大鼠脑缺血再灌流后海马氨基酸水平变化与神经元损害的关系探讨

目的探讨脑缺血再灌流后海马氨基酸递质变化与神经元损害的关系。方法建立大鼠前脑缺血再灌流模型,测定海马ＣＡ１区和ＣＡ３／齿状回区游离氨基酸含量,观察阻断隔－海马通路对海马神经元损害和氨基酸水平的影响。结果（１）海马结构中仅ＣＡ１区神经元明显损害,但ＣＡ１区和ＣＡ３／齿状回区的Ｇｌｕ、Ａｓｐ和ＧＡＢＡ含量无差异。（２）阻断隔－海马通路可明显减轻海马神经元损害,但对海马氨基酸水平变化无影响。结论脑缺血再灌流后,氨基酸递质水平的异常变化不是海马ＣＡ１区神经元选择性易损的唯一决定因素,隔－海马通路末梢释放的神经递质也参与海马神经元损害过程。     

Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.     

Severe alcohol-induced neuronal deficits in the hippocampus and neocortex of neonatal mice genetically deficient for neuronal nitric oxide synthase (nNOS)

Alcohol can severely damage the developing brain, and neuronal loss is a critical component of this injury. Thus, identification of molecular factors that ameliorate alcohol-induced neuronal loss is of great importance. Previous in vitro work has demonstrated that nitric oxide (NO) protects neurons against alcohol toxicity. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes NO in neurons, have an increased vulnerability to alcohol-induced neuronal loss in the neocortex and hippocampus. Wildtype mice and nNOS-/- mice received ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (P) 4-9 and were sacrificed on P10. The number of hippocampal CA1 and CA3 pyramidal cells, dentate gyrus granule cells, and neocortical neurons were determined using stereological methods. Alcohol pharmacokinetics did not differ between wildtype and nNOS-/- strains. Alcohol induced dose-dependent reductions in all four neuronal populations, and the losses were substantially more severe in the nNOS-/- mice than in wildtype. Furthermore, the threshold dose of alcohol to induce cell death was lower in the nNOS-/- mice than in the wildtype mice for all neuronal populations. While nNOS deficiency worsened alcohol-induced neuronal losses, the magnitude of this exacerbation varied among brain regions and depended on alcohol dose. These results demonstrate that nNOS deficiency decreases the ability of developing neurons in vivo to survive the toxic effects of alcohol and strengthen the hypothesis that NO exerts a neuroprotective effect against alcohol toxicity in the developing brain.     

p53 potentiates hippocampal neuronal death caused by global ischemia.

Although p53 controls cell death after various stresses, its role in neuronal death after brain ischemia is poorly understood. To address this issue, we subjected p53-deficient (p53-/- and p53+/-) mice (backcrossed for 12 generations with C57BL/6 mice) and wild-type mice (p53+/+) to transient global ischemia by the three-vessel occlusion method. Despite similar severity of ischemia, as shown by anoxic depolarization and cortical blood flow, neuronal death in the hippocampal cornus ammonis (CA)1 region was much more extensive in p53+/+ than in p53-/- mice (surviving neuronal count, 9.3%+/-3.0% versus 61.3%+/-34.0% of nonischemic p53+/+ controls, respectively, P<0.0037). In p53+/- mice, a similar trend was also observed, though not statistically significant (43.5% of nonischemic p53+/+ controls). In p53+/+ mice, p53-like immunoreactivity in hippocampal CA1 neurons was enhanced at 12 h after ischemia, and messenger ribonucleic acid for Bax, a direct downstream target of p53, was also increased. These results indicate that p53 potentiates ischemic neuronal death in vivo and suggest that this molecule could be a therapeutic target in neuronal death after cerebral ischemia.     

Pattern of neuronal death in the rat hippocampus after status epilepticus. Relationship to calcium binding protein content and ischemic vulnerability.

