Intact CA1 Research Articles

CDDO-Me Abrogates Aberrant Mitochondrial Elongation in Clasmatodendritic Degeneration by Regulating NF-κB-PDI-Mediated S-Nitrosylation of DRP1

Clasmatodendrosis is a kind of astroglial degeneration pattern which facilitates excessive autophagy. Although abnormal mitochondrial elongation is relevant to this astroglial degeneration, the underlying mechanisms of aberrant mitochondrial dynamics are still incompletely understood. Protein disulfide isomerase (PDI) is an oxidoreductase in the endoplasmic reticulum (ER). Since PDI expression is downregulated in clasmatodendritic astrocytes, PDI may be involved in aberrant mitochondrial elongation in clasmatodendritic astrocytes. In the present study, 26% of CA1 astrocytes showed clasmatodendritic degeneration in chronic epilepsy rats. 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl or RTA 402) and SN50 (a nuclear factor-κB (NF-κB) inhibitor) ameliorated the fraction of clasmatodendritic astrocytes to 6.8 and 8.1% in CA1 astrocytes, accompanied by the decreases in lysosomal-associated membrane protein 1 (LAMP1) expression and microtubule-associated protein 1A/1B light-chain 3 (LC3)-II/LC3-I ratio, indicating the reduced autophagy flux. Furthermore, CDDO-Me and SN50 reduced NF-κB S529 fluorescent intensity to 0.6- and 0.57-fold of vehicle-treated animal level, respectively. CDDO-Me and SN50 facilitated mitochondrial fission in CA1 astrocytes, independent of dynamin-related protein 1 (DRP1) S616 phosphorylation. In chronic epilepsy rats, total PDI protein, S-nitrosylated PDI (SNO-PDI), and SNO-DRP1 levels were 0.35-, 0.34- and 0.45-fold of control level, respectively, in the CA1 region and increased CDDO-Me and SN50. Furthermore, PDI knockdown resulted in mitochondrial elongation in intact CA1 astrocytes under physiological condition, while it did not evoke clasmatodendrosis. Therefore, our findings suggest that NF-κB-mediated PDI inhibition may play an important role in clasmatodendrosis via aberrant mitochondrial elongation.

Influence of Different Sevoflurane Concentrations on Postoperative Cognitive Function in Aged Rats.

Postoperative cognitive dysfunction may be associated with neuroinflammation, and sevoflurane suppresses surgery-induced inflammation. We hypothesized that low concentrations of sevoflurane would result in more impaired postoperative cognitive function compared to high concentrations. Aged male Sprague-Dawley rats (n = 21, 17-22 months) were randomly assigned to 1 of 3 groups: control (C), sevoflurane 2% (S2), and sevoflurane 4% (S4). Rats in the S2 and S4 groups underwent open femoral fracture and intramedullary fixation of the left hind limb under 2 hours of sevoflurane anesthesia. Neurological outcomes were evaluated using the Morris water maze (MWM) test, and histopathological outcomes were assessed 28 days after surgery. The S2 group showed prolonged swimming latency compared to S4 on day 7 (difference of means, 34.4; 95% confidence interval [CI], 2.57-66.3; P = .031) and compared to the C group on day 9 (difference of means, -33.4; 95% CI, -65.3 to -1.55; P = .037). The intact CA1 cells in the S2 group were significantly less than those in the C and S4 groups (H statistic, 10.87; P = .006 versus C; P = .033 versus S4). We found that low concentrations of sevoflurane prolonged the swimming latency of the MWM compared to high concentrations and reduced intact CA1 hippocampal neurons in aged rats. These results suggest that low-concentration sevoflurane anesthesia may be more detrimental than high concentration for spatial cognitive function and postoperative impairment of hippocampal CA1 cells in aged rats.

Varying Dietary Component Ratios and Lingonberry Supplementation May Affect the Hippocampal Structure of ApoE-/- Mice.

