Excitotoxic stimulation activates distinct pathogenic and protective expression signatures in the hippocampus.

Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N-methyl-d-aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology-specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24-h post-insult. Egr-1 and NOR-1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA-induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up-regulated pathogenic genes, down-regulated pro-survival genes) and opposing compensatory responses (down-regulated pathogenic genes, up-regulated pro-survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF-κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF-κB inhibitor MG-132 indeed altered NMDA-mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95.

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

Repetitive optogenetic stimulation of glutamatergic neurons: An alternative to NMDA treatment for generating locomotor activity in spinalized zebrafish larvae.

N-methyl-d-aspartate (NMDA) application has conventionally been used to activate spinal networks to induce locomotion in spinalized animals. We recently described an alternative approach in which application of continuous blue light activates channelrhodopsin-2 in vesicular glutamate transporter 2a (vglut2a)-expressing spinal neurons to produce organized, rhythmic locomotor activity in spinally-transected larval zebrafish. This technique arguably enhances research validity, because endogenous glutamate is released into existing synapses instead of activating only a subset of glutamatergic (NMDA) receptors with an exogenous compound. Here, we explored the viability of this approach in the context of using it for longer-term experiments. Fictive swimming was induced through repetitive application of 10-s blue light stimuli to spinalized preparations for up to 60 min at intervals of 1, 3, or 15 min. Locomotor activity was maintained throughout the experimental timecourse, demonstrating the robustness of the system. Although locomotor bursts remained organized into episodes of activity, the number of bursts elicited during each successive stimulus decreased. This was in contrast to NMDA bath application, in which bursts became less episodically organized while the overall number of bursts remained unchanged. The efficacy of the repetitive optogenetic stimulation paradigm was demonstrated through application of exogenous dopamine, which reversibly decreased the number of bursts produced per stimulus compared with untreated preparations. Finally, increasing the stimulus interval to 15 min lessened, but did not eliminate locomotor fatigue from repetitive activation. Altogether, we established repetitive optogenetic stimulation of vglut2a-expressing neurons as a viable alternative to NMDA application for activation of the zebrafish spinal locomotor network.

Perineural high-mobility group box 1 induces mechanical hypersensitivity through activation of spinal microglia: Involvement of glutamate-NMDA receptor dependent mechanism in spinal dorsal horn.

High mobility box 1 (HMGB1), a damage-associated molecular pattern, has crucial roles in induction of neuropathic pain. Upregulation of HMGB1 around the injured sciatic nerve contributes to mechanical hypersensitivity following partial sciatic nerve ligation (PSNL) of mice. However, central mechanisms mediating perineural HMGB1-induced nociceptive hypersensitivity, especially within the spinal dorsal horn, have not been determined. The current study shows that perineural treatment of naïve mice with recombinant HMGB1, which mimics increased HMGB1 around the injured sciatic nerve of PSNL mice, significantly induced activation of microglia, but not astrocytes, in the spinal dorsal horn. Intraperitoneal injection of minocycline, a microglial inhibitor, ameliorated perineural rHMGB1-induced mechanical hypersensitivity. In addition, blockade of spinal N-methyl-D-aspartate (NMDA) receptors significantly prevented perineural rHMGB1-induced mechanical hypersensitivity and microglial activation. In contrast, non-NMDA receptors, neurokinin 1 receptor, colony-stimulating factor 1 receptor and P2Y12 receptor were not involved in perineural rHMGB1-induced mechanical hypersensitivity. Furthermore, repeated perineural treatment with an anti-HMGB1 antibody blocked activation of spinal microglia in PSNL mice. Collectively, the current findings demonstrate that increased HMGB1 around injured sciatic nerve might induce nociceptive hypersensitivity through activation of spinal microglia. Thus, HMGB1-dependent mechanisms between the injured sciatic nerve and spinal dorsal horn could be crucial in induction of neuropathic pain.

Tyrosine triple mutated AAV2-BDNF gene therapy in an inner retinal injury model induced by intravitreal injection of N-methyl-D-aspartate (NMDA).

