Role of UCHL1 in axonal injury and functional recovery after cerebral ischemia.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a unique brain-specific deubiquitinating enzyme. Mutations in and aberrant function of UCHL1 have been linked to many neurological disorders. UCHL1 activity protects neurons from hypoxic injury, and binding of stroke-induced reactive lipid species to the cysteine 152 (C152) of UCHL1 unfolds the protein and disrupts its function. To investigate the role of UCHL1 and its adduction by reactive lipids in inhibiting repair and recovery of function following ischemic injury, a knock-in (KI) mouse expressing the UCHL1 C152A mutation was generated. Neurons derived from KI mice had less cell death and neurite injury after hypoxia. UCHL1 C152A KI and WT mice underwent middle cerebral artery occlusion (MCAO) or sham surgery. White matter injury was significantly decreased in KI compared with WT mice 7 d after MCAO. Histological analysis revealed decreased tissue loss at 21 d after injury in KI mice. There was also significantly improved sensorimotor recovery in postischemic KI mice. K63- and K48-linked polyubiquitinated proteins were increased in penumbra of WT mouse brains but not in KI mouse brains at 24 h post MCAO. The UCHL1 C152A mutation preserved excitatory synaptic drive to pyramidal neurons and their excitability in the periinfarct zone; axonal conduction velocity recovered by 21 d post MCAO in KI mice in corpus callosum. These results demonstrate that UCHL1 activity is an important determinant of function after ischemia and further demonstrate that the C152 site of UCHL1 plays a significant role in functional recovery after stroke.

TrkB-mediated activation of the phosphatidylinositol-3-kinase/Akt cascade reduces the damage inflicted by oxygen-glucose deprivation in area CA3 of the rat hippocampus.

The selective vulnerability of hippocampal area CA1 to ischemia-induced injury is a well-known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen-glucose deprivation-reperfusion (OGD-RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain-derived neurotrophic factor (BDNF) or 7,8-dihydroxyflavone (7,8-DHF) exhibited reduced depression of the electrical activity triggered by OGD-RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD-RP. The protective effect of TrkB activation was limited to area CA3, as OGD-RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3-kinase/Akt (PI-3-K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD-RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase-3 activity in slices pretreated with BDNF and exposed to OGD-RP. We propose that PI-3-K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Critical involvement of postsynaptic protein kinase activation in long-term potentiation at hippocampal mossy fiber synapses on CA3 interneurons.

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs). Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI(6-22) failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI(6-22) induced posttetanic potentiation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

Properties and functional implications of I (h) in hippocampal area CA3 interneurons.

The present study examines the biophysical properties and functional implications of I (h) in hippocampal area CA3 interneurons with somata in strata radiatum and lacunosum-moleculare. Characterization studies showed a small maximum h-conductance (2.6 ± 0.3 nS, n = 11), shallow voltage dependence with a hyperpolarized half-maximal activation (V (1/2) = -91 mV), and kinetics characterized by double-exponential functions. The functional consequences of I (h) were examined with regard to temporal summation and impedance measurements. For temporal summation experiments, 5-pulse mossy fiber input trains were activated. Blocking I (h) with 50 µM ZD7288 resulted in an increase in temporal summation, suggesting that I (h) supports sensitivity of response amplitude to relative input timing. Impedance was assessed by applying sinusoidal current commands. From impedance measurements, we found that I (h) did not confer theta-band resonance, but flattened the impedance-frequency relations instead. Double immunolabeling for hyperpolarization-activated cyclic nucleotide-gated proteins and glutamate decarboxylase 67 suggests that all four subunits are present in GABAergic interneurons from the strata considered for electrophysiological studies. Finally, a model of I (h) was employed in computational analyses to confirm and elaborate upon the contributions of I (h) to impedance and temporal summation.

High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons.

Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 µM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 µM produced no further decrease in MF EPSC amplitude compared with 20 µM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 µM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40 Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs.

Passive and active shaping of unitary responses from associational/commissural and perforant path synapses in hippocampal CA3 pyramidal cells.