ObjectiveThis study aimed to investigate and compare the morphological and biochemical characteristics of the hippocampus and the spatial memory of young adult ApoE–/– mice on a standard chow diet, a low-fat diet (LFD), a high-fat diet (HFD), and an HFD supplemented with lingonberries.MethodsEight-week-old ApoE–/– males were divided into five groups fed standard chow (Control), an LFD (LF), an HFD (HF), and an HFD supplemented with whole lingonberries (HF+WhLB) or the insoluble fraction of lingonberries (HF+InsLB) for 8 weeks. The hippocampal cellular structure was evaluated using light microscopy and immunohistochemistry; biochemical analysis and T-maze test were also performed. Structural synaptic plasticity was assessed using electron microscopy.ResultsApoE–/– mice fed an LFD expressed a reduction in the number of intact CA1 pyramidal neurons compared with HF+InsLB animals and the 1.6–3.8-fold higher density of hyperchromic (damaged) hippocampal neurons relative to other groups. The LF group had also morphological and biochemical indications of astrogliosis. Meanwhile, both LFD- and HFD-fed mice demonstrated moderate microglial activation and a decline in synaptic density. The consumption of lingonberry supplements significantly reduced the microglia cell area, elevated the total number of synapses and multiple synapses, and increased postsynaptic density length in the hippocampus of ApoE–/– mice, as compared to an LFD and an HFD without lingonberries.ConclusionOur results suggest that, in contrast to the inclusion of fats in a diet, increased starch amount (an LFD) and reduction of dietary fiber (an LFD/HFD) might be unfavorable for the hippocampal structure of young adult (16-week-old) male ApoE–/– mice. Lingonberries and their insoluble fraction seem to provide a neuroprotective effect on altered synaptic plasticity in ApoE–/– animals. Observed morphological changes in the hippocampus did not result in notable spatial memory decline.

Low Dose Teucrium Polium Hydro-Alcoholic Extract Treatment Effects on Spatial Memory and Hippocampal Neuronal Count of Rat Aβ25-35 Model of Alzheimer’s Disease

: Alzheimer’s disease (AD) is a multifactorial and debilitating condition without precise etiology and definite cure burdened with high monetary and human resources to the society around the globe. Given that Iranian traditional medicine has a valuable treasure of medicinal plants with multi-dimensional effects, we designed this study to examine one week daily low single-dose treatment protocol of 3 mg/kg Teucrium polium (TP) hydro-alcoholic extract effects on rat’s spatial memory performance and CA1 hippocampal neuronal count in rat Aβ25-35 as an AD model. Animals were bilaterally microinjected with single 10 µg/2 µg intrahippocampal Aβ25-35 fragment for the AD induction. After a two-week recovery period, daily intraperitoneal injection of TP for a week was initiated in Aβ-received TP-treated (treated) and TP-received animals (control). Spatial memory performance and CA1 neuronal count were evaluated. TP significantly reduced time latency and distance swum during the first three days of the training protocol compared with Aβ-received rats (sham). Probe reference test findings, however, were not statistically different in treated, sham, and control groups. Intact CA1 hippocampal neuronal count showed a significant increase in Aβ-received TP-treated (case) rats in comparison with Aβ-received (sham) animals. It seems that the least reported low dose TP has the active chemical components capable of memory enhancing effects in Morris water maze (MWM) setting and potential neuronal survival property in hippocampal tissue. In spite of myriad claims against TP use, we should reconsider the matter and do more research to find the exact active ingredients of various species of the plant plus possible underlying mechanisms for ensuring potential use for human clinical trials in AD and other central nervous system-related diseases.

Intraischemic Modest Hypothermia Does Not Prevent Onset of Locomotor Inactivity After Transient Forebrain Ischemia in Rats.