Purpose: Glaucoma is a group of chronic optic neuropathies characterized by the degeneration of retinal ganglion cells (RGCs) and their axons, and they ultimately cause blindness. Because neuroprotection using neurotrophic factors against RGC loss has been proven a beneficial strategy, extensive attempts have been made to perform gene transfer of neurotrophic proteins. This study used the inner retinal injury mouse model to evaluate the neuroprotective effect of tyrosine triple mutated and self-complementary adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF; tm-scAAV2-BDNF). Methods: C57BL/6J mice were intravitreally injected with 1 µl of tm-scAAV2-BDNF and its control AAV at a titer of 6.6 E+13 genome copies/ml. Three weeks later, 1 µl of 2 mM N-methyl-D-aspartate (NMDA) was administered in the same way as the viral injection. Six days after the NMDA injection, we assessed the dark-adapted electroretinography (ERG). Mice were sacrificed at one week after the NMDA injection, followed by RNA quantification, protein detection, and histopathological analysis. Results: The RNA expression of BDNF in retinas treated with tm-scAAV2-BDNF was about 300-fold higher than that of its control AAV. Meanwhile, the expression of recombinant BDNF protein increased in retinas treated with tm-scAAV2-BDNF. In addition, histological analysis revealed that tm-scAAV2-BDNF prevented thinning of the inner retina. Furthermore, b-wave amplitudes of the tm-scAAV2-BDNF group were significantly higher than those of the control vector group. Histopathological and electrophysiological evaluations showed that tm-scAAV2-BDNF treatment offered significant protection against NMDA toxicity. Conclusions: Results showed that tm-scAAV2-BDNF-treated retinas were resistant to NMDA injury, while retinas treated with the control AAV exhibited histopathological and functional changes after the administration of NMDA. These results suggest that tm-scAAV2-BDNF is potentially effective against inner retinal injury, including normal tension glaucoma.

Outer retinal involvement in N-methyl-D-aspartate-induced inner retinal injury in rabbits assessed by optical coherence tomography.

This study was aimed to investigate morphological alteration of the retina with N-methyl-D-aspartate (NMDA)-induced injury in rabbits by optical coherence tomography (OCT). The right and left eyes of a total of 12 rabbits received single-intravitreal injection of vehicle and NMDA, respectively. Four out of the 12 animals underwent OCT and quantification of plasma microRNA repeatedly (4, 48, and 168 hr after dosing), followed by ocular histopathology at the end of the study. Ocular histopathology was also conducted in the eyes collected 4 or 48 hr after dosing from 4 animals at each time period. OCT revealed hyper-reflective ganglion cell complex and thickened inner retina in NMDA-treated eyes 4 hr after dosing; the inner retina shifted to thinning at later time points. The eyes given NMDA also exhibited greater thickness of the outer retina, which contains photoreceptors, after treatment, and thickened and obscured ellipsoid zone 168 hr after dosing. The plasma levels of miR-182 and miR-183, which are known to be highly expressed in photoreceptors, were higher 4 hr after dosing than pre-dosing values. Histopathologically, NMDA-induced inner retinal damage was confirmed: single-cell necrosis was observed in the ganglion cell layer and the inner nuclear layer 4 hr after dosing, the incidence of which decreased thereafter. At 168 hr after dosing, reduced number of ganglion cells was noted. No change was histopathologically observed in the outer retina. In conclusion, our results suggest involvement of photoreceptors in NMDA-induced inner retinal injury. Additionally, OCT revealed acute inner retinal findings suggestive of temporary edema.

Modulation of NR1 receptor by CaMKIIα plays an important role in chronic itch development in mice.

