotropin-releasing hormone (TRH) has a critical role in the central regulation of thyroid-stimulating hormone (TSH) from the anterior pituitary, and subsequently, thyroid hormone secretion from the thyroid gland. In addition to its role in the regulation of HPT axis, TRH is a potent regulator of prolactin (PRL) secretion by stimulating PRL secretion either directly from lactotrophs or indirectly via its action on the tuberoinfundibular dopamine (TIDA) neurons. In rodents, the TRH neurons which regulate TSH and thyroid hormone secretion, called hypophysiotropic TRH neurons, are in the medial subdivision of the parvicellular paraventricular nucleus (PVN). In humans, the PVN also contains a large population of TRH neurons, especially in its medial part, but the location of hypophysiotropic TRH neurons is not yet known. In addition to regulating TSH and PRL secretion, TRH also functions as a neurotransmitter/neuromodulator. In rodents and teleosts, TRH axons densely innervate TIDA neurons to inhibit tyrosine hydroxylase (TH) biosynthesis, neuronal firing, and dopamine turnover which may contribute to increasing PRL secretion. No such connections have been reported in humans, although dopaminergic neurons express TRH receptors and TRH also regulates PRL secretion. The objectives of this study were to map TRH-IR and TH-IR structures in the human hypothalamus with single-label light microscopic immunocytochemistry and study their interaction with double-label light microscopic immunocytochemistry. We show that TRH-IR nerve terminals densely surround TH-IR neurons (perikarya and dendrites) in the infundibulum of the human hypothalamus. The micrographs illustrating these juxtapositions were taken by Olympus BX45 microscope equipped with a digital camera and with 100X oil immersion objective. Composite images were created from the consecutive micrographs if the neurons were larger than the frame of the camera, using Adobe Photoshop software. As no gaps between TRH-IR and TH-IR elements were seen, these contacts may be functional synapses by which TRH regulates the activity of dopaminergic neurons and subsequently TSH and PRL secretion.

Brain γ-Tocopherol Levels Are Associated with Presynaptic Protein Levels in Elderly Human Midfrontal Cortex.

BACKGROUND: Higher vitamin E intake has been widely related to lower risks of cognitive decline and dementia. Animal models suggest that this relationship might be (partially) explained by the protection of vitamin E against presynaptic protein oxidation. OBJECTIVE: In this cross-sectional study, we aimed to examine the associations between brain tocopherols and presynaptic protein levels in elderly humans. METHODS: We examined associations of α- and γ-tocopherol brain levels with presynaptic protein levels in 113 deceased participants (age 88.5±6.0 years, 45 (40%) female) from the prospective Memory and Aging project. Three distinct presynaptic proteins, a SNARE protein composite, a synaptotagmin synaptophysin composite and the protein-protein interaction between synaptosomal-associated protein 25 (SNAP-25), and syntaxin were measured in two cortical brain regions. Linear regression models assessed associations of brain tocopherols with presynaptic protein levels. RESULTS: Higher brain γ-tocopherol levels were associated with higher levels of the SNARE protein composite, complexin-I, complexin-II, the synaptotagmin synaptophysin composite, and septin-5 in the midfrontal cortex (B(SE) = 0.272 to 0.412 (0.084 to 0.091), p < 0.001 to 0.003). When additionally adjusted for global Alzheimer's disease pathology, cerebral infarcts, and Lewy body disease pathology, these associations remained largely similar. No associations were found between α-tocopherol and presynaptic protein levels. CONCLUSION: In this cross-sectional study, we found higher brain γ-tocopherol levels were associated with presynaptic protein levels in the midfrontal cortex. These results are consistent with a proposed role of vitamin E to maintain presynaptic protein levels.

Presynaptic glutamatergic transmission and feedback system of oxytocinergic neurons in the hypothalamus of a rat model of adjuvant arthritis.