Although associational/commissural (A/C) and perforant path (PP) inputs to CA3b pyramidal cells play a central role in hippocampal mnemonic functions, the active and passive processes that shape A/C and PP AMPA and NMDA receptor-mediated unitary EPSP/EPSC (AMPA and NMDA uEPSP/uEPSC) have not been fully characterized yet. Here we find no differences in somatic amplitude between A/C and PP for either AMPA or NMDA uEPSPs. However, larger AMPA uEPSCs were evoked from proximal than from distal A/C or PP. Given the space-clamp constraints in CA3 pyramidal cells, these voltage clamp data suggest that the location-independence of A/C and PP AMPA uEPSP amplitudes is achieved in part through the activation of voltage dependent conductances at or near the soma. Moreover, similarity in uEPSC amplitudes for distal A/C and PP points to the additional participation of unclamped active conductances. Indeed, the pharmacological blockade of voltage-dependent conductances eliminates the location-independence of these inputs. In contrast, the location-independence of A/C and PP NMDA uEPSP/uEPSC amplitudes is maintained across all conditions indicating that propagation is not affected by active membrane processes. The location-independence for A/C uEPSP amplitudes may be relevant in the recruitment of CA3 pyramidal cells by other CA3 pyramidal cells. These data also suggest that PP excitation represents a significant input to CA3 pyramidal cells. Implication of the passive data on local synaptic properties is further investigated in the companion paper with a detailed computational model.

A computer model of unitary responses from associational/commissural and perforant path synapses in hippocampal CA3 pyramidal cells.

Despite the central position of CA3 pyramidal cells in the hippocampal circuit, the experimental investigation of their synaptic properties has been limited. Recent slice experiments from adult rats characterized AMPA and NMDA receptor unitary synaptic responses in CA3b pyramidal cells. Here, excitatory synaptic activation is modeled to infer biophysical parameters, aid analysis interpretation, explore mechanisms, and formulate predictions by contrasting simulated somatic recordings with experimental data. Reconstructed CA3b pyramidal cells from the public repository NeuroMorpho.Org were used to allow for cell-specific morphological variation. For each cell, synaptic responses were simulated for perforant pathway and associational/commissural synapses. Means and variability for peak amplitude, time-to-peak, and half-height width in these responses were compared with equivalent statistics from experimental recordings. Synaptic responses mediated by AMPA receptors are best fit with properties typical of previously characterized glutamatergic receptors where perforant path synapses have conductances twice that of associational/commissural synapses (0.9 vs. 0.5 nS) and more rapid peak times (1.0 vs. 3.3 ms). Reanalysis of passive-cell experimental traces using the model shows no evidence of a CA1-like increase of associational/commissural AMPA receptor conductance with increasing distance from the soma. Synaptic responses mediated by NMDA receptors are best fit with rapid kinetics, suggestive of NR2A subunits as expected in mature animals. Predictions were made for passive-cell current clamp recordings, combined AMPA and NMDA receptor responses, and local dendritic depolarization in response to unitary stimulations. Models of synaptic responses in active cells suggest altered axial resistivity and the presence of synaptically activated potassium channels in spines.

Functional expression of TrkB receptors on interneurones and pyramidal cells of area CA3 of the rat hippocampus.

The dentate gyrus and hippocampal area CA3 region of the mammalian brain contains the highest levels of brain-derived neurotrophic factor (BDNF) and its canonical membrane receptor, tropomyosin-related kinase B (TrkB). Therefore, the present study examines the expression and physiological responses triggered by activation of TrkB on hippocampal area CA3 interneurones and pyramidal cells of the rat hippocampus. Triple immunolabelling for TrkB, glutamate decarboxylase 67, and the calcium-binding proteins parvalbumin, calbindin or calretinin confirms the somatic expression of TrkB in all CA3 sublayers. TrkB-positive interneurones with fast-spiking discharge are restricted to strata oriens and lucidum, whereas regular-spiking interneurones are found in the strata lucidum, radiatum and lacunosum-moleculare. Activation of TrkB receptors with 7,8-dihydroxyflavone (DHF) modulates amplitude and frequency of spontaneous synaptic currents recorded from CA3 interneurones. Furthermore, the isolated excitatory postsynaptic currents (EPSC) of CA3 interneurones evoked by the mossy fibres (MF) or commissural/associational (C/A) axons, show input-specific synaptic potentiation in response to TrkB stimulation. On CA3 pyramidal cells, stimulation with DHF potentiates the MF synaptic transmission and increases the MF-EPSP - spike coupling. The latter exhibits a dramatic increase when picrotoxin is bath perfused after DHF, indicating that local interneurones restrain the excitability mediated by activation of TrkB. Therefore, we propose that release of BDNF on area CA3 reshapes the output of this hippocampal region by simultaneous activation of TrkB on GABAergic interneurones and pyramidal cells.