Although modest hypothermia of 35°C has been demonstrated to provide histological neuroprotection in a rodent model of cerebral ischemia, the long-term behavioral outcome is still not clear. This study was designed to investigate whether modest hypothermia of 35°C provides sustained histological and behavioral neuroprotection following transient forebrain ischemia in rats. Male Sprague-Dawley rats were randomly assigned to one of three groups: sham, control, and modest hypothermia group. Each group contained eight rats. Ten-minute transient forebrain ischemia was produced by bilateral carotid artery occlusion plus hemorrhagic hypotension (mean arterial pressure = 40 mmHg). The hypothermic group was cooled to 35°C in preischemic period, and the cooling was continued for 1 hour postischemia. To evaluate behavioral outcome, spontaneous alternation behavior and locomotor activity were assessed using Y-maze test on a weekly basis. The rats were sacrificed after 28 days, and the number of intact neurons per 1 mm in the hippocampal CA1 subfield was counted microscopically. There was significant difference between the control [19(24.5)/mm: median (interquartile range)] and hypothermia groups [116(24)/mm; p < 0.01] in the intact CA1 neuron count. In the control and modest hypothermia groups, the locomotor activities were gradually decreased, and reached significantly lower levels in comparison with the sham group at 14 days postischemia. This study indicates that intraischemic modest hypothermia provided long-term histological neuroprotection, but did not reverse the onset of locomotor inactivity in a rat transient forebrain ischemia model.

G9a/GLP Complex Acts as a Bidirectional Switch to Regulate Metabotropic Glutamate Receptor-Dependent Plasticity in Hippocampal CA1 Pyramidal Neurons.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is conventionally considered to be solely dependent on local protein synthesis. Given the impact of epigenetics on memory, the intriguing question is whether epigenetic regulation influences mGluR-LTD as well. G9a/GLP histone lysine methyltransferase complex is crucial for brain development and goal-directed learning as well as for drug-addiction. In this study, we analyzed whether the epigenetic regulation by G9a/GLP complex affects mGluR-LTD in CA1 hippocampal pyramidal neurons of 5-7 weeks old male Wistar rats. In hippocampal slices with intact CA1 dendritic regions, inhibition of G9a/GLP activity abolished mGluR-LTD. The inhibition of this complex upregulated the expression of plasticity proteins like PKMζ, which mediated the prevention of mGluR-LTD expression by regulating the NSF-GluA2-mediated trafficking of AMPA receptors towards the postsynaptic site. G9a/GLP inhibition during the induction of mGluR-LTD also downregulated the protein levels of phosphorylated-GluA2 and Arc. Interestingly, G9a/GLP inhibition could not impede the mGluR-LTD when the cell-body was severed. Our study highlights the role of G9a/GLP complex in intact neuronal network as a bidirectional switch; when turned on, it facilitates the expression of mGluR-LTD, and when turned off, it promotes the expression of long-term potentiation.

Differential modification of the phospholipid profile by transient ischemia in rat hippocampal CA1 and CA3 regions

The hippocampal CA1 region is most susceptible to cerebral ischemia in both rodents and humans, whereas CA3 is remarkably resistant. Here, we investigated the possible role of membrane lipids in differential susceptibility in these regions. Transient ischemia was induced in rats via bilateral occlusion of common carotid arteries and membrane lipids were analyzed by mass spectrometry. While lipid profile differences between the intact CA1 and CA3 were rather minor, ischemia caused significant pyramidal cell death with concomittant reduction of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, plasmalogen and sphingomyelin only in CA1. The phospholipid loss was evenly distributed in most molecular species. Ischemia also significantly increased cell death mediator ceramides only in CA1. Our data suggests that differential susceptibility to ischemia between CA1 and CA3 is not linked to their unique phospholipid profile. Also, selective activation of phospholipase A2, which primarily releases polyunsaturated fatty acids, might not be characteristic to cell death in CA1.

Insulin Reduces Neuronal Excitability by Turning on GABAA Channels that Generate Tonic Current

Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABAA channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocamal slices that insulin (1 nM) “turns on” new extrasynaptic GABAA channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC50) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABAA antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.