The neurohypophysial hormone oxytocin (OXT) is synthesized in the hypothalamic paraventricular and supraoptic nuclei. Recently, some studies have considered OXT to be important in sensory modulation and that the OXT protein is upregulated by acute and chronic nociception. However, the mechanism by which OXT is upregulated in neurons is unknown. In this study, we examined the resting membrane potentials and excitatory postsynaptic currents in OXT-ergic neurons in the paraventricular nucleus in adjuvant arthritis rat model, a model of chronic inflammation, using whole-cell patch-clamping. Transgenic rats expressing OXT and monomeric red fluorescent protein 1 (mRFP1) fusion protein to visualize the OXT-ergic neurons were used, and the OXT-mRFP1 transgenic rat model of adjuvant arthritis was developed by injection of heat-killed Mycobacterium butyricum. Furthermore, the feedback system of synthesized OXT was also examined using the OXT receptor antagonist L-368,899. We found that the resting membrane potentials and frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-monomeric red fluorescent protein 1 neurons in the paraventricular nucleus were significantly increased in adjuvant arthritis rats. Furthermore, L-368,899 dose-dependently increased the frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-ergic neurons. Following bath application of the GABAA receptor antagonist picrotoxin and the cannabinoid receptor 1 antagonist AM 251, L-368,899 still increased the frequency of miniature excitatory postsynaptic currents. However, following bath application of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, L-368,899 did not alter the miniature excitatory postsynaptic current frequency. Thus, it is suggested that OXT-ergic neuron activity is upregulated via an increase in glutamate release, and that the upregulated OXT neurons have a feedback system with released endogenous OXT. It is possible that nitric oxide, but not GABA, may contribute to the feedback system of OXT neurons in chronic inflammation.

Morphological plasticity of the tuberoinfundibular dopaminergic neurones in the rat during the oestrous cycle and lactation.

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones are critical with respect to regulating prolactin secretion from the anterior pituitary. Under most physiological conditions, they are stimulated by prolactin to release dopamine into the median eminence which subsequently suppresses further prolactin secretion from the lactotrophs. During lactation, the TIDA neurones are known to undergo both electrophysiological and neurochemical changes that alleviate this negative-feedback, thus allowing circulating prolactin levels to rise. The present study aimed to determine whether TIDA neurone morphology, most notably spine density, is also modified during lactation. This was achieved by stereotaxically injecting the arcuate nucleus of female, tyrosine hydroxylase-promoter driven Cre-recombinase transgenic rats with Cre-dependent adeno-associated virus-expressing Brainbow. This resulted in the highly specifici transfection of between 10% and 30% of the TIDA neurones, thus allowing the morphologies on multiple individual neurones to be examined in a single hypothalamic slice. The transfected neurones exhibited a range of complex forms, including a diversity of soma and location of axonal origin. Neuronal spine counting showed that the density of somatic, but not dendritic, spines was significantly higher during lactation than at any other reproductive stage. There was also a significant fall in somatic spine density across the oestrous cycle from dioestrus to oestrus. Although the functional characteristics of the additional somatic spines have not been determined, if, as might be expected, they represent an increased excitatory input to the TIDA neurones, this could have important physiological implications by perhaps supporting altered neurotransmitter release at their neuroendocrine terminals. Enhanced excitatory input may, for example, favour the release of the opioid peptide enkephalin rather than dopamine, which is potentially significant because the expression of the peptide is known to increase in the TIDA neurones during lactation and, in contrast to dopamine, it stimulates rather than inhibits prolactin secretion from the pituitary.

Presynaptic dopamine deficit in minimally conscious state patients following traumatic brain injury.

Dopaminergic stimulation has been proposed as a treatment strategy for post-traumatic brain injured patients in minimally conscious state based on a clinical trial using amantadine, a weak dopamine transporter blocker. However, a specific contribution of dopaminergic neuromodulation in minimally conscious state is undemonstrated. In a phase 0 clinical trial, we evaluated 13 normal volunteers and seven post-traumatic minimally conscious state patients using 11C-raclopride PET to estimate dopamine 2-like receptors occupancy in the striatum and central thalamus before and after dopamine transporter blockade with dextroamphetamine. If a presynaptic deficit was observed, a third and a fourth 11C-raclopride PET were acquired to evaluate changes in dopamine release induced by l-DOPA and l-DOPA+dextroamphetamine. Permutation analysis showed a significant reduction of dopamine release in patients, demonstrating a presynaptic deficit in the striatum and central thalamus that could not be reversed by blocking the dopamine transporter. However, administration of the dopamine precursor l-DOPA reversed the presynaptic deficit by restoring the biosynthesis of dopamine from both ventral tegmentum and substantia nigra. The advantages of alternative pharmacodynamic approaches in post-traumatic minimally conscious state patients should be tested in clinical trials, as patients currently refractory to amantadine might benefit from them.

