CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy.

CaV3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that CaV3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional CaV3.2 channels primarily incorporate into synapses, replace existing CaV3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCaV3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCaV3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of CaV3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.

The orexinergic neurons receive synaptic input from C1 cells in rats.

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N-methyl transferase-immunoreactive (PNMT-ir) axons were detected among orexin-ir somata, and close appositions between PNMT-ir axonal varicosities and orexin-ir profiles were observed. The existence of synapses between PNMT-ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2-mCherry under the control of the catecholaminergic neuron-selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre-dependent adeno-associated vector and TH-Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT-ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin-ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense-core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1-orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep.

Monosynaptic glutamatergic activation of locus coeruleus and other lower brainstem noradrenergic neurons by the C1 cells in mice.

The C1 neurons, located in the rostral ventrolateral medulla (VLM), are activated by pain, hypotension, hypoglycemia, hypoxia, and infection, as well as by psychological stress. Prior work has highlighted the ability of these neurons to increase sympathetic tone, hence peripheral catecholamine release, probably via their direct excitatory projections to sympathetic preganglionic neurons. In this study, we use channelrhodopsin-2 (ChR2) optogenetics to test whether the C1 cells are also capable of broadly activating the brain's noradrenergic system. We selectively expressed ChR2(H134R) in rostral VLM catecholaminergic neurons by injecting Cre-dependent adeno-associated viral vectors into the brain of adult dopamine-ß-hydroxylase (DßH)(Cre/0) mice. Most ChR2-expressing VLM neurons (75%) were immunoreactive for phenylethanolamine N-methyl transferease, thus were C1 cells, and most of the ChR2-positive axonal varicosities were immunoreactive for vesicular glutamate transporter-2 (78%). We produced light microscopic evidence that the axons of rostral VLM (RVLM) catecholaminergic neurons contact locus coeruleus, A1, and A2 noradrenergic neurons, and ultrastructural evidence that these contacts represent asymmetric synapses. Using optogenetics in tissue slices, we show that RVLM catecholaminergic neurons activate the locus coeruleus as well as A1 and A2 noradrenergic neurons monosynaptically by releasing glutamate. In conclusion, activation of RVLM catecholaminergic neurons, predominantly C1 cells, by somatic or psychological stresses has the potential to increase the firing of both peripheral and central noradrenergic neurons.

C1 neurons: the body's EMTs.

The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension.

Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent adeno-associated virus (AAV2) DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-ß-hydroxylase Cre transgenic mice (DßH(Cre/0)), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive(ir) axonal varicosities were tyrosine hydroxylase (TH)-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry colocalization was virtually absent when AAV2 was injected into the RVLM of DßH(Cre/0);VGLUT2(flox/flox) mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DßH(Cre/0) mice or into the raphe of ePet(Cre/0) mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase and VGLUT2 immunoreactivities were highly colocalized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DßH(Cre/0) mice made predominantly asymmetric synapses with choline acetyl-transferase-ir DMV neurons. Photostimulation of ChR2-positive axons in DßH(Cre/0) mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/s (current-clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine, and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic PGNs monosynaptically and this connection uses glutamate as an ionotropic transmitter.

Pre-Bötzinger complex receives glutamatergic innervation from galaninergic and other retrotrapezoid nucleus neurons.