Intractable scratching is the characteristic of chronic itch, which represents a great challenge in clinical practice. However, the mechanism underlying chronic itch development is largely unknown. In the present study, we investigated the role of NMDA receptor in acute itch and in development of chronic itch. A mouse model was developed by painting DNFB to induce allergic contact dermatitis (ACD). We found that the expression of pNR1, which is a subunit of NMDA receptor, was significantly increased in the dorsal root ganglion in the DNFB model. The DNFB-evoked spontaneous scratching was blocked by the NMDA antagonist D-AP-5, the calcium-calmodulin-dependent protein kinase (CaMK) inhibitor KN-93, a CaMKIIα siRNA and the PKC inhibitor LY317615. Moreover, activation of PKC did not reverse the CaMKIIα knockdown-induced decrease in scratching, suggesting that PKC functions upstream of CaMKIIα. Thus, our study indicates that modulation of NR1 receptor by CaMKIIα plays an important role in the development of chronic itch.

Activation of medial orbitofrontal cortex abolishes fear extinction and interferes with fear expression in rats.

Pavlovian fear conditioning and extinction procedures have long been used to study the regulation of learned fear. The amygdala is vital for the association of cues and fear expression, whereas the medial prefrontal cortex (mPFC) is critical for fear regulation after extinction. The medial orbitofrontal cortex (mOFC) has an extensive connection with the fear circuit. In human studies, emotional regulation disorders, such as obsessive-compulsive disorder and post-traumatic stress disorder, are often linked to an abnormality in the orbitofrontal cortex (OFC). Therefore, in a series of experiments, we examined whether abnormal mOFC activities interfere with the regulation of learned fear. The mOFC of rats was pharmacologically activated with N-methyl-D-aspartate (NMDA) during the acquisition, early consolidation, or retrieval phase of fear extinction. Under mOFC activation, there was a general initial suppression of the fear response followed by the development of nonspecific fear expression. Moreover, pre-extinction activation of the mOFC abolished extinction acquisition, causing an up-shift in the fear response during the retrieval test. Nonetheless, immediate post-extinction activation of the mOFC did not interfere with extinction consolidation. Overall, our results suggested that mOFC activation abolished fear extinction acquisition and interfered with fear expression.

Cannabidiol-induced panicolytic-like effects and fear-induced antinociception impairment: the role of the CB1 receptor in the ventromedial hypothalamus.

RATIONALE: The behavioural effects elicited by chemical constituents of Cannabis sativa, such as cannabidiol (CBD), on the ventromedial hypothalamus (VMH) are not well understood. There is evidence that VMH neurons play a relevant role in the modulation of unconditioned fear-related defensive behavioural reactions displayed by laboratory animals. OBJECTIVES: This study was designed to explore the specific pattern of distribution of the CB1 receptors in the VMH and to investigate the role played by this cannabinoid receptor in the effect of CBD on the control of defensive behaviours and unconditioned fear-induced antinociception. METHODS: A panic attack-like state was triggered in Wistar rats by intra-VMH microinjections of N-methyl-D-aspartate (NMDA). One of three different doses of CBD was microinjected into the VMH prior to local administration of NMDA. In addition, the most effective dose of CBD was used after pre-treatment with the CB1 receptor selective antagonist AM251, followed by NMDA microinjections in the VMH. RESULTS: The morphological procedures demonstrated distribution of labelled CB1 receptors on neuronal perikarya situated in dorsomedial, central and ventrolateral divisions of the VMH. The neuropharmacological approaches showed that both panic attack-like behaviours and unconditioned fear-induced antinociception decreased after intra-hypothalamic microinjections of CBD at the highest dose (100 nmol). These effects, however, were blocked by the administration of the CB1 receptor antagonist AM251 (100 pmol) in the VMH. CONCLUSION: These findings suggest that CBD causes panicolytic-like effects and reduces unconditioned fear-induced antinociception when administered in the VMH, and these effects are mediated by the CB1 receptor-endocannabinoid signalling mechanism in VMH.

Regional Differences Within the Anterior Cingulate Cortex in the Generation Versus Suppression of Pain Affect in Rats.