The pattern of hippocampal cell death has been studied following hippocampal seizure activity and status epilepticus induced by 110-min stimulation of the perforant pathway in awake rats. The order of vulnerability of principal cells in the different hippocampal subfields--as determined by silver impregnation--was found to be very similar to the pattern found in ischemia; i.e., dentate hilus greater than CA1, subiculum greater than CA3c greater than CA3a,b greater than dentate granule cells. The hilar somatostatin-containing cells were the most vulnerable cell type, whereas all other subpopulations of nonprincipal neurons--visualized by immunocytochemistry for the calcium binding proteins parvalbumin and calbindin--were remarkably resistant. Pyramidal cells in the CA3 region containing neither of the examined calcium binding proteins were more resistant to overexcitation than CA1 pyramidal cells, most of which do contain calbindin. This indicates that no simple relationship exists between vulnerability in status epilepticus and neuronal calcium binding protein content, and that local and/or systemic hypoxia during status epilepticus may be responsible for the ischemic pattern of cell death.     

Poor correlation between delayed neuronal death induced by transient forebrain ischemia,and immunoreactivity for parvalbumin and calbindin D-28k in developing gerbil hippocampus

In the normal developing hippocampus of the gerbil, parvalbumin-immunoreactive neurons first appear in the stratum pyramidale of CA3 at postnatal day 15 (P15), and in CA2 and hilus of the dentate gyrus from P21 onwards. Immunoreactive terminals also follow the same sequence from CA3 to CA1 to reach adult patterns by the end of the 1st month. Calbindin D-28k immunoreactivity is seen in the external part of the upper blade of the dentate gyrus at P5, and progresses to the granule cell and molecular layers of the whole gyrus by P15, except for a thin band of immature cells located at the base of the granule cell layer which are calbindin negative. Calbindin immunoreactivity in mossy fibers progresses from the external to the hilar region of CA3 during the same period. A few immunoreactive cells are also found in the stratum radiatum/lacunare of the CA3, but no calbindin-immunoreactive cells are observed in the CA1 and CA2 subfields. The adult pattern of calbindin immunoreactivity is reached at P21. Vulnerability following transient forebrain ischemia for 20 min was examined in the hippocampal formation of gerbils during postnatal development. No cellular damage was seen in animals aged 7 days. Dying cells were observed at the base of the granule cell layer of the dentate gyrus in animals aged 15, 21 and 30 days. Pyramidal cells in the CA3 subfield were also sensitive to ischemia in gerbils aged 15 days, and less frequently in animals aged 21 days. The adult pattern of cellular damage, characterized by selective vulnerability of the CA1 subfield, was seen from day 30 onwards. These findings show that the pattern of selective vulnerability following transient forebrain ischemia is different in young and adult gerbils, and suggest that little, if any, correlation exists between resistance to delayed cellular damage and parvalbumin and calbindin D-28k content in the hippocampus of young gerbils.Supported in part by grant FIS 93-131 and a grant from the Fundacio Pi i Synyer (to A.T.)     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

The temporal evolution of neuronal damage from pilocarpine-induced status epilepticus

The temporal evolution of irreversible neuronal damage from pilocarpine-induced seizures was studied by light microscopy. Neuronal cell death was judged on a 0–3 scale by estimating the percentage of acidophilic neurons in each of 23 brain regions. In addition, in the dorsal dentate hilus (CA4), quantitative cell counts of normal and acidophilic neurons were also performed. A few dead neurons (grade 0.5 damage) appeared in ventral hippocampal CA1 and CA3 regions after 20-min status epilepticus (SE). Slight-to-mild damage (grades 0.5–1.5) occurred in 14 and 12 brain regions after 40-min and 1-h SE respectively, and slight-to-moderate damage (grades 0.5–2.0) was found in 15 regions after 3-h SE. Twenty-four h and 72 h after 3-h SE, there was slight-to-severe damage (grade 0.5–3.0) in 22 and 21 regions respectively. Three-h SE produced more severe damage to 7 brain regions compared to 1-h SE, and 16 regions had more pronounced neuronal injury 24 h after rather than 0–4 h after 3-h SE. Eight brain regions had less damage 72 h compared to 24 h after SE, probably because of progressive neuronal lysis and dropout, but in mediodorsal and lateroposterior thalamic nuclei damage worsened from 24 to 72 h after SE. Neuronal cell counting revealed 20% acidophilic neurons in dorsal dentate hilus after 40-min SE and no difference between the 1-h and 3-h seizure groups (31% vs. 43% acidophilic neurons respectively). Among the 3 groups of rats with 3-h SE and varying recovery periods, the 24-h and 72-h recovery groups had higher percentages of acidophilic neurons (65% and 54% respectively) than the 0–4-h group (43%). Finally, the hippocampal CA2 region and dentate granule cell layer and the caudate-putamen, considered resistant to seizure-induced cell injury, were all damaged from SE lasting 40 min or more.     

Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons' death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.     

Differential effects of Bcl‐2 overexpression on hippocampal CA1 neurons and dentate granule cells following hypoxic ischemia in adult mice

In contrast to its known anti‐apoptotic activity in sympathetic neurons, immortal neuronal cell lines, and primary cultured immature neurons of the central nervous system (CNS), the role of Bcl‐2 in CNS neurons in the adult brain is poorly understood. In the present study, we examined effects of overexpression of Bcl‐2 on selective neuronal death of the hippocampal CA1 neurons and the dentate granule cells induced by hypoxic ischemia in adult transgenic mice overexpressing human Bcl‐2 under the control of neuron‐specific enolase (NSE‐hbcl‐2). At the light microscopic level, numbers of TUNEL‐positive cells with pyknotic nuclei were observed in the CA1 subfield of NSE‐hbcl‐2 transgenic mice, as well as that of wild‐type mice, after hypoxic ischemic insult, although the onset of neuronal death was apparently delayed in NSE‐hbcl‐2 transgenic mice. The electron microscopic studies showed that morphological changes of the degenerating CA1 neurons from both groups were clearly distinct from ordinary apoptosis. In contrast, a significant amount of degenerating dentate granule cells from wild‐type but not from transgenic mice had typical apoptotic nuclei by the treatment. The activation of caspase‐3 was detected in the dentate granule cells but not that of the CA1 neurons. These results indicate that the overexpression of Bcl‐2 effectively suppressed dentate granule cell apoptosis but only delayed cell death of the CA1 neurons induced by hypoxic ischemia, suggesting the occurrence of a non‐apoptotic, caspase‐3–independent mechanism for neuronal death in the CA1 subfield. J. Neurosci. Res. 57:1–12, 1999. © 1999 Wiley‐Liss, Inc.     

Continuous cytosine-b-D-arabinofuranoside infusion reduces ectopic granule cells in adult rat hippocampus with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus

Brief or prolonged seizures induce various patterns of plasticity. Axonal or dendritic remodelling and development of ectopic granule cells have been described in the hilus and molecular layer of the adult rodent hippocampus. Hippocampal cell proliferation also occurs after seizures. However, whether the seizure-induced cell proliferation plays a pathological or reparative role in the epileptic brain is unknown. In this study, we attempted to suppress the seizure-induced cell proliferation with the antimitotic agent cytosine-b-D-arabinofuranoside (Ara-C) and to examine the development of spontaneous recurrent seizures (SRS). Experimental status epilepticus was induced with pilocarpine, and Ara-C or vehicle alone was infused continuously with an osmotic minipump. SRS were video-monitored. BrdU immunohistochemistry was used for the spatial and temporal analysis of hippocampal cell proliferation, and double labelling with NeuN, calbindin and GFAP antibodies was performed for the differentiation of BrdU-positive cells. Timm staining was also performed for evaluation of mossy fibre sprouting (MFS). With continuous Ara-C infusion, the likelihood of developing SRS was decreased and, during the latent period, the development of ectopic granule cells in the hilus and new glia in the CA1 area was reduced when compared with the vehicle-infused group, while MFS was not altered. The results suggest that the hippocampal cell proliferation plays a pro-epileptogenic role rather than a compensatory role, and that the epileptogenic process may be associated with the generation of new glia in the CA1 area and/or new neurons in the dentate gyrus, particularly the ectopically located hilar granule cells.     

Regional distribution of fluoro-jade B staining in the hippocampus following traumatic brain injury

Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons. We were interested in applying FJB staining in a well-characterized model of traumatic brain injury (TBI) in order to estimate the total number of neurons in different regions of the hippocampus that die after a mild or moderate injury. Rats were subjected to a mild or moderate unilateral cortical contusion (1.0- or 1.5-mm displacement from the cortical surface) with a controlled cortical impacting device. Animals were allowed to survive for 1, 2, or 7 days and the total number of FJB-positive neurons in hippocampal areas CA1, CA3, and the dentate gyrus granule layer was estimated using sterological methods. The region that had the highest number of FJP-positive neurons after TBI was the dentate gyrus. In both 1- and 1.5-mm injuries, FJB-positive granule cells were observed throughout the rostro-caudal extent of the dentate. In contrast, labeled pyramidal neurons of area CA3 were most numerous after the 1.5-mm injury. The area that had the fewest number of FJB-labeled cells was area CA1 with only scattered neurons seen in the 1.5-mm group. In both injury groups and in all hippocampal regions, more FJB-positive neurons were seen at the earlier times post injury (1 and 2 days) than at 7 days. FJB appears to be a reliable marker for neuronal vulnerability following TBI.     