Area CA3 interneurons receive two spatially segregated mossy fiber inputs.

Area CA3 receives two extrinsic excitatory inputs, the mossy fibers (MF), and the perforant path (PP). Interneurons with somata in str. lacunosum moleculare (L-M) of CA3 modulate the influence of the MF and PP on pyramidal cell activity by providing strong feed-forward inhibitory influence to pyramidal cells. Here we report that L-M interneurons receive two separate MF inputs, one to the dorsal dendrites from the suprapyramidal blade of the dentate gyrus (MF(SDG)), and a second to ventral dendrites from the str. lucidum (MF(SL)). Responses elicited from MF(SDG) and MF(SL) stimulation sites have strong paired-pulse facilitation, similar DCG-IV sensitivity, amplitude, and decay kinetics but target spatially segregated domains on the interneuron dendrites. These data demonstrate that certain interneuron subtypes are entrained by two convergent MF inputs to spatially separated regions of the dendritic tree. This anatomical arrangement could make these interneurons considerably more responsive to the excitatory drive from dentate granule cells. Furthermore, temporal summation is linear or slightly sublinear between PP and MF(SL) but supralinear between PP and MF(SDG). This specific boosting of the excitatory drive to interneurons from the SDG location may indicate that L-M interneurons could be specifically involved in the processing of the associational component of the recognition memory.

Bidirectional Hebbian plasticity at hippocampal mossy fiber synapses on CA3 interneurons.

Hippocampal area CA3 is critically involved in the formation of nonoverlapping neuronal subpopulations ("pattern separation") to store memory representations as distinct events. Efficient pattern separation relies on the strong and sparse excitatory input from the mossy fibers (MFs) to pyramidal cells and feedforward inhibitory interneurons. However, MF synapses on CA3 pyramidal cells undergo long-term potentiation (LTP), which, if unopposed, will degrade pattern separation because MF activation will now recruit additional CA3 pyramidal cells. Here, we demonstrate MF LTP in stratum lacunosum-moleculare (L-M) interneurons induced by the same stimulation protocol that induces MF LTP in pyramidal cells. This LTP was NMDA receptor (NMDAR) independent and occurred at MF Ca(2+)-impermeable AMPA receptor synapses. LTP was prevented by with voltage clamping the postsynaptic cell soma during high-frequency stimulation (HFS), intracellular injections of the Ca(2+) chelator BAPTA (20 mm), or bath applications of the L-type Ca(2+) channel blocker nimodipine (10 microm). We propose that MF LTP in L-M interneurons preserves the sparsity of pyramidal cell activation, thus allowing CA3 to maintain its role in pattern separation. In the presence of the mGluR1alpha antagonist LY367385 [(S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] (100 microm), the same HFS that induces MF LTP in naive slices triggered NMDAR-independent MF LTD. This LTD, like LTP, required activation of the L-type Ca(2+) channel and also was induced after blockade of IP(3) receptors with heparin (4 mg/ml) or the selective depletion of receptor-gated Ca(2+) stores with ryanodine (10 or 100 microm). We conclude that L-M interneurons are endowed with Ca(2+) signaling cascades suitable for controlling the polarity of MF long-term plasticity induced by joint presynaptic and postsynaptic activities.

Functional maturation of excitatory synapses in layer 3 pyramidal neurons during postnatal development of the primate prefrontal cortex.