The anterior cingulate cortex (ACC) modulates emotional responses to pain. Whereas, the caudal ACC (cACC) promotes expression of pain affect, the rostral ACC (rACC) contributes to its suppression. Both subdivisions receive glutamatergic innervation, and the present study evaluated the contribution of N-methyl-d-aspartic acid (NMDA) receptors within these subdivisions to rats' expression of pain affect. Vocalizations that follow a brief noxious tail shock (vocalization afterdischarges, VAD) are a validated rodent model of pain affect. The threshold current for eliciting VAD was increased in a dose-dependent manner by injecting NMDA into the rACC, but performance (latency, amplitude, and duration) at threshold was not altered. Alternately, the threshold current for eliciting VAD was not altered following injection of NMDA into the cACC, but its amplitude and duration at threshold were increased in a dose-dependent manner. These effects were limited to Cg1 of the rACC and cACC, and blocked by pretreatment of the ACC with the NMDA receptor antagonist d-2-amino-5-phosphonovalerate. These findings demonstrate that NMDA receptor agonism within the cACC and rACC either increases or decreases emotional responses to noxious stimulation, respectively. PERSPECTIVE: NMDA receptor activation of the rostral and caudal ACC respectively inhibited or enhanced rats' emotional response to pain. These findings mirror those obtained from human neuroimaging studies; thereby, supporting the use of this model system in evaluating the contribution of ACC to pain affect.

Adenosine and NMDA Receptors Modulate Neuroprotection-Induced NMDA Preconditioning in Mice.

The severity score of quinolinic acid (QA)-induced seizures was investigated after N-methyl-D-aspartate (NMDA) preconditioning associated with adenosine receptors. Also, the levels of adenosine A1 and A2A receptors and subunits of NMDA receptors in the hippocampi of mice were determined to define components of the resistance mechanism. Adult CF-1 mice were treated intraperitoneally with saline or NMDA (75 mg/kg), and some mice were treated intracerebroventricularly (i.c.v.) with 0.1 pmol of adenosine receptor antagonists 8-cyclopentyltheophylline (CPT; receptor A1) or ZM241385 (receptor A2A) 0, 1, or 6 h after NMDA administration. These adenosine receptor antagonists were administered to block NMDA's protective effect. Seizures and their severity scores were evaluated during convulsions induced by QA (36.8 nmol) that was administered i.c.v. 24 h after NMDA. The cell viability and content of subunits of the NMDA receptors were analyzed 24 h after QA administration. NMDA preconditioning reduced the maximal severity 6 displayed in QA-administered mice, inducing protection in 47.6% of mice after QA-induced seizures. CPT increased the latency of seizures when administered 0 or 6 h, and ZM241385 generated the same effect when administered 6 h after NMDA administration. The GluN1 content was lower in the hippocampi of the QA mice and the NMDA-preconditioned animals without seizures. GluN2A content was unaltered in all groups. The results demonstrated the components of resistance evoked by NMDA, in which adenosine receptors participate in a time-dependent mode. Similarly, the reduction on GluN1 expression in the hippocampus may contribute to this effect during the preconditioning period.

Regulatory role of hippocampal PI3K and mTOR signaling pathway in NMDA-induced infant spasm rats.

Objectives: To explore the role of mTOR signaling pathway in modulating epileptogenesis in an N-methyl-D-aspartic acid (NMDA)-induced infant spasm (IS) rat model. Methods: After inducing IS successfully, the phosphorylation status of PI3K, Akt, mTOR and S6K of brain and hippocampus tissues was assessed using western blot and immunochemistry analysis, respectively. The possible mechanism of mTOR signaling pathway was evaluated by the, inhibitors for mTOR and PI3K, rapamycin and wortmannin, respectively. The inhibitors were injected into the intraperitoneal space of the rats to examine the effects of PI3K and mTOR in IS rat model. Results: The phosphorylated levels of mTOR and PI3K in hippocampus increased significantly (P < 0.05) 7 days after IS induction in rats. After administration of wortmannin, the phosphorylated levels of PI3K and mTOR decreased. However, only the phosphorylated level of mTOR decreased obviously after rapamycin administration. No obvious neurogenesis was found after IS induction. Discussion: Results of the present study suggest that hippocampal PI3K may be another potential target for IS treatment.