The retrotrapezoid nucleus (RTN) contains CO(2) -responsive neurons that regulate breathing frequency and amplitude. These neurons (RTN-Phox2b neurons) contain the transcription factor Phox2b, vesicular glutamate transporter 2 (VGLUT2) mRNA, and a subset contains preprogalanin mRNA. We wished to determine whether the terminals of RTN-Phox2b neurons contain galanin and VGLUT2 proteins, to identify the specific projections of the galaninergic subset, to test whether RTN-Phox2b neurons contact neurons in the pre-Bötzinger complex, and to identify the ultrastructure of these synapses. The axonal projections of RTN-Phox2b neurons were traced by using biotinylated dextran amine (BDA), and many BDA-ir boutons were found to contain galanin immunoreactivity. RTN galaninergic neurons had ipsilateral projections that were identical with those of this nucleus at large: the ventral respiratory column, the caudolateral nucleus of the solitary tract, and the pontine Kölliker-Fuse, intertrigeminal region, and lateral parabrachial nucleus. For ultrastructural studies, RTN-Phox2b neurons (galaninergic and others) were transfected with a lentiviral vector that expresses mCherry almost exclusively in Phox2b-ir neurons. After spinal cord injections of a catecholamine neuron-selective toxin, there was a depletion of C1 neurons in the RTN area; thus it was determined that the mCherry-positive terminals located in the pre-Bötzinger complex originated almost exclusively from the RTN-Phox2b (non-C1) neurons. These terminals were generally VGLUT2-immunoreactive and formed numerous close appositions with neurokinin-1 receptor-ir pre-Bötzinger complex neurons. Their boutons (n = 48) formed asymmetric synapses filled with small clear vesicles. In summary, RTN-Phox2b neurons, including the galaninergic subset, selectively innervate the respiratory pattern generator plus a portion of the dorsolateral pons. RTN-Phox2b neurons establish classic excitatory glutamatergic synapses with pre-Bötzinger complex neurons presumed to generate the respiratory rhythm.

Activity patterns govern synapse-specific AMPA receptor trafficking between deliverable and synaptic pools.

In single neurons, glutamatergic synapses receiving distinct afferent inputs may contain AMPA receptors (-Rs) with unique subunit compositions. However, the cellular mechanisms by which differential receptor transport achieves this synaptic diversity remain poorly understood. In lateral geniculate neurons, we show that retinogeniculate and corticogeniculate synapses have distinct AMPA-R subunit compositions. Under basal conditions at both synapses, GluR1-containing AMPA-Rs are transported from an anatomically defined reserve pool to a deliverable pool near the postsynaptic density (PSD), but further incorporate into the PSD or functional synaptic pool only at retinogeniculate synapses. Vision-dependent activity, stimulation mimicking retinal input, or activation of CaMKII or Ras signaling regulated forward GluR1 trafficking from the deliverable pool to the synaptic pool at both synapses, whereas Rap2 signals reverse GluR1 transport at retinogeniculate synapses. These findings suggest that synapse-specific AMPA-R delivery involves constitutive and activity-regulated transport steps between morphological pools, a mechanism that may extend to the site-specific delivery of other membrane protein complexes.

Ras signaling mechanisms underlying impaired GluR1-dependent plasticity associated with fragile X syndrome.

Fragile X syndrome, caused by the loss of FMR1 gene function and loss of fragile X mental retardation protein (FMRP), is the most commonly inherited form of mental retardation. The syndrome is characterized by associative learning deficits, reduced risk of cancer, dendritic spine dysmorphogenesis, and facial dysmorphism. However, the molecular mechanism that links loss of function of FMR1 to the learning disability remains unclear. Here, we report an examination of small GTPase Ras signaling and synaptic AMPA receptor (AMPA-R) trafficking in cultured slices and intact brains of wild-type and FMR1 knock-out mice. In FMR1 knock-out mice, synaptic delivery of GluR1-, but not GluR2L- and GluR4-containing AMPA-Rs is impaired, resulting in a selective loss of GluR1-dependent long-term synaptic potentiation (LTP). Although Ras activity is upregulated, its downstream MEK (extracellular signal-regulated kinase kinase)-ERK (extracellular signal-regulated kinase) signaling appears normal, and phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB; or Akt) signaling is compromised in FMR1 knock-out mice. Enhancing Ras-PI3K-PKB signaling restores synaptic delivery of GluR1-containing AMPA-Rs and normal LTP in FMR1 knock-out mice. These results suggest aberrant Ras signaling as a novel mechanism for fragile X syndrome and indicate manipulating Ras-PI3K-PKB signaling to be a potentially effective approach for treating patients with fragile X syndrome.