Positive allosteric activation of GABAA receptors bi-directionally modulates hippocampal glutamate plasticity and behaviour

Long-term BZ (benzodiazepine) anxiolytic therapy increases the risk of physical dependence manifested as withdrawal anxiety. BZ-induced potentiation of GABA(A)R (gamma-aminobutyric acid type-A receptor) function by 1-week oral administration of FZP (flurazepam) bi-directionally modulates excitatory glutamatergic synaptic transmission in hippocampal CA1 neurons during drug withdrawal. Previous electrophysiological studies on acutely isolated and intact CA1 neurons, as well as immunofluorescence and post-embedding immunogold electron microscopy studies, suggest increased synaptic insertion of GluR (glutamate receptor) 2-lacking AMPARs (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors) in 2-day FZP-withdrawn rats. Preliminary studies indicated a similar increase in GluR1, then phospho-Ser(831)-GluR1, as well as CaMKIIalpha (Ca(2+)/calmodulin-dependent protein kinase IIalpha), but not phospho-Thr(286)-CaMKII levels at the same time point. In our studies, whole-cell recordings in hippocampal slices revealed that AMPAR mEPSC [miniature EPSC (excitatory postsynaptic current)] amplitude was increased in 1-day FZP-withdrawn rats followed by an increase in estimated single-channel conductance in 2-day-FZP-withdrawn rats. Enhanced conductance was no longer observed in slices pre-incubated for 2 h in the CaMKII inhibitor KN-93, but not the inactive analogue KN-92. To evaluate whether CaMKII-mediated AMPA potentiation could occlude LTP (long-term potentiation), LTP was induced by TBS (theta burst stimulation) and recorded using whole-cell and extracellular techniques. LTP was induced in both groups, but only maintained for <15 min in 2-day FZP-withdrawn rats. LTP was fully restored after 7-day withdrawal. Despite the lack of LTP maintenance, impairment of object recognition, place and context was not observed in 2-day-FZP-withdrawn rats. Since L-VGCC (L-type voltage-gated calcium channel) current density was doubled on drug withdrawal and up to 2 days, Ca(2+) entry through L-VGCCs and perhaps subsequently through Ca(2+)-permeable AMPARs are proposed to be responsible for enhanced CaMKIIalpha levels and AMPAR potentiation. Mechanisms associated with several different models of activity-dependent plasticity may underlie BZ physical dependence.

Caveolin‐1 knockout mice are resistant to the neuroprotective effect of ischemic preconditioning

Caveolin‐1 (Cav‐1) is thought to be a structural component of caveolae in numerous cells. Although no caveolae exist in neurons, Cav‐1 is expressed and may contribute to signaling. We recently showed the involvement of Cav‐1 in protection of isolated neurons. No studies have assessed the role of Cav‐1 in in vivo models of cerebral protection. We hypothesized that ischemic preconditioning, where a sublethal stress protect from a more lethal stress, is absent in Cav‐1 knockout mice. We induced global cerebral ischemia by bilateral carotid artery occlusion (BCAO) with cerebral blood flow (CBF) monitoring. Mice were divided into the following groups: sham, lethal ischemia (LI: BCAO for 12 min), and preconditioning (PC: BCAO for 3 min followed by 24 hr recovery prior to LI; in both wild‐type and Cav‐1 knockout mice). At 3 days following final surgery, the percent of intact CA1 neurons was calculated. Additionally, the plasticity of the posterior communicating arteries on both sides was scored. The percent of intact CA1 neuron in PC group was nearly 3‐fold higher than that in LI group (67.9 ± 7.1 %; PC, 25.8 ± 7.0 %; LI, p&lt;0.001). However, in the PC Cav‐1 knockout mice, similar CA1 neuron loss to the LI group was observed (31.9 ± 9.7 %; PC of Cav‐1 knockout, 25.8 ± 7.0 %; LI of wild‐type, p&gt;0.05). The minimum CBF after BCAO was correlated to the plasticity score. There was a tendency that plasticity score was increased relative to aging in Cav‐1 knockout mice. In conclusion our data suggest that 1) Cav‐1 is important to neuronal protection in vivo and 2) collateral formation appears to be linked to loss of Cav‐1 gene expression and age. Cav‐1 might serve as a unique target for neuronal preconditioning agents.

