Rapid hippocampal network adaptation to recurring synchronous activity--a role for calcineurin.

Neuronal networks are thought to gradually adapt to altered neuronal activity over many hours and days. For instance, when activity is increased by suppressing synaptic inhibition, excitatory synaptic transmission is reduced. The underlying compensatory cellular and molecular mechanisms are thought to contribute in important ways to maintaining normal network operations. Seizures, due to their massive and highly synchronised discharging, probably challenge the adaptive properties of neurons, especially when seizures are frequent and intense - a condition common in early childhood. In the experiments reported here, we used rat and mice hippocampal slice cultures to explore the effects that recurring seizure-like activity has on the developing hippocampus. We found that developing networks adapted rapidly to recurring synchronised activity in that the duration of seizure-like events was reduced by 42% after 4 h of activity. At the same time, the frequency of spontaneous excitatory postsynaptic currents in pyramidal cells, the expression of biochemical biomarkers for glutamatergic synapses and the branching of pyramidal cell dendrites were all dramatically reduced. Experiments also showed that the reduction in N-methyl-D-aspartate receptor subunits and postsynaptic density protein 95 expression were N-methyl-D-aspartate receptor-dependent. To explore calcium signaling mechanisms in network adaptation, we tested inhibitors of calcineurin, a protein phosphatase known to play roles in synaptic plasticity and activity-dependent dendrite remodeling. We found that FK506 was able to prevent all of the electrophysiological, biochemical, and anatomical changes produced by synchronised network activity. Our results show that hippocampal pyramidal cells and their networks adapt rapidly to intense synchronised activity and that calcineurin play an important role in the underlying processes.

Demographic Characteristics, Survival and Prognostic Factors for Mortality in Cats with Primary Immune-Mediated Hemolytic Anemia.

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) is uncommon in cats, but may result in severe disease. Demographic predispositions for development of the disease and prognostic factors for mortality have not been investigated previously. HYPOTHESIS/OBJECTIVES: To explore possible demographic predispositions for development of primary IMHA in cats and to investigate possible prognostic factors for mortality. ANIMALS: 107 client-owned cats with IMHA, of which 72 had primary IMHA and 35 had secondary IMHA, and 9,194 control cats. METHODS: Data were collected retrospectively from records of cats with IMHA, defined by the presence of anemia and concurrent autoagglutination, ghost cells without oxidative damage on fresh blood smear, positive titer in a direct antiglobulin test, or evidence of phagocytosis of erythroid precursors in bone marrow. Odds ratios were calculated to assess the risk of development of primary IMHA in different demographic groups and Cox proportional hazards analysis was conducted to evaluate prognostic factors. RESULTS: No sex or breed predisposition was identified for the development of primary IMHA in comparison to the control cats, but cats in the age range 2.1-5.9 years were predisposed. Higher total bilirubin concentration and age were significant negative prognostic factors and higher lymphocyte numbers and serum globulin concentration were positive prognostic factors in a multivariable model. CONCLUSIONS AND CLINICAL IMPORTANCE: Young adult cats were more likely to develop primary IMHA than other groups, but no apparent male predisposition was identified in this study, contrary to previous reports. Several prognostic factors were identified, which may be helpful in guiding clinical practice in the future.

Blockade of neuronal activity during hippocampal development produces a chronic focal epilepsy in the rat.

During brain development, neuronal activity can transform neurons characterized by widely ranging axonal projections to ones with more restricted patterns of synaptic connectivity. Previous studies have shown that an exuberant outgrowth of local recurrent excitatory axons occurs in hippocampal area CA3 during postnatal weeks 2 and 