Effects of epoxygenases on the nonadrenergic noncholinergic relaxant responses induced by electrical field stimulation in rabbit corpus cavernosum.

We aimed to investigate the effects of epoxygenases on electrical field stimulation (EFS)-mediated nitric oxide (NO)-dependent and NO-independent nonadrenergic noncholinergic (NANC) relaxations in isolated rabbit corpus cavernosum. The tissues of 20 male adult albino rabbits (2.5-3 kg) were suspended in organ baths containing aerated Krebs solution, and isometric contractions were recorded. EFS-mediated NANC relaxations were obtained on phenylephrin (3 × 10-5  M)-contracted tissues in the presence of guanethidine (10-6  M) and atropine (10-6  M). Miconazole (10-9 -10-4  M), 17-octadecynoic acid (ODYA) (10-10 -10-5  M), 14,15-epoxyeicosatrienoic acid (EET) (10-11 -10-8  M), 11,12-EET (10-12 -3 × 10-8  M) and 20-hydroxyeicosatetraenoic acid (HETE) (10-11 -3 × 10-8  M) were added cumulatively (n = 5-7 for each set of experiments). For NO-independent relaxations, Nω -nitro-l-arginine methyl ester (l-NAME) (10-4  M) was added before a group of experiments. Depending on the concentration, miconazole, 17-ODYA, 14,15-EET, 11,12-EET, and 20-HETE significantly enhanced both NO-dependent and NO-independent EFS-mediated relaxations (p < 0.05). Epoxygenases showed similar effect on NO-dependent and NO-independent relaxant responses except 20-HETE which caused significantly more enhanced relaxation on NO-dependent responses (p < 0.05). No drug caused a significant relaxation response on tissues contracted with phenylephrine. Epoxygenases contribute to EFS-mediated NO-dependent and NO-independent NANC relaxations by presynaptic mechanisms, offering a new treatment alternative for erectile dysfunction which needs to be explored in further in vivo, molecular and clinical studies.

Increase in Glutamatergic Terminals in the Striatum Following Dopamine Depletion in a Rat Model of Parkinson's Disease.

Dopaminergic neuron degeneration is known to give rise to dendrite injury and spine loss of striatal neurons, however, changes of intrastriatal glutamatergic terminals and their synapses after 6-hydroxydopamine (6OHDA)-induced dopamine (DA)-depletion remains controversial. To confirm the effect of striatal DA-depletion on the morphology and protein levels of corticostriatal and thalamostriatal glutamatergic terminals and synapses, immunohistochemistry, immuno-electron microscope (EM), western blotting techniques were performed on Parkinson's disease rat models in this study. The experimental results of this study showed that: (1) 6OHDA-induced DA-depletion resulted in a remarkable increase of Vesicular glutamate transporter 1 (VGlut1) + and Vesicular glutamate transporter 2 (VGlut2)+ terminal densities at both the light microscope (LM) and EM levels, and VGlut1+ and VGlut2+ terminal sizes were shown to be enlarged by immuno-EM; (2) Striatal DA-depletion resulted in a decrease in both the total and axospinous terminal fractions of VGlut1+ terminals, but the axodendritic terminal fraction was not significantly different from the control group. However, total, axospinous and axodendritic terminal fractions for VGlut2+ terminals declined significantly after striatal DA-depletion. (3) Western blotting data showed that striatal DA-depletion up-regulated the expression levels of the VGlut1 and VGlut2 proteins. These results suggest that 6OHDA-induced DA-depletion affects corticostriatal and thalamostriatal glutamatergic synaptic inputs, which are involved in the pathological process of striatal neuron injury induced by DA-depletion.

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

Chronic inflammatory pain decreases the glutamate vesicles in presynaptic terminals of the nucleus accumbens.

