Amygdala Kisspeptin Neurons: Putative Mediators of Olfactory Control of the Gonadotropic Axis.

Kisspeptins and their receptors are potent regulators of the gonadotropic axis. Kisspeptin neurons are found mainly in the hypothalamic arcuate nucleus and the anteroventral periventricular nucleus. However, there is also a third population of kisspeptin neurons, located in the amygdala. We used fluorescence immunohistochemistry to quantify and localize the amygdala kisspeptin neurons and to reveal close apposition and putative innervations by vasopressinergic and tyrosine hydroxylase-positive dopaminergic neurons. Using microinjections of retro- and anterograde tracers, and viral transfection systems in rats and transgenic mice, we showed reciprocal connectivity between the accessory olfactory bulb and the amygdala kisspeptin neurons. In vitro recordings indicate an inhibitory action of kisspeptin on mitral cells in the accessory olfactory bulb. Using viral specific-cell gene expression in transgenic mice in combination with double immunofluorescence histochemistry, we found that the amygdala kisspeptin neurons also project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area. Our neuroanatomical and electrophysiological data suggest that amygdala kisspeptin neurons integrate social behaviour and odour information into GnRH neurons in the preoptic area to coordinate the gonadotropic axis and the appropriate output behaviour to odour cues.

COX-2-Derived Prostaglandin E2 Produced by Pyramidal Neurons Contributes to Neurovascular Coupling in the Rodent Cerebral Cortex.

Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE2) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE2 is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets. SIGNIFICANCE STATEMENT: Brain function critically depends on a permanent spatiotemporal match between neuronal activity and blood supply, known as NVC. In the cerebral cortex, prostaglandins are major contributors to NVC. However, their biochemical identity remains elusive and their cellular origins are still under debate. Although astrocytes can induce vasodilations through the release of prostaglandins, the recruitment of this pathway during sensory stimulation is questioned. Using multidisciplinary approaches from single-cell reverse transcriptase-PCR, mass spectrometry, to ex vivo and in vivo pharmacology and optogenetics, we provide compelling evidence identifying PgE2 as the main prostaglandin in NVC, pyramidal neurons as their main cellular source and the vasodilatory EP2 and EP4 receptors as their main targets. These original findings will certainly change the current view of NVC.

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.

Pyramidal neurons are "neurogenic hubs" in the neurovascular coupling response to whisker stimulation.

The whisker-to-barrel cortex is widely used to study neurovascular coupling, but the cellular basis that underlies the perfusion changes is still largely unknown. Here, we identified neurons recruited by whisker stimulation in the rat somatosensory cortex using double immunohistochemistry for c-Fos and markers of glutamatergic and GABAergic neurons, and investigated in vivo their contribution along with that of astrocytes in the evoked perfusion response. Whisker stimulation elicited cerebral blood flow (CBF) increases concomitantly with c-Fos upregulation in pyramidal cells that coexpressed cyclooxygenase-2 (COX-2) and GABA interneurons that coexpressed vasoactive intestinal polypeptide and/or choline acetyltransferase, but not somatostatin or parvalbumin. The evoked CBF response was decreased by blockade of NMDA (MK-801, -37%), group I metabotropic glutamate (MPEP+LY367385, -40%), and GABA-A (picrotoxin, -31%) receptors, but not by GABA-B, VIP, or muscarinic receptor antagonism. Picrotoxin decreased stimulus-induced somatosensory evoked potentials and CBF responses. Combined blockade of GABA-A and NMDA receptors yielded an additive decreasing effect (-61%) of the evoked CBF compared with each antagonist alone, demonstrating cooperation of both excitatory and inhibitory systems in the hyperemic response. Blockade of prostanoid synthesis by inhibiting COX-2 (indomethacin, NS-398), expressed by ∼40% of pyramidal cells but not by astrocytes, impaired the CBF response (-50%). The hyperemic response was also reduced (-40%) after inhibition of astroglial oxidative metabolism or epoxyeicosatrienoic acids synthesis. These results demonstrate that changes in pyramidal cell activity, sculpted by specific types of inhibitory GABA interneurons, drive the CBF response to whisker stimulation and, further, that metabolically active astrocytes are also required.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Neural connectivity between the hypothalamic supramammillary nucleus and appetite- and motivation-related regions of the rat brain.

The supramammillary nucleus (SuM) has an emerging role in appetite control. We have shown that the rat SuM is activated during hunger or food anticipation, or by ghrelin administration. In the present study, we characterised the connectivity between the SuM and key appetite- and motivation-related nuclei in the rat. In adult wild-type rats, or rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (TH-Cre rats), we used c-Fos immunohistochemistry to visualise and correlate the activation of medial SuM (SuMM) with activation in the lateral hypothalamic area (LH), the dorsomedial hypothalamus (DMH) or the ventral tegmental area (VTA) after voluntary consumption of a high-sugar, high-fat food. To determine neuroanatomical connectivity, we used retrograde and anterograde tracing methods to specifically investigate the neuronal inputs and outputs of the SuMM. After consumption of the food there were positive correlations between c-Fos expression in the SuMM and the LH, DMH and VTA (P = 0.0001, 0.01 and 0.004). Using Fluoro-Ruby as a retrograde tracer, we demonstrate the existence of inputs from the LH, DMH, VTA and ventromedial hypothalamus (VMH) to the SuMM. The SuMM showed reciprocal inputs to the LH and DMH, and we identified a TH-positive output from SuMM to DMH. We co-labelled retrogradely-labelled sections for TH in the VMH, or for TH, orexin and melanin-concentrating hormone in the LH and DMH. However, we did not observe any colocalisation of immunoreactivity with any retrogradely-labelled cells. Viral mapping in TH-Cre rats confirms the existence of a reciprocal SuMM-DMH connection and shows that TH-positive cells project from the SuMM and VTA to the lateral septal area and cingulate cortex, respectively. These data provide evidence for the connectivity of the SuMM to brain regions involved in appetite control, and form the foundation for functional and behavioural studies aiming to further characterise the brain circuitry controlling eating behaviours.