Anti-apoptotic signaling and failure of apoptosis in the ischemic rat hippocampus

Several anti-apoptotic proteins are induced in CA1 neurons after transient forebrain ischemia (TFI), but fail to protect the majority of these cells from demise. Correlating cell death morphologies (apoptosis-like and necrosis-like death) with immunohistochemistry (IHC), we investigated whether anti-apoptosis contributes to survival, compromises apoptosis effector functions and/or delays death in CA1 neurons 1-7 days after TFI. As surrogate markers for bioenergetic failure, the IHC of respiratory chain complex (RCC) subunits was investigated. Dentate granule cell (DGC) apoptosis following colchicine injection severed as a reference for classical apoptosis. Heat shock protein 70 (Hsp70), neuronal apoptosis inhibitory protein (NAIP) and manganese superoxide dismutase (MnSOD) were upregulated in the majority of intact CA1 neurons paralleling the occurrence of CA1 neuronal death (days 3-7) as well as in a proportion of apoptosis-(<50%) and necrosis-like (<30%) CA1 neurons. Colchicine did not provoke an anti-apoptotic response in DGC at all. In addition, more than 70% of apoptosis- and necrosis-like CA1 neurons had completely lost their RCC subunits suggesting bioenergetic failure; by contrast, following colchicine injection, 88% of all apoptotic DGC presented RCC subunits. Thus, anti-apoptotic proteins may, in a subset of ischemic CA1 neurons, prevent cell death, while in others, affected by pronounced energy failure, they may cause secondary necrosis.

Propagation of low calcium non-synaptic induced epileptiform activity to the contralateral hippocampus in vivo

Recent experiments show that non-synaptic epileptiform activity can be induced by high K + and low Ca 2+ solution in vivo in the hippocampal CA1 region when synaptic transmission is blocked. However, the ability of this type of epileptiform activity to propagate to other brain areas is unknown. Presumably, this epileptiform activity should propagate and project along the axons to remote brain areas. This hypothesis was tested in vivo by inducing non-synaptic seizures in the left hippocampus and by recording spontaneous and evoked field potentials in both left and right hippocampi. The results show that one type of non-synaptic epileptiform activity, late bursts, observed in the left exposed CA1 and CA3 regions could propagate to the contralateral intact CA1 and induce seizures with onsets of high-frequency rhythm. A cut of the commissural fibers near the midline of the brain prevented this propagation. In addition, the measurement of time delays between the exposed left CA3 and contralateral right CA1, as well as between the two recording electrodes in the right CA1, showed that the burst activity propagated through the commissural pathways. Experimental data also showed that these late bursts in the left hippocampus were first generated in the Schaffer collaterals of the CA1 region, traveled to the ipsilateral CA3 region and then propagated through the commissural fibers to the other side. These results suggest that non-synaptic epileptiform activity can propagate along axon projections to intact brain area causing seizure activity. This non-synaptic activity propagating through axonal pathway provides a possible mechanism for the generation of high-frequency low-amplitude onset activity observed commonly in human epileptic EEGs.

Hypoxic excitability changes and sodium currents in hippocampus CA1 neurons.

1. The objective of the present study was to distinguish if inhibition of neuronal activity by hypoxia is related to a block of voltage-gated Na+ channels. 2. The effect of chemical hypoxia induced by cyanide (0.5 mM, 10 min perfusion) was studied with patch-clamp technique in visualized intact CA1 pyramidal neurons in rat brain slices. Action potentials were elicited in whole cell current-clamp recordings and the threshold was estimated by current pulses of 50-ms duration and incremental amplitudes (n = 31). The effect of cyanide on the Na+ current and conductance was studied in voltage clamp recordings from cell-attached patches (n = 13). 3. Cyanide perfusion during 10 min increased the threshold for excitation by 73 +/- 79 pA (p = 0.001), which differed from the effect in control cells (11 +/- 41 pA, ns). The change in current threshold was correlated to a change in membrane potential (r = -0.88, p < 0.0001). Cyanide had no significant effect on the peak amplitude, duration, or rate of rise of the action potential. 4. Cyanide perfusion did not change the Na+ current size, but caused a small decrease in ENa (-17 +/- 22 mV, ns) and a slight increase in Na+ conductance (+14 +/- 26%, ns), which differed (p = 0.045) from controls (-19 +/- 23 %, ns). 5. In conclusion, chemical hypoxia does not cause a decrease in Na+ conductance. The decreased excitability during hypoxia can be explained by an increase in the current threshold, which is correlated with the effect on the membrane potential.