Reward system has been proved to be important to nociceptive behavior, and the nucleus accumbens (NAc) is a key node in reward circuitry. It has been further revealed that dopamine system modulates the NAc to influence the pain sensation, whereas the role of glutamatergic projection in the NAc in the modulation of chronic pain is still elusive. In this study, we used a complete Freund's adjuvant-induced chronic inflammatory pain model to explore the changes of the glutamatergic terminals in the NAc, and we found that following the chronic inflammation, the protein level of vesicular glutamate transporter1 (VGLUT1) was significantly decreased in the NAc. Immunofluorescence staining further showed a reduced expression of VGLUT1-positive terminals in the dopamine receptor 2 (D2R) spiny projection neurons of NAc after chronic inflammatory pain. Furthermore, using a whole-cell recording in double transgenic mice, in which dopamine receptor 1- and D2R-expressing neurons can be visualized, we found that the frequency of spontaneous excitatory postsynaptic currents was significantly decreased and paired-pulse ratio of evoked excitatory postsynaptic currents was increased in D2R neurons, but not in dopamine receptor 1 neurons in NAc of complete Freund's adjuvant group. Moreover, the abnormal expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex contributed to the reduced formation of glutamate vesicles. Hence, our results demonstrated that decreased glutamate release in the indirect pathway of the NAc may be a critical mechanism for chronic pain and provided a novel evidence for the presynaptic mechanisms in chronic pain regulation.

Dietary therapy restores glutamatergic input to orexin/hypocretin neurons after traumatic brain injury in mice.

Study Objectives: In previous work, dietary branched-chain amino acid (BCAA) supplementation, precursors to de novo glutamate and γ-aminobutyric acid (GABA) synthesis, restored impaired sleep-wake regulation and orexin neuronal activity following traumatic brain injury (TBI) in mice. TBI was speculated to reduce orexin neuronal activity through decreased regional excitatory (glutamate) and/or increased inhibitory (GABA) input. Therefore, we hypothesized that TBI would decrease synaptic glutamate and/or increase synaptic GABA in nerve terminals contacting orexin neurons, and BCAA supplementation would restore TBI-induced changes in synaptic glutamate and/or GABA. Methods: Brain tissue was processed for orexin pre-embed diaminobenzidine labeling and glutamate or GABA postembed immunogold labeling. The density of glutamate and GABA immunogold within presynaptic nerve terminals contacting orexin-positive lateral hypothalamic neurons was quantified using electron microscopy in three groups of mice (n = 8 per group): Sham/noninjured controls, TBI without BCAA supplementation, and TBI with BCAA supplementation (given for 5 days, 48 hr post-TBI). Glutamate and GABA were also quantified within the cortical penumbral region (layer VIb) adjacent to the TBI lesion. Results: In the hypothalamus and cortex, TBI decreased relative glutamate density in presynaptic terminals making axodendritic contacts. However, BCAA supplementation only restored relative glutamate density within presynaptic terminals contacting orexin-positive hypothalamic neurons. BCAA supplementation did not change relative glutamate density in presynaptic terminals making axosomatic contacts, or relative GABA density in presynaptic terminals making axosomatic or axodendritic contacts, within either the hypothalamus or cortex. Conclusions: These results suggest TBI compromises orexin neuron function via decreased glutamate density and highlight BCAA supplementation as a potential therapy to restore glutamate density to orexin neurons.

Parvalbumin-producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice.

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor-related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)-producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor-related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno-associated virus vector combined with immunodetection of pre- and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway-specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Prostaglandin E2 modulates presynaptic regulation of GnRH neurons via EP4 receptors in accordance with estrogen milieu.

Prostaglandin E2 (PGE2) promotes gonadotropin secretion by regulating the activity of neurons that release gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, the mechanisms of action of PGE2 at these neurons have yet to be fully explored. We examined the effects of PGE2 on the generation of miniature excitatory postsynaptic currents (mEPSCs) at GnRH neurons as measured by whole-cell, patch-clamp recordings. GnRH neurons were identified in slices prepared from the preoptic areas of female GnRH-EGFP rats. Exposure to PGE2 significantly increased the frequency, but not the amplitude, of the mEPSCs generated on the day of proestrus, but neither frequency nor amplitude was altered on day 1 of diestrus. These data suggest that the action of PGE2 on mEPSC frequency varies depending on the stage of estrous. An estrogen-dependence of PGE2's action was further supported by the increased frequency, but not amplitude, of mEPSCs generated at GnRH neurons prepared from estrogen-primed ovariectomized rats. Conversely, PGE2 had no effect on mEPSC frequency or amplitude at GnRH neurons in cholesterol-treated rats. Subsequent experiments to identify candidate receptors for PG2E's action revealed that exposure to a PGE2 receptor 4 (EP4) agonist, but not EP1 or EP2 agonists, mimicked the effects achieved by PGE2 exposure. These effects of mEPSCs could be reversed using an EP4 antagonist, illustrating the specificity of the effect. Collectively, these data demonstrate that PGE2 can alter excitatory synaptic neurotransmission at GnRH neurons via EP4 signaling at presynaptic site(s) in an estrogen-dependent fashion during proestrus.

