Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors

Increasing attention is being focused on the use of polypeptide-based N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of nervous system disorders. In our study on Achyranthes bidentata Blume, we identified an NMDA receptor subtype 2B (NR2B) antagonist that exerts distinct neuroprotective actions. This antagonist is a 33 amino acid peptide, named bidentatide, which contains three disulfide bridges that form a cysteine knot motif. We determined the neuroactive potential of bidentatide by evaluating its in vitro effects against NMDA-mediated excitotoxicity. The results showed that pretreating primary cultured hippocampal neurons with bidentatide prevented NMDA-induced cell death and apoptosis via multiple mechanisms that involved intracellular Ca2+ inhibition, NMDA current inhibition, and apoptosis-related protein expression regulation. These mechanisms were all dependent on bidentatide-induced inhibitory regulation of NR2B-containing NMDA receptors; thus, bidentatide may contribute to the development of neuroprotective agents that would likely possess the high selectivity and safety profiles inherent in peptide drugs.

Computational Evidence for a Competitive Thalamocortical Model of Spikes and Spindle Activity in Rolandic Epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy syndrome, characterized by sleep-activated epileptiform spikes and seizures and cognitive deficits in school age children. Recent evidence suggests that this disease may be caused by disruptions to the Rolandic thalamocortical circuit, resulting in both an abundance of epileptiform spikes and a paucity of sleep spindles in the Rolandic cortex during non-rapid eye movement sleep (NREM); electrographic features linked to seizures and cognitive symptoms, respectively. The neuronal mechanisms that support the competitive shared thalamocortical circuitry between pathological epileptiform spikes and physiological sleep spindles are not well-understood. In this study we introduce a computational thalamocortical model for the sleep-activated epileptiform spikes observed in RE. The cellular and neuronal circuits of this model incorporate recent experimental observations in RE, and replicate the electrophysiological features of RE. Using this model, we demonstrate that: (1) epileptiform spikes can be triggered and promoted by either a reduced NMDA current or h-type current; and (2) changes in inhibitory transmission in the thalamic reticular nucleus mediates an antagonistic dynamic between epileptiform spikes and spindles. This work provides the first computational model that both recapitulates electrophysiological features and provides a mechanistic explanation for the thalamocortical switch between the pathological and physiological electrophysiological rhythms observed during NREM sleep in this common epileptic encephalopathy.

Forced swim stress dynamically alters neural activity and bi-directionally modulates NMDA receptors in the prefrontal cortex

Repeated exposure to stress results in progressively divergent effects on cognitive behaviors that are dependent on the integrity of networks in the medial prefrontal cortex (mPFC). To investigate molecular mechanisms responsive to variable repetition of mild stress, we measured persistent neural activity, in vitro, from mPFC slices in mice that had been repetitively exposed to 10 minutes of forced swim stress for 3-10 days. 3-day short-term stress facilitated persistent neural activity by increasing event duration while 10 days suppressed event duration and amplitude. These dynamic changes were accompanied by a similar bi-directional modulation of the NMDA/AMPA receptor current ratio, an important synaptic mechanism for sustaining the persistency of neural activity. Specifically, short-term stress led to potentiated NMDA currents with slower decay kinetics, and extended stress produced smaller currents with faster decay. The inhibitory action of ifenprodil, a specific blocker of NR2B-containing NMDA receptors, was more effective in NMDA current suppression following light stress and less effective after longer stress compared to naive controls. Persistent activity and glutamate receptor balance in the neocortex have been linked to working memory and impulse control. Therefore, these results could provide insight for generating therapeutic strategies to prevent or reverse stress-induced cognitive deficits.

Astrocytic modulation of glutamatergic synaptic transmission is reduced in NTS of rats submitted to short-term sustained hypoxia

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [−183 ± 122 pA ( n = 10) vs. −353 ± 101 pA ( n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA ( n = 7) vs. 102 ± 37 pA ( n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): −353 ± 101 pA vs. aCSF + FAC: −369 ± 76 pA ( n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA ( n = 10)]. SH increased AMPA current density [control: −6 ± 3.5 pA/pF ( n = 6) vs. SH: −20 ± 12 pA/pF ( n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.

Contributions of space-clamp errors to apparent time-dependent loss of Mg2+ block induced by NMDA

N-methyl-d-aspartate receptors (NMDARs) govern synaptic plasticity, development, and neuronal response to insult. Prolonged activation of NMDARs such as during an insult may activate secondary currents or modulate Mg2+ sensitivity, but the conditions under which these occur are not fully defined. We reexamined the effect of prolonged NMDAR activation in juvenile mouse hippocampal slices. NMDA (10 μM) elicited current with the expected negative-slope conductance in the presence of 1.2 mM Mg2+. However, several minutes of continued NMDA exposure elicited additional inward current at −70 mV. A higher concentration of NMDA (100 µM) elicited the current more rapidly. The additional current was not dependent on Ca2+, network activity, or metabotropic NMDAR function and did not persist on agonist removal. Voltage ramps revealed no alteration of either reversal potential or NMDA-elicited conductance between −30 mV and +50 mV. The result was a more linear NMDA current-voltage relationship. The current linearization was also induced in interneurons and in mature dentate granule neurons but not immature dentate granule cells, dissociated cultured hippocampal neurons, or nucleated patches excised from CA1 pyramidal neurons. Comparative simulations of NMDA application to a CA1 pyramidal neuron and to a cultured neuron revealed that linearization can be explained by space-clamp errors arising from gradual recruitment of distal dendritic NMDARs. We conclude that persistent secondary currents do not strongly contribute to NMDAR responses in juvenile mouse hippocampus and careful discernment is needed to exclude contributions of clamp artifacts to apparent secondary currents. NEW & NOTEWORTHY We report that upon sustained activation of NMDARs in juvenile mouse hippocampal neurons there is apparent loss of Mg2+ block at negative membrane potentials. However, the phenomenon is explained by loss of dendritic voltage clamp, leading to a linear current-voltage relationship. Our results give a specific example of how spatial voltage errors in voltage-clamp recordings can readily be misinterpreted as biological modulation.

Variability of quantal NMDA to AMPA current ratio in nucleus tractus solitarii neurons

The ratio between AMPA and NMDA receptors is a key factor governing integrative and plastic properties of excitatory glutamatergic synapses. To determine whether the respective proportions of AMPA and NMDA receptors are similar or vary across a neuron's synapse, we analyzed the variability of NMDA and AMPA currents in quantal responses recorded from neurons located in the nucleus tractus solitarii. We found that the average NMDA to AMPA current ratio strongly differed between recorded neurons and that most of the intra-neuronal current ratio variability was attributable to fluctuations in NMDA current. We next performed computer simulations with a Monte Carlo model of a glutamatergic synapse to estimate the part of AMPA and NMDA currents fluctuations induced by stochastic factors. We found that NMDA current variability mainly resulted from strong channel noise with few influence of release variations. On the contrary, partly because of the presence of subconductance states, AMPA receptor channel noise was low and AMPA current fluctuations tightly reflected changes in the amount of glutamate released. We next showed that these two factors, channel noise and fluctuations in glutamate release, were sufficient to explain the observed variability of the NMDA to AMPA current ratio in quantal events recorded from the same neuron. We therefore concluded that the proportion of AMPA and NMDA receptors was similar, or roughly similar, across synapses onto the same target cell.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca2+ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca2+ levels (NMDA-ΔCa2+) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa2+ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa2+ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca2+ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca2+ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa2+ signaling during HF are discussed.