Persistence of neuronal representations through time and damage in the hippocampus.

How do neurons encode long-term memories? Bilateral imaging of neuronal activity in the mouse hippocampus reveals that, from one day to the next, ~40% of neurons change their responsiveness to cues, but thereafter only 1% of cells change per day. Despite these changes, neuronal responses are resilient to a lack of exposure to a previously completed task or to hippocampus lesions. Unlike individual neurons, the responses of which change after a few days, groups of neurons with inter- and intrahemispheric synchronous activity show stable responses for several weeks. The likelihood that a neuron maintains its responsiveness across days is proportional to the number of neurons with which its activity is synchronous. Information stored in individual neurons is relatively labile, but it can be reliably stored in networks of synchronously active neurons.

Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia.

Ischemia-reperfusion injuries produce reactive oxygen species that promote the peroxide lipid oxidation process resulting in the production of an endogenic lipid peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces cell death. We synthesized a novel 4-HNE scavenger - a carnosine-hydrazide derivative, l-carnosine hydrazide (CNN) - and examined its neuroprotective effect in a model of transient ischemia. PC-12 cells were pre-incubated with various doses (0-50 mmol/L) of CNN for 30 min, followed by incubation with 4-HNE (250 µM). An MTT assay was performed 24 h later to examine cell survival. Transient ischemia was induced by bilateral common carotid artery occlusion (BCCO) in the Mongolian gerbil. Animals were assigned to sham-operated (n = 6), placebo-treated (n = 12), CNN pre-treated (20 mg/kg; n = 12), CNN post-treated (100 mg/kg; n = 11), and histidyl hydrazide (a previously known 4-HNE scavenger) post-treated (100 mg/kg; n = 7) groups. Heat shock protein 70 immunoreactivity in the hippocampal CA1 region was evaluated 24 h later, while delayed neuronal death using 4-HNE staining was evaluated 7 days later. Pre-incubation with 30 mmol/L CNN completely inhibited 4-HNE-induced cell toxicity. CNN prevented delayed neuronal death by >60% in the pre-treated group (p < 0.001) and by >40% in the post-treated group (p < 0.01). Histidyl hydrazide post-treatment elicited no protective effect. CNN pre-treatment resulted in high heat shock protein 70 and low 4-HNE immunoreactivity in CA1 pyramidal neurons. Higher 4-HNE immunoreactivity was also found in the placebo-treated animals than in the CNN pre-treated animals. Our novel compound, CNN, elicited highly effective 4-HNE scavenging activity in vitro. Furthermore, CNN administration both pre- and post-BCCO remarkably reduced delayed neuronal death in the hippocampal CA1 region via its induction of heat shock protein 70 and scavenging of 4-HNE.

Hippocampal area CA1 and remote memory in rats.

Hippocampal lesions often produce temporally graded retrograde amnesia (TGRA), whereby recent memory is impaired more than remote memory. This finding has provided support for the process of systems consolidation. However, temporally graded memory impairment has not been observed with the watermaze task, and the findings have been inconsistent with context fear conditioning. One possibility is that large hippocampal lesions indirectly disrupt (by retrograde degeneration) the function of areas that project to the hippocampus that are important for task performance or thought to be important for storing consolidated memories. We developed a discrete lesion targeting area CA1, the sole output of the hippocampus to neocortex, and tested the effects of this lesion on recent and remote memory in the watermaze task, in context fear conditioning, and in trace fear conditioning. In all three tasks, recent and remote memory were similarly impaired after CA1 lesions. We discuss factors that help to illuminate these findings and consider their relevance to systems consolidation.

Enhanced expression of the calcium-sensing receptor in reactive astrocytes following ischemic injury in vivo and in vitro.