Ischemia Leads to Apoptosis—and Necrosis‐like Neuron Death in the Ischemic Rat Hippocampus

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.

Ischemia-Induced Degeneration of CA1 Pyramidal Cells Decreases Seizure Severity in a Subgroup of Epileptic Gerbils and Affects Parvalbumin Immunoreactivity of CA1 Interneurons

Mongolian gerbils are epilepsy-prone animals. In adult gerbils two major groups can be differentiated according to their seizure behavior: Highly seizure-sensitive gerbils exhibit facial and forelimb clonus or generalized tonic-clonic seizures from the first test on, while kindled-like gerbils are seizure free for the first three to six consecutive tests, later develop forelimb myoclonus, and eventually progress to generalized tonic-clonic seizures. In the hippocampus, seizure history of the individual animal is mirrored in the intensity in which GABAergic neurons are immunostained for the calcium-binding protein parvalbumin: they lose parvalbumin with increasing seizure incidence. In a first step to clarify the influence of hippocampal projection neurons on spontaneous seizure behavior and related parvalbumin expression, we induced degeneration of the CA1 pyramidal cells by transient forebrain ischemia. This results in a decreased seizure sensitivity in highly seizure-sensitive gerbils. The kindling-like process, however, is not permanently blocked by the ischemic nerve cell loss, suggesting that an intact CA1 field is not a prerequisite for the development of seizure behavior. The seizure-induced loss of parvalbumin from the ischemia-resistant interneurons recovers after ischemia. Thus, changes in parvalbumin content brought about by repeated seizures are not permanent but can rather be modulated by novel stimuli.

Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome)

Living brain slices are usually cut with razor blades, which compress a ca. 50-μm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100–1800 bar was ejected through nozzles of 35–100 μm to cut 300–500-μm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400–600 bar and with nozzle diameters of 60–80 μm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 μm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg 2+-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

Protective effect of γ-glutamylethylamide (theanine) on ischemic delayed neuronal death in gerbils

We examined the protective effect of γ-glutamylethylamide (theanine) on ischemic delayed neuronal death in field CA1 of the gerbil hippocampus. One microliter of theanine from each three concentrations (50, 125 and 500 μM) was administered through the lateral ventricle 30 min before ischemia. Transient forebrain ischemia was induced by bilateral occlusion of the common carotid arteries for 3 min under careful control of brain temperature at approximately 37°C. Seven days after ischemia, the number of intact CA1 neurons in the hippocampus was assessed. Ischemia-induced neuronal death in hippocampal CA1 region was significantly prevented in a dose-dependent manner in the theanine-pretreated groups. These findings indicate that theanine might be useful clinically for preventing ischemic neuronal damage.

Noninvasive Measurements of the Membrane Potential and GABAergic Action in Hippocampal Interneurons