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation.

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons.

Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro.

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.

O-GlcNAc transferase regulates excitatory synapse maturity.

Experience-driven synaptic plasticity is believed to underlie adaptive behavior by rearranging the way neuronal circuits process information. We have previously discovered that O-GlcNAc transferase (OGT), an enzyme that modifies protein function by attaching ß-N-acetylglucosamine (GlcNAc) to serine and threonine residues of intracellular proteins (O-GlcNAc), regulates food intake by modulating excitatory synaptic function in neurons in the hypothalamus. However, how OGT regulates excitatory synapse function is largely unknown. Here we demonstrate that OGT is enriched in the postsynaptic density of excitatory synapses. In the postsynaptic density, O-GlcNAcylation on multiple proteins increased upon neuronal stimulation. Knockout of the OGT gene decreased the synaptic expression of the AMPA receptor GluA2 and GluA3 subunits, but not the GluA1 subunit. The number of opposed excitatory presynaptic terminals was sharply reduced upon postsynaptic knockout of OGT. There were also fewer and less mature dendritic spines on OGT knockout neurons. These data identify OGT as a molecular mechanism that regulates synapse maturity.

An Ultrastructural Study of the Thalamic Input to Layer 4 of Primary Motor and Primary Somatosensory Cortex in the Mouse.

The traditional classification of primary motor cortex (M1) as an agranular area has been challenged recently when a functional layer 4 (L4) was reported in M1. L4 is the principal target for thalamic input in sensory areas, which raises the question of how thalamocortical synapses formed in M1 in the mouse compare with those in neighboring sensory cortex (S1). We identified thalamic boutons by their immunoreactivity for the vesicular glutamate transporter 2 (VGluT2) and performed unbiased disector counts from electron micrographs. We discovered that the thalamus contributed proportionately only half as many synapses to the local circuitry of L4 in M1 compared with S1. Furthermore, thalamic boutons in M1 targeted spiny dendrites exclusively, whereas ∼9% of synapses were formed with dendrites of smooth neurons in S1. VGluT2+ boutons in M1 were smaller and formed fewer synapses per bouton on average (1.3 vs 2.1) than those in S1, but VGluT2+ synapses in M1 were larger than in S1 (median postsynaptic density areas of 0.064 µm2 vs 0.042 µm2). In M1 and S1, thalamic synapses formed only a small fraction (12.1% and 17.2%, respectively) of all of the asymmetric synapses in L4. The functional role of the thalamic input to L4 in M1 has largely been neglected, but our data suggest that, as in S1, the thalamic input is amplified by the recurrent excitatory connections of the L4 circuits. The lack of direct thalamic input to inhibitory neurons in M1 may indicate temporal differences in the inhibitory gating in L4 of M1 versus S1.SIGNIFICANCE STATEMENT Classical interpretations of the function of primary motor cortex (M1) emphasize its lack of the granular layer 4 (L4) typical of sensory cortices. However, we show here that, like sensory cortex (S1), mouse M1 also has the canonical circuit motif of a core thalamic input to the middle cortical layer and that thalamocortical synapses form a small fraction (M1: 12%; S1: 17%) of all asymmetric synapses in L4 of both areas. Amplification of thalamic input by recurrent local circuits is thus likely to be a significant mechanism in both areas. Unlike M1, where thalamocortical boutons typically form a single synapse, thalamocortical boutons in S1 usually formed multiple synapses, which means they can be identified with high probability in the electron microscope without specific labeling.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.