We recently demonstrated that the G protein-coupled calcium-sensing receptor (CaSR) is associated with the pathogenesis of ischemic stroke and may be involved in vascular remodeling and astrogliosis. To further substantiate the involvement of CaSR in the astroglial reaction common to ischemic insults, we investigated the temporal and cell type-specific expression patterns of CaSR in the hippocampus after transient forebrain ischemia. CaSR was constitutively expressed in neurons of the pyramidal and granule cell layers, whereas increased CaSR immunoreactivity was observed in reactive astrocytes, but not in activated microglia or macrophages, in the CA1 region of the post-ischemic hippocampus. Astroglial induction of CaSR expression was evident on days 3-7 after reperfusion and appeared to increase progressively through day 28, at which time CaSR expression was prominent in astrocytes with a highly reactive hypertrophic phenotype and elevated levels of glial fibrillary acidic protein. This expression pattern was supported by results of immunoblot analyses. Furthermore, CaSR expression was upregulated in rat primary cortical astrocytes exposed to oxygen-glucose deprivation, which undergo reactive gliosis-like changes. Thus, our results demonstrate that selective and long-lasting astroglial induction of CaSR expression is a common characteristic of ischemic injury and suggest its involvement in the ischemia-induced astroglial reaction.

Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats.

BACKGROUND: Dynamin-related protein 1 (Drp1) is a mitochondrial fission protein that, upon phosphorylation at serine 616 (p-Drp1(Ser616)), plays a pivotal role in neuronal death after ischemia. In the present study, we hypothesized that peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury induced by global ischemia in rats. RESULTS: We found that pretreatment of the rats with Mdivi-1, a selective Drp1 inhibitor, decreased the level of transient global ischemia (TGI)-induced p-Drp1(Ser616) and reduced cellular contents of oxidized proteins, activated caspase-3 expression as well as the extent of DNA fragmentation. Delivery of siRNA against Drp1 attenuated the expression of p-Drp1(Ser616) that was accompanied by alleviation of the TGI-induced protein oxidation, activated caspase-3 expression and DNA fragmentation in hippocampal proteins. Exogenous application of pioglitazone, a PPARγ agonist, reduced the p-Drp1(Ser616) expression, decreased TGI-induced oxidative stress and activated caspase-3 expression, lessened the extents of DNA fragmentation, and diminished the numbers of TUNEL-positive neuronal cells; all of these effects were reversed by GW9662, a PPARγ antagonist. CONCLUSIONS: Our findings thus indicated that inhibition of TGI-induced p-Drp1(Ser616) expression by Drp1 inhibitor and Drp1-siRNA can decrease protein oxidation, activated caspase-3 expression and neuronal damage in the hippocampal CA1 subfield. PPARγ agonist, through PPARγ-dependent mechanism and via decreasing p-Drp1(Ser616) expression, can exert anti-oxidative and anti-apoptotic effects against ischemic neuronal injury.

Neuroprotection against traumatic brain injury by xenon, but not argon, is mediated by inhibition at the N-methyl-D-aspartate receptor glycine site.

BACKGROUND: Xenon, the inert anesthetic gas, is neuroprotective in models of brain injury. The authors investigate the neuroprotective mechanisms of the inert gases such as xenon, argon, krypton, neon, and helium in an in vitro model of traumatic brain injury. METHODS: The authors use an in vitro model using mouse organotypic hippocampal brain slices, subjected to a focal mechanical trauma, with injury quantified by propidium iodide fluorescence. Patch clamp electrophysiology is used to investigate the effect of the inert gases on N-methyl-D-aspartate receptors and TREK-1 channels, two molecular targets likely to play a role in neuroprotection. RESULTS: Xenon (50%) and, to a lesser extent, argon (50%) are neuroprotective against traumatic injury when applied after injury (xenon 43±1% protection at 72 h after injury [N=104]; argon 30±6% protection [N=44]; mean±SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50%) prevents development of secondary injury up to 48 h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50±5% reduction in secondary injury at 72 h after injury [N=104]; argon 34±8% reduction [N=44]; mean±SEM). Glycine reverses the neuroprotective effect of xenon, but not argon, consistent with competitive inhibition at the N-methyl-D-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Xenon inhibits N-methyl-D-aspartate receptors and activates TREK-1 channels, whereas argon, krypton, neon, and helium have no effect on these ion channels. CONCLUSIONS: Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-D-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.

Why is CA3 more vulnerable than CA1 in experimental models of controlled cortical impact-induced brain injury?

One interesting finding of controlled cortical impact (CCI) experiments is that the CA3 region of the hippocampus, which is positioned further from the impact than the CA1 region, is reported as being more injured. The current literature has suggested a positive correlation between brain tissue stretch and neuronal cell loss. However, it is counterintuitive to assume that CA3 is stretched more during CCI injury. Recent mechanical studies of the brain have reported on a level of spatial heterogeneity not previously appreciated-the finding that CA1 was significantly stiffer than all other regions tested and that CA3 was one of the most compliant. We hypothesized that mechanical heterogeneity of anatomical structures could underlie the proposed heterogeneous mechanical response and hence the pattern of cell death. As such, we developed a three-dimensional finite element (FE) rat brain model representing detailed hippocampal structures and simulated various CCI experiments. Four groups of material properties based on recent experiments were tested. In group 1, hyperelastic material properties were assigned to various hippocampal structures, with CA3 more compliant than CA1. In group 2, linear viscoelastic material properties were assigned to hippocampal structures, with CA3 more compliant than CA1. In group 3, the hippocampus was represented by homogenous linear viscoelastic material properties. In group 4, a homogeneous nonlinear hippocampus was adopted. Simulation results demonstrated that for CCI with a 5-mm diameter, flat shape impactor, CA3 experienced increased tensile strains over a larger area and to a greater magnitude than did CA1 for group 1, which best explained why CA3 is more sensitive to CCI injury. However, for groups 2-4, the total volume with high strain (>30%) in CA3 was smaller than that in CA1. The FE rat brain model, with detailed hippocampal structures presented here, will help to engineer desired experimental neurotrauma models by virtually characterizing brain biomechanics before testing.

Homeostatic increase in excitability in area CA1 after Schaffer collateral transection in vivo.

PURPOSE: Epilepsy is a significant long-term consequence of traumatic brain injury (TBI) and is likely to result from multiple mechanisms. One feature that is common to many forms of TBI is denervation. We asked whether chronic partial denervation in vivo would lead to a homeostatic increase in the excitability of a denervated cell population. METHODS: To answer this question, we took advantage of the unique anatomy of the hippocampus where the input to the CA1 neurons, the Schaffer collaterals, could be transected in vivo with preservation of their outputs and only minor cell death. KEY FINDINGS: We observed a delayed increase in neuronal excitability, as apparent in extracellular recordings from hippocampal brain slices prepared 14 days (but not 3 days) post lesion. Although population spikes in slices from control and lesioned animals were comparable under resting conditions, application of solutions that were mildly proconvulsive (high K(+) , low Mg(2+) , low concentrations of bicuculline) produced increases in the number of population spikes in slices from lesioned rats, but not in slices from unlesioned sham controls. Denervation did not produce changes in several markers of γ-aminobutyric acid (GABA)ergic synaptic inhibition, including the number of GABAergic neurons, α1 GABA(A) receptor subunits, the vesicular GABA transporter, or miniature inhibitory postsynaptic currents. SIGNIFICANCE: We conclude that chronic partial denervation does lead to a delayed homeostatic increase in neuronal excitability, and may, therefore, contribute to the long-term neurologic consequences of TBI.

Effects of excitotoxic lesions of the CA1 region of the hippocampus on acquisition of a place and cue water maze task.

The aim of the present study was to investigate the effects of excitotoxic lesions of the dorsal CA1 region of the hippocampus on acquisition of a place and cue water maze task. The ibotenic acid injections into CA1 produced removal of the pyramidal cells in CA1, while saving most of the pyramidal cells in CA3 and granule cells in DG intact. In conditions of visible platform training trials, differences in the platform reaching latency between the animals of different groups, were not found. When testing was performed in conditions of submerged platform, the latency of the platform finding was significantly increased (P<0.05). This fact certifies for deficit of the place learning strategy in the CA1-lesioned rats. Decreased place-bias in CA1-lesioned rats in hidden platform training trials compared to the sham-operated rats was significant, but in testing trials when required to choose between the spatial location they had learned and the visible platform in a new location majority of them swam first to the old spatial location. Decreased place-bias in CA1-lesioned rats compared to the sham-operated rats was not significant. We suggest that spatial learning deficits observed after dorsal hippocampal lesions cannot be accounted solely to the loss of dorsal hippocampal CA1 region cells.

Experimental mild traumatic brain injury induces functional alteration of the developing hippocampus.

It is estimated that approximately 1.5 million Americans suffer a traumatic brain injury (TBI) every year, of which approximately 80% are considered mild injuries. Because symptoms caused by mild TBI last less than half an hour by definition and apparently resolve without treatment, the study of mild TBI is often neglected resulting in a significant knowledge gap for this wide-spread problem. In this work, we studied functional (electrophysiological) alterations of the neonatal/juvenile hippocampus after experimental mild TBI. Our previous work reported significant cell death after in vitro injury >10% biaxial deformation. Here we report that biaxial deformation as low as 5% affected neuronal function during the first week after in vitro mild injury of hippocampal slice cultures. These results suggest that even very mild mechanical events may lead to a quantifiable neuronal network dysfunction. Furthermore, our results highlight that safe limits of mechanical deformation or tolerance criteria may be specific to a particular outcome measure and that neuronal function is a more sensitive measure of injury than cell death. In addition, the age of the tissue at injury was found to be an important factor affecting posttraumatic deficits in electrophysiological function, indicating a relationship between developmental status and vulnerability to mild injury. Our findings suggest that mild pediatric TBI could result in functional deficits that are more serious than currently appreciated.

Distinct roles for dorsal CA3 and CA1 in memory for sequential nonspatial events.

Previous studies have suggested that dorsal hippocampal areas CA3 and CA1 are both involved in representing sequences of events that compose unique episodes. However, it is uncertain whether the contribution of CA3 is restricted to spatial information, and it is unclear whether CA1 encodes order per se or contributes by an active maintenance of memories of sequential events. Here, we developed a new behavioral task that examines memory for the order of sequential nonspatial events presented as trial-unique odor pairings. When the interval between odors within a studied pair was brief (3 sec), bilateral dorsal CA3 lesions severely disrupted memory for their order, whereas dorsal CA1 lesions did not affect performance. However, when the inter-item interval was extended to 10 sec, CA1 lesions, as well as CA3 lesions, severely disrupted performance. These findings suggest that the role of CA3 in sequence memory is not limited to spatial information, but rather appears to be a fundamental property of CA3 function. In contrast, CA1 becomes involved when memories for events must be held or sequenced over long intervals. Thus, CA3 and CA1 are both involved in memory for sequential nonspatial events that compose unique experiences, and these areas play different roles that are distinguished by the duration of time that must be bridged between key events.

The velocity-related firing property of hippocampal place cells is dependent on self-movement.

Hippocampal place cells have the interesting property of increasing their firing rate when a freely moving animal increases its running speed through the cell's place field. A previous study from this laboratory showed that this movement-related firing property is disrupted by lesions of the perirhinal cortex (PrhC). It is possible, therefore, that PrhC lesions disrupt speed-modulated sensory information such as optic flow or motor efferent or proprioceptive input that might be available to the hippocampus from the PrhC. To test this hypothesis, rats with single unit recording electrodes implanted in the CA1 region of the hippocampus received different levels of optic flow stimulation in both a freely moving and a passive movement condition. The effects of PrhC lesions were also tested. Although increasing the amount of optic flow information available decreased place field size, it had no discernable effect on the movement-firing rate relationship in the place cells of control animals run in the free-movement condition. In lesioned animals the relationship was disrupted, replicating our previous results. In the passive movement condition many place cells stopped firing. In those cells that did fire, however, the movement-firing rate relationship was no longer evident. These data indicate that the movement-firing rate relationship is not driven by vestibular or optic flow cues, but rather depends on either motor efferent or proprioceptive input, or that it results from some other form of input that may be modulated by self-motion, such as from the vibrissae.

The role of the dorsal CA1 and ventral CA1 in memory for the temporal order of a sequence of odors.

Memory for the temporal order of a sequence of odors was assessed in male rats. A sequence of five odors mixed in sand was presented in digging cups one at a time to each rat in a sequence that varied on each trial. A reward was buried in each cup. Following the fifth odor, two of the previous five odors were presented simultaneously and the rat needed to choose the odor that occurred earliest in the sequence to receive a reward. Temporal separations of 1, 2, or 3 were used which represented the number of odors that occurred between the two odors in the sequence. Once pre-operative criterion was reached, rats received a control, dorsal CA1 (dCA1), or ventral CA1 (vCA1) lesion and were retested on the task. On post-operative trials, only the vCA1 group was impaired relative to both control and dCA1 groups. All groups of rats could discriminate between the odors. The data suggest that the vCA1, but not dorsal CA1, is involved in separating sensory events (odors) in time so that one odor can be remembered separate from another odor.

Expression of protein phosphatase 2B (calcineurin) subunit A isoforms in rat hippocampus after traumatic brain injury.

Calcineurin (CaN) is a calcium/calmodulin-dependent phosphatase directly activated by calcium as a result of neuronal activation that is important for neuronal function. CaN subunit isoforms are implicated in long-term potentiation (LTP), long-term depression (LTD), and structural plasticity. CaN inhibitors are also beneficial to cognitive outcomes in animal models of traumatic brain injury (TBI). There are known changes in the CaN A (CnA) subunit following fluid percussion injury (FPI). The CnA subunit has two isoforms: CnAalpha and CnAbeta. The effect of moderate controlled cortical impact (CCI) on distribution of CnA isoforms was examined at 2 h and 2 weeks post-injury. CnA distribution was assayed by immunohistochemistry and graded for non-parametric analysis. Acutely CnA isoforms showed reduced immunoreactivity in stratum radiatum processes of the ipsilateral CA1 and CA1-2. There was also a significant alteration in the immunoreactivity of both CnA isoforms in the ipsilateral dentate gyrus, predominantly within the hidden blade. Alterations in CnA isoform regional distribution within the CA1, CA1-2, and dentate gyrus may have significant implications for persistent hippocampal dysfunction following TBI, including dysfunction in hippocampal plasticity. Understanding alterations in CnA isoform distribution may help improve the targeting of current therapeutic interventions and/or the development of new treatments for TBI.

Measurement of cerebral reactive hyperemia at the initial post-ischemia reperfusion stage under normothermia and moderate hypothermia in rats.

Inhibition of the initial events occurring immediately after ischemia-reperfusion seems to be beneficial for reducing the extent of subsequent chronic neuronal cell injury. We investigated the effects of moderate hypothermia (32 degrees C) commencing 30 min before ischemia on reactive hyperemia by measuring cerebral blood flow (CBF) with a laser-Doppler flowmeter at the initial ischemia-reperfusion stage (60 min) following 10 min of global cerebral ischemia in rats. In normothermia, CBF was increased to approximately 240% and decreased thereafter, although it remained at approximately 150% after 60 min of ischemia-reperfusion. In contrast, hypothermia increased CBF to more than 270% after ischemia-reperfusion, then recovered to the basal level within 30 min. The period of reactive hyperemia under normothermia tended to be shortened by pre-administration of an NMDA antagonist, in a manner similar to hypothermia. Furthermore, hypothermia inhibited the presence of cells with caspase-3-like immunoreactivity in the hippocampal CA1 sector after 8 h of ischemia-reperfusion. Our findings indicate that hypothermia tends to shorten the period of reactive hyperemia during the initial ischemia-reperfusion stage. This phenomenon may be partly associated with activation of NMDA receptors and a beneficial effect of hypothermia in resisting progression of the neurotoxic cascade in the first 8 h after ischemia-reperfusion.