Neurotransmitters affect the membrane potential (Vm) of target cells by modulating the activity of receptor-linked ion channels. The direction and amplitude of the resulting transmembrane current depend on the resting level of Vm and the gradient across the membrane of permeant ion species. Vm, in addition, governs the activation state of voltage-gated channels. Knowledge of the exact level of Vm is therefore crucial to evaluate the nature of the neurotransmitter effect. However, the traditional methods to measure Vm, with microelectrodes or the whole-cell current-clamp technique, have the drawback that the recording pipette is in contact with the cytoplasm, and dialysis with the pipette solution alters the ionic composition of the interior of the cell. Here we describe a novel technique to determine the Vm of an intact cell from the reversal potential of K+ currents through a cell-attached patch. Applying the method to interneurons in hippocampal brain slices yielded more negative values for Vm than subsequent whole-cell current-clamp measurements from the same cell, presumably reflecting the development of a Donnan potential between cytoplasm and pipette solution in the whole-cell mode. Cell-attached Vm measurements were used to study GABAergic actions in intact CA1 interneurons. In 1- to 3-week-old rats, bath-applied GABA inhibited these cells by stabilizing Vm at a level depending on contributions from both GABAA and GABAB components. In contrast, in 1- to 4-d-old animals, only GABAA receptors were activated resulting in a depolarizing GABA response.

Dantrolene is cytoprotective in two models of neuronal cell death.

The neuroprotective effects of dantrolene, an inhibitor of calcium release from intracellular stores, were investigated in a model of cell death induced by calcium release from endoplasmic reticulum in vitro. Thapsigargin (50 nM), a selective inhibitor of endoplasmic reticular Ca(2+)-ATPase, significantly increased the cytosolic Ca2+ concentration to 230% over basal levels, induced DNA fragmentation, and reduced cell viability from 94% in control cells to 41% after a 24-h treatment in GT1-7 hypothalamic neurosecretory cells. Pretreatment with dantrolene for 30 min significantly inhibited elevation of cytosolic Ca2+ levels, DNA fragmentation, and GT1-7 cell death induced by thapsigargin in a dose-dependent manner. To determine if dantrolene would also be protective in an in vivo model of neurodegeneration, it was administered intravenously immediately following a 5-min global cerebral ischemia in gerbils, and the number of intact hippocampal CA1 pyramidal neurons was counted 7 days later. The effects of dantrolene on brain and rectal temperature were monitored in a separate experiment. Dantrolene significantly increased the number of intact CA1 pyramidal neurons from 40% (untreated ischemic animals) to 67 (10 mg/kg), 78 (25 mg/kg), or 83% (50 mg/kg) of values in sham controls (all p < 0.001). No significant changes in brain or rectal temperature were detected for 4 h following 50 mg/kg dantrolene. These results suggest that abnormal Ca2+ release from intracellular stores can induce neuronal death and that such a mechanism may contribute to delayed hippocampal neuronal death after cerebral ischemia. Dantrolene may be a potentially useful drug for neuroprotection after cerebral ischemia.

Ketanserin reduces neuronal calcium accumulation and cell death in the hippocampus of the mongolian gerbil after transient forebrain ischemia

Excessive neuronal activity combined with an increased release of neurotransmitters is supposed to contribute to the delayed neuronal degeneration in animal models of transient cerebral ischemia. Since evidence is accumulating that serotonin (5-HT) exerts an excitatory effect on neurons via 5-HT 2 receptors we tested the hypothesis that 5-HT 2 receptor antagonists could protect neurons in the gerbil after transient bilateral carotid occlusion. In a first series of experiments, the 5-HT 2 receptor antagonist ketanserin was injected intraperitoneally 15 min prior to 5 min of forebrain ischemia and given twice daily on the following 3 days. At a dose of 10 mg/kg i.p., the number of intact hippocampal CA1 neurons was significantly higher than in the saline-treated group and reached 74% of the sham-operated controls. In addition, the degree of neuronal damage correlated with an increased intracellular Ca 2+ content in CA1 pyramidal neurons as revealed by arsenazo (III) staining with a procedure modified for paraffin sections. In a second series of experiments, ketanserin (10 mg/kg) was injected at various times after onset of ischemia. Up to a period of 90 min after ischemia, the number of intact CA1 pyramidal cells in ketanserin-treated animals was still significantly higher than in the saline-treated group. The results indicate that 5-HT 2 receptor antagonists may protect neurons against ischemic damage even when the treatment is started after onset of ischemia. It remains to be investigated whether the neuroprotective effect of ketanserin is due to a neuronal action or to an inhibition of cerebrovascular vasopasm.