Transplantation of Retinal Ganglion Cells Derived from Male Germline Stem Cell as a Potential Treatment to Glaucoma.

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

How can methylprednisolone work on epileptic spasms with malformation of cortical development?

Although steroids are suggested as the treatment of choice for infantile spasms, the mechanism of action is still unclear. Using a rat model of malformation of cortical development with refractory infantile spasms, we evaluated the efficacy of methylprednisolone on spasms susceptibility and behaviors. Additionally, we investigated the in vivo electrophysiological and neurochemical changes of the brain after methylprednisolone treatment. Infant rats with prenatal exposure of methylazoxymethanol at gestational day 15 were used. After a single dose of methylprednisolone or three different doses of methylprednisolone for 3 days, spasms were triggered by intraperitoneal injection of N-methyl-d-aspartic acid. In rats with 3 days of methylprednisolone pretreatment and their controls, behavioral testing was performed at postnatal day 15. In vivo magnetic resonance imaging was conducted at postnatal day 15 after 3 days of methylprednisolone treatment. The rats with single methylprednisolone pretreatment showed significantly delayed onset of spasms and multiple doses of methylprednisolone significantly suppressed the development of spasms in a dose-dependent manner. After multiple methylprednisolone pretreatment and a cluster of N-methyl-d-aspartic acid-induced spasms, the rats showed significantly increased freezing behaviors to conditioned stimuli. Glutamate-weighted chemical exchange saturation transfer revealed significant elevation of glutamate concentration in the cortices of the rats with multiple methylprednisolone pretreatments. Methylprednisolone pretreatment could attenuate N-methyl-d-aspartic acid-induced spasms with in vivo neurochemical and electrophysiological changes, which indicates this steroid's action on the brain and in epilepsy.

Modulation of NMDA-mediated intrinsic membrane properties of ascending commissural interneurons in neonatal rat spinal cord.

In this paper, the modulation of ascending commissural interneurons by N-methyl-D-aspartate was investigated in neonatal rats by using retrograde labeling and whole-cell patch clamp. Data shows these interneurons can be divided into three types (single spike, phasic, and tonic) based on their firing patterns. A hyperpolarization-activated nonselective cation current and persistent inward current are expressed in these interneurons. The parameters studied (n = 48) include: resting membrane potential (-59.2 ± 0.8 mV), input resistance (964.4 ± 49.3 MΩ), voltage threshold (-39.5 ± 0.6 mV), rheobase (13.5 ± 0.7 pA), action potential height (55.6 ± 2.2 mV), action potential half-width (2.8 ± 0.1 ms), afterhyperpolarization magnitude (16.1 ± 1.2 mV) and half-decay (217.9 ± 10.7 ms). 10 µM N-methyl-D-aspartate increases excitability of ascending commissural interneurons by depolarizing the membrane potential, hyperpolarizing voltage threshold, reducing rheobase, and shifting the frequency-current relationship to the left. N-methyl-Daspartate enhances persistent inward currents but reduces hyperpolarization-activated nonselective cation currents. This research uncovers unique ionic and intrinsic properties of ascending commissural interneurons which can be modulated by major excitatory neurotransmitters such as N-methyl-D-aspartate to potentially facilitate left-right alternation during locomotion.

Specific inter-stimulus interval effect of NMDA receptor activation in the insular cortex during conditioned taste aversion.