In the primate dorsolateral prefrontal cortex (DLPFC), the density of excitatory synapses decreases by 40-50% during adolescence. Although such substantial circuit refinement might underlie the adolescence-related maturation of working memory performance, its functional significance remains poorly understood. The consequences of synaptic pruning may depend on the properties of the eliminated synapses. Are the synapses eliminated during adolescence functionally immature, as is the case during early brain development? Or do maturation-independent features tag synapses for pruning? We examined excitatory synaptic function in monkey DLPFC during postnatal development by studying properties that reflect synapse maturation in rat cortex. In 3-month-old (early postnatal) monkeys, excitatory inputs to layer 3 pyramidal neurons had immature properties, including higher release probability, lower alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate (NMDA) ratio, and longer duration of NMDA-mediated synaptic currents, associated with greater sensitivity to the NMDA receptor subunit B (NR2B) subunit-selective antagonist ifenprodil. In contrast, excitatory synaptic inputs in neurons from preadolescent (15 months old) and adult (42 or 84 months old) monkeys had similar functional properties. We therefore conclude that the contribution of functionally immature synapses decreases significantly before adolescence begins. Thus, remodeling of excitatory connectivity in the DLPFC during adolescence may occur in the absence of widespread maturational changes in synaptic strength.

Oxygen-Glucose Deprivation Differentially Affects Neocortical Pyramidal Neurons and Parvalbumin-Positive Interneurons.

Stroke is a devastating brain disorder. The pathophysiology of stroke is associated with an impaired excitation-inhibition balance in the area that surrounds the infarct core after the insult, the peri-infarct zone. Here we exposed slices from adult mouse prefrontal cortex to oxygen-glucose deprivation and reoxygenation (OGD-RO) to study ischemia-induced changes in the activity of excitatory pyramidal neurons and inhibitory parvalbumin (PV)-positive interneurons. We found that during current-clamp recordings, PV-positive interneurons were more vulnerable to OGD-RO than pyramidal neurons as indicated by the lower percentage of recovery of PV-positive interneurons. However, neither the amplitude of OGD-induced depolarization observed in current-clamp mode nor the OGD-associated current observed in voltage-clamp mode differed between the two cell types. Large amplitude, presumably action-potential dependent, spontaneous postsynaptic inhibitory currents recorded from pyramidal neurons were less frequent after OGD-RO than in control condition. Disynaptic inhibitory postsynaptic currents (dIPSCs) in pyramidal neurons produced predominantly by PV-positive interneurons were reduced by OGD-RO. Following OGD-RO, dendrites of PV-positive interneurons exhibited more pathological beading than those of pyramidal neurons. Our data support the hypothesis that the differential vulnerability to ischemia-like conditions of excitatory and inhibitory neurons leads to the altered excitation-inhibition balance associated with stroke pathophysiology.

Coincidence detection of convergent perforant path and mossy fibre inputs by CA3 interneurons.

We performed whole-cell recordings from CA3 s. radiatum (R) and s. lacunosum-moleculare (L-M) interneurons in hippocampal slices to examine the temporal aspects of summation of converging perforant path (PP) and mossy fibre (MF) inputs. PP EPSPs were evoked from the s. lacunosum-moleculare in area CA1. MF EPSPs were evoked from the medial extent of the suprapyramidal blade of the dentate gyrus. Summation was strongly supralinear when examining PP EPSP with MF EPSP in a heterosynaptic pair at the 10 ms ISI, and linear to sublinear at longer ISIs. This pattern of nonlinearities suggests that R and L-M interneurons act as coincidence detectors for input from PP and MF. Summation at all ISIs was linear in voltage clamp mode demonstrating that nonlinearities were generated by postsynaptic voltage-dependent conductances. Supralinearity was not detected when the first EPSP in the pair was replaced by a simulated EPSP injected into the soma, suggesting that the conductances underlying the EPSP boosting were located in distal dendrites. Supralinearity was selectively eliminated with either Ni2+ (30 microm), mibefradil (10 microm) or nimodipine (15 microm), but was unaffected by QX-314. This pharmacological profile indicates that supralinearity is due to recruitment of dendritic T-type Ca2+channels by the first subthreshold EPSP in the pair. Results with the hyperpolarization-activated (Ih) channel blocker ZD 7288 (50 microm) revealed that Ih restricted the time course of supralinearity for coincidently summed EPSPs, and promoted linear to sublinear summation for asynchronous EPSPs. We conclude that coincidence detection results from the counterbalanced activation of T-type Ca2+ channels and inactivation of Ih.

Mitochondrial GSH Systems in CA1 Pyramidal Cells and Astrocytes React Differently during Oxygen-Glucose Deprivation and Reperfusion.