Alterations of potassium currents in ischemia-vulnerable and ischemia-resistant neurons in the hippocampus after ischemia

CA1 pyramidal neurons in the hippocampus die 2-3 days following transient forebrain ischemia, whereas CA3 pyramidal neurons and granule cells in the dentate gyrus remain viable. Excitotoxicity is the major cause of ischemic cell death, and potassium currents play important roles in regulating the neuronal excitability. The present study compared the changes of potassium currents in acutely dissociated hippocampal neurons at different intervals after ischemia. In CA1 neurons, the amplitude of rapid inactivating potassium currents (I(A)) was significantly increased at 14 h and returned to control levels at 38 h after ischemia; the rising slope and decay time constant of I(A) were accordingly increased after ischemia. The activation curve of I(A) in CA1 neurons shifted to the depolarizing direction at 38 h after ischemia. In granule cells, the amplitude and rising slope of I(A) were significantly increased at 38 h after ischemia; the inactivation curves of I(A) shifted toward the depolarizing direction accordingly at 38 h after ischemia. The I(A) remained unchanged in CA3 neurons after ischemia. The amplitudes of delayed rectifier potassium currents (I(Kd)) in CA1 neurons were progressively increased after ischemia. No significant difference in I(Kd) was detected in CA3 and granule cells at any time points after reperfusion. These results indicated that the voltage dependent potassium currents in hippocampal neurons were differentially altered after cerebral ischemia. The up-regulation of I(A) in dentate granule cells might have protective effects. The increase of I(Kd) in CA1 neurons might be associated with the neuronal damage after ischemia.     

Glutamate receptor 1-immunopositive neurons in the gliotic CA1 area of the mouse hippocampus after pilocarpine-induced status epilepticus

Significant reduction in glutamate receptor 1 (GluR1)- and GluR2/3-immunopositive neurons was demonstrated in the hilus of the dentate gyrus in mice killed on days 1, 7 and 60 after pilocarpine-induced status epilepticus (PISE). In addition, GluR1 and GluR2/3 immunostaining in the strata oriens, radiatum and lacunosum moleculare of areas CA1-3 decreased drastically on days 7 and 60 after PISE. Neuronal loss observed in the above regions may account, at least in part, for a decrease in GluR immunoreactivity. By contrast, many GluR1-immunopositive neurons were observed in the gliotic area of CA1. Of these, about 42.8% were immunopositive for markers for hippocampal interneurons, namely calretinin (7.6%), calbindin (12.8%) and parvalbumin (22.4%). GluR1 or GluR2/3 and BrdU double-labelling showed that the GluR1- and GluR2/3-immunopositive neurons at 60 days after PISE were neurons that had survived rather than newly generated neurons. Furthermore, anterograde tracer and double-labelling studies performed on animals at 60 days after PISE indicated a projection from the hilus of the dentate gyrus to gliotic areas in both CA3 and CA1, where the projecting fibres apparently established connections with GluR1-immunopositive neurons. The projection to CA1 was unexpected. These novel findings suggest that the intrinsic hippocampal neuronal network is altered after PISE. We speculate that GluR1-immunopositive neurons in gliotic CA1 act as a bridge between dentate gyrus and subiculum contributing towards epileptogenesis.     