Taste memory recognition is crucial for species survival; thus, the acquisition of conditioned taste aversion (CTA) protects animals against consuming poisons or toxins. In nature, food and poison are confined in the same edible item; however, in the laboratory these food constituents are usually presented separately for experimental analysis. The taste, or conditioned stimulus (CS), can be hours apart from the gastric malaise, or unconditioned stimulus (US); this extended inter-stimulus interval (ISI) allows the analysis of a particular learning phase. Evidence indicates a relevant function of glutamatergic activity in the insular cortex (IC) throughout the ISI. N-methyl-D-aspartate receptors (NMDAR) are crucial during CTA acquisition and retrieval. However, the exact participation of NMDAR in the IC during the ISI has not been demonstrated. Thus, the aim of this work was to evaluate the effects of temporal NMDAR activation during four time frames throughout the ISI of conditioned sugar aversion with bilateral injections of NMDA at a physiological dose (1 µg/µl) in the IC, given (1) immediately before or (2) immediately after sugar presentation, or (3) immediately before or (4) immediately after LiCl i.p. injection. The results showed that NMDAR activation in the IC had a specific ISI effect during CTA acquisition, increasing aversive memory formation and delaying extinction only after CS presentation. Overall, these results demonstrate that NMDAR in the IC have a particular enhancing associative effect after CS and suggest that there is a precise coincidence in neurochemical events in the IC that correlates with the stimulus to be associated and the glutamate NMDAR activity that must be finely tuned in the ISI during CTA acquisition.

Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity.

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

Restoring retinal neurovascular health via substance P.

Regulation of vascular permeability plays a major role in the pathophysiology of visually threatening conditions such as retinal vein occlusion and diabetic retinopathy. Principally, several factors such as vascular endothelial growth factor (VEGF), are up-regulated or induced in response to hypoxia thus adversely affecting the blood-retinal barrier (BRB), resulting in retinal edema and neovascularisation. Furthermore, current evidence supports a dysregulation of the inner retinal neural-vascular integrity as a critical factor driving retinal ganglion cell (RGC) death and visual loss. The principal objective of this study was to interrogate whether Substance P (SP), a constitutive neurotransmitter of amacrine and ganglion cells, may protect against N-methyl-d-aspartate (NMDA)-induced excitotoxic apoptosis of ganglion cells and VEGF-induced vessel leakage in the retina. Tight junctional protein expression and a Vascular Permeability Image Assay were used to determine vascular integrity in vitro. The protective effect of SP on RGC was established in ex vivo retinal explants and in vivo murine models. After NMDA administration, a reduction in TUNEL+ cells and a maintained number of Brn-3a+ cells were found, indicating an inhibition of RGC apoptosis mediated by SP. Additionally, SP maintained endothelial tight junctions and decreased VEGF-induced vascular permeability. In conclusion, administration of SP protects against NMDA apoptosis of RGC and VEGF-induced endothelial barrier breakdown.

Acupuncture reduces nicotine-induced norepinephrine release in the hypothalamus via the solitary NMDA receptor/NOS pathway.

Noradrenergic projections from the nucleus tractus solitarius (NTS) to the hypothalamic paraventricular nucleus (PVN) are involved in nicotine (Nic) dependence. Nic induces hypothalamic norepinephrine (NE) release through N-methyl-d-aspartate receptors (NMDARs) and nitric oxide in the NTS. However, acupuncture attenuates Nic withdrawal-induced anxiety. Therefore, this study investigated the effects of acupuncture on Nic-induced hypothalamic NE release. Rats received an intravenous infusion of Nic (90 µg/kg, over 60 s) and extracellular NE levels in the PVN were determined by in vivo microdialysis. Immediately after Nic administration, the rats were bilaterally treated with acupuncture at acupoint HT7 (Shen-Men) or PC6 (Nei-Guan), or a non-acupoint (tail) for 60 s. Acupuncture at HT7, but not at PC6 or the tail, significantly reduced Nic-induced NE release. However, this was abolished by a post-acupuncture infusion of either NMDA or sodium nitroprusside into the NTS. Additionally, acupuncture at HT7, but not the control points, prevented Nic-induced plasma corticosterone secretion and inhibited Nic-induced increases in the phosphorylation of neuronal nitric oxide synthase (nNOS) and endothelial NOS in the NTS. These findings suggest that acupuncture at HT7 reduces Nic-induced NE release in the PVN via inhibition of the solitary NMDAR/NOS pathway.