Pyramidal cells and astrocytes have differential susceptibility to oxygen-glucose deprivation and reperfusion (OGD-RP). It is known that excessive reactive oxygen species (ROS) in mitochondria initiates cell death, while glutathione (GSH) is one of the major defenses against ROS. Although it is known that astrocytes contain a higher concentration of GSH than neurons, and that astrocytes can provide neurons with GSH, we are unaware of a detailed and quantitative examination of the dynamic changes in the mitochondrial GSH system in the two cell types during OGD-RP. Here, we determined mitochondrial membrane potential and the degrees of oxidation of the mitochondrially targeted roGFP-based sensors for hydrogen peroxide (OxDP) and GSH (OxDG). We also developed a method to estimate the mitochondrial GSH (mGSH) concentration in single cells in the CA1 region of organotypic hippocampal slice cultures at several time-points during OGD-RP. We find that mitochondrial membrane potential drops in pyramidal cells during OGD while it is relatively stable in astrocytes. In both types of cell, the mitochondrial membrane potential decreases during RP. During OGD-RP, mitochondrial peroxide levels are the same. Astrocytic mGSH is more than four times higher than pyramidal cell mGSH (3.2 vs 0.7 mM). Astrocytic mGSH is drained from mitochondria during OGD, whereas in pyramidal cells it remains fairly constant. OxDGSH prior to and during OGD is lower (less oxidized) in pyramidal cells than in astrocytes, but the two nearly converge during RP. The larger changes of redox status in the GSH system in pyramidal cells than astrocytes is an upstream sign of the higher mortality of the pyramidal cells after facing an insult. The pattern of [mGSH] changes in the two cell types could be recognized as another mechanism by which astrocytes protect neurons from transient, extreme conditions.

Differences in Reperfusion-Induced Mitochondrial Oxidative Stress and Cell Death Between Hippocampal CA1 and CA3 Subfields Are Due to the Mitochondrial Thioredoxin System.

AIMS: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. RESULTS: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen-glucose deprivation (OGD)-reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitochondrial thioredoxin (Trx) system in CA3. Inhibition of TrxR eliminates the differences in redox status and cell death between the regions. Overexpression of cytosolic Trx1 does not influence mitochondrial H2O2 production. INNOVATION: Real-time changes of mitochondrial H2O2 and GSH in tissue cultures during early RP, and also during controlled production of superoxide and peroxide, reveal significant differences between CA1 and CA3. The mitochondrial Trx system is responsible for the observed differences during RP as well as for delayed cell death 18 h afterward. CONCLUSION: Greater mitochondrial Trx efficacy in CA3 pyramidal cells results in less vulnerability to ischemia/reperfusion because of the less oxidizing environment in CA3 mitochondria during RP. Antioxid. Redox Signal. 27, 534-549.

Early maintenance of hippocampal mossy fiber--long-term potentiation depends on protein and RNA synthesis and presynaptic granule cell integrity.

The neural substrates of memory likely include long-term potentiation (LTP) of synaptic strength that results from high-frequency stimulation (HFS) of the afferent pathway. The mechanisms that underlie the maintenance of LTP include RNA and protein synthesis, although the contribution of these molecular events typically does not become essential until several hours after LTP induction. We here show that, different from this pattern, (1) LTP maintenance at the mossy fiber (MF) input to CA3 pyramidal cells in the hippocampus depends on protein and RNA synthesis soon after LTP induction, and (2) some of these molecular events are controlled by signaling from the presynaptic granule cell soma. Bath application of the protein synthesis inhibitor emetine or cycloheximide 1 hr after MF LTP induction in hippocampal slices caused loss of MF potentiation. In contrast, application of emetine 1 hr after LTP induction at the commissural-associational input to CA3 pyramidal cells had no effect on this form of LTP. Administration of emetine or the RNA synthesis inhibitor actinomycin-D before delivery of HFS to MF input also caused a rapid decay of MF potentiation, although neither drug had an effect on the amplitude or the time-constant of decay of post-tetanic potentiation (PTP). Similarly, transection of MF axons near granule cell somas had no effect on baseline or PTP parameters but caused loss of potentiation at a rate comparable with that after actinomycin-D application. These results indicate that the mechanisms that underlie MF LTP maintenance differ from those involved in LTP maintenance at other glutamatergic synapses.

Mediodorsal thalamic afferents to layer III of the rat prefrontal cortex: synaptic relationships to subclasses of interneurons.

The mediodorsal nucleus of the thalamus (MD) represents the main subcortical structure that projects to the prefrontal cortex (PFC) and it regulates key aspects of the cognitive functions of this region. Within the PFC, GABA local circuit neurons shape the activity patterns and hence the "memory fields" of pyramidal cells. Although the connections between the MD and PFC are well established, the ultrastructural relationships between projecting fibers from the MD and different subclasses of GABA cells in the PFC are not known. In order to address this issue in the rat, we examined MD axons labeled by tract-tracing in combination with immunogold-silver to identify different calcium-binding proteins localized within separate populations of interneurons. Electron micrographic examination of PFC sections from these animals revealed that MD terminals made primarily asymmetric synapses onto dendritic spines and less commonly onto dendritic shafts. Most of the dendrites receiving MD synaptic input were immunoreactive for parvalbumin (ParV), whereas MD synapses onto dendrites labeled for calretinin or calbindin were less frequent. We also observed that some MD terminals were themselves immunoreactive for calcium-binding proteins, again more commonly for ParV. These results suggest that the MD exerts a dual influence on PFC pyramidal cells: direct inputs onto spines and an indirect influence mediated via synapses onto each subclass of interneurons. The apparent preferential input to ParV cells endows MD afferents with a strong indirect inhibitory influence on pyramidal neuron activity by virtue of ParV cell synapses onto soma, proximal dendrites, and axon initial segments.

Optimized real-time monitoring of glutathione redox status in single pyramidal neurons in organotypic hippocampal slices during oxygen-glucose deprivation and reperfusion.

A redox-sensitive Grx1-roGFP2 fusion protein was introduced by transfection into single pyramidal neurons in the CA1 subfield of organotypic hippocampal slice cultures (OHSCs). We assessed changes in the GSH system in neuronal cytoplasm and mitochondria during oxygen-glucose deprivation and reperfusion (OGD/RP), an in vitro model of stroke. Pyramidal cells in a narrow range of depths below the surface of the OHSC were transfected by gene gun or single-cell electroporation with cyto- or mito-Grx1-roGFP2. To mimic the conditions of acute stroke, we developed an optimized superfusion system with the capability of rapid and reproducible exchange of the solution bathing the OHSCs. Measurements of pO2 as a function of tissue depth show that in the region containing the transfected cells, the pO2 is well-controlled. We also found that the pO2 changes on the same time scale as changes in intracranial pressure, cerebral blood flow, and pO2 during acute stroke. Determining the reduction potential, EGSH, from the ratiometric fluorescence signal requires an absolute intensity measurement during calibration of the Grx1-roGFP2. Using the signal from cotransfected tdTomato as an internal standard during calibration improves quantitative measurements of Grx1-roGFP2 redox status and allows EGSH to be determined. EGSH becomes more reducing during OGD and more oxidizing during RP in mitochondria while changes in cytoplasm are not significant compared with controls.

Multiple forms of long-term synaptic plasticity at hippocampal mossy fiber synapses on interneurons.

The hippocampal mossy fiber (MF) pathway originates from the dentate gyrus granule cells and provides a powerful excitatory synaptic drive to neurons in the dentate gyrus hilus and area CA3. Much of the early work on the MF pathway focused on its electrophysiological properties, and ability to drive CA3 pyramidal cell activity. Over the last ten years, however, a new focus on the synaptic interaction between granule cells and inhibitory interneurons has emerged. These data have revealed an immense heterogeneity of long-term plasticity at MF synapses on various interneuron targets. Interestingly, these studies also indicate that the mechanisms of MF long-term plasticity in some interneuron subtypes may be more similar to pyramidal cells than previously appreciated. In this review, we first define the synapse types at each of the interneuron targets based on the receptors present. We then describe the different forms of long-term plasticity observed, and the mechanisms underlying each form as they are currently understood. Finally we highlight various open questions surrounding MF long-term plasticity in interneurons, focusing specifically on the induction and maintenance of LTP, and what the functional impact of persistent changes in efficacy at MF-interneuron synapses might be on the emergent properties of the inhibitory network dynamics in area CA3. This article is part of a Special Issue entitled 'Synaptic Plasticity & Interneurons'.