Acute, but reversible, kainic acid-induced DNA damage in hippocampal CA1 pyramidal cells of p53-deficient mice

p53 plays an essential role in mediating apoptotic responses to cellular stress, especially DNA damage. In a kainic acid (KA)-induced seizure model in mice, hippocampal CA1 pyramidal cells undergo delayed neuronal death at day 3-4 following systemic KA administration. We previously demonstrated that CA1 neurons in p53(-/-) animals are protected from such apoptotic neuronal loss. However, extensive morphological damage associated with DNA strand breaks in CA1 neurons was found in a fraction of p53(-/-) animals at earlier time points (8?h to 2?days). No comparable acute damage was observed in wild-type animals. Stereological counting confirmed that there was no significant loss of CA1 pyramidal cells in p53(-/-) animals at 7?days post-KA injection. These results suggest that seizure-induced DNA strand breaks are accumulated to a greater extent but do not lead to apoptosis in the absence of p53. In wild-type animals, therefore, p53 appears to stimulate DNA repair and also mediate apoptosis in CA1 neurons in this excitotoxicity model. These results also reflect remarkable plasticity of neurons in recovery from injury.     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Cracking down on inhibition: selective removal of GABAergic interneurons from hippocampal networks

Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic--but not glutamatergic--synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin(+), calretinin(+), parvalbumin/system A transporter 1(+), somatostatin(+), vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB(1) cannabinoid receptor(+) and neuropeptide Y(+) local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1(+) excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.     

Topiramate reduces neuronal injury after experimental status epilepticus

Prolonged seizures are associated with injury to vulnerable neurons, particularly in the hippocampus. Identification of compounds that attenuate injury after prolonged seizures could be of value in the management of refractory status epilepticus. We hypothesized that topiramate, an anticonvulsant with multiple mechanisms of action, would attenuate hippocampal neuronal injury when given after experimental status epilepticus. Limbic status epilepticus was induced in adult male Wistar rats for 140 min by unilateral hippocampal electrical stimulation. Rats then received intraperitoneal injections of either vehicle (n=6) or topiramate at 20 mg/kg (n=6), 40 mg/kg (n=7) or 80 mg/kg (n=7). Three days later, hippocampal sections were processed for neuronal degeneration using a silver impregnation stain. Seizure-induced damage was assessed by measuring the density of silver staining in hippocampal regions CA1, CA3 and dentate hilus. Administration of topiramate at each dose was associated with a significant reduction in staining density bilaterally in area CA1 and the dentate hilus. Reduction in staining density in area CA3 was seen contralateral to the side of stimulation at the two higher topiramate doses only. The results indicate that administration of topiramate after experimental status epilepticus can attenuate seizure-induced hippocampal neuronal injury.     

Hippocampal injury,atrophy, synaptic reorganization,and epileptogenesis after perforant pathway stimulation‐induced status epilepticus in the mouse

Prolonged dentate granule cell discharges produce hippocampal injury and chronic epilepsy in rats. In preparing to study this epileptogenic process in genetically altered mice, we determined whether the background strain used to generate most genetically altered mice, the C57BL/6 mouse, is vulnerable to stimulation‐induced seizure‐induced injury. This was necessary because C57BL/6 mice are reportedly resistant to the neurotoxic effects of kainate‐induced seizures, which we hypothesized to be related to strain differences in kainate's effects, rather than genetic differences in intrinsic neuronal vulnerability. Bilateral perforant pathway stimulation‐induced granule cell discharge for 4 hours under urethane anesthesia produced degeneration of glutamate receptor subunit 2 (GluR2)‐positive hilar mossy cells and peptide‐containing interneurons in both FVB/N (kainate‐vulnerable) and C57BL/6 (kainate‐resistant) mice, indicating no strain differences in neuronal vulnerability to seizure activity. Granule cell discharge for 2 hours in C57BL/6 mice destroyed most GluR2‐positive dentate hilar mossy cells, but not peptide‐containing hilar interneurons, indicating that mossy cells are the neurons most vulnerable to this insult. Stimulation for 24 hours caused extensive hippocampal neuron loss and injury to the septum and entorhinal cortex, but no other detectable damage. Mice stimulated for 24 hours developed hippocampal sclerosis, granule cell mossy fiber sprouting, and chronic epilepsy, but not the granule cell layer hypertrophy (granule cell dispersion) produced by intrahippocampal kainate. These results demonstrate that perforant pathway stimulation in mice reliably reproduces the defining features of human mesial temporal lobe epilepsy with hippocampal sclerosis. Experimental studies in transgenic or knockout mice are feasible if electrical stimulation is used to produce controlled epileptogenic insults. J. Comp. Neurol. 515:181–196, 2009. © 2009 Wiley‐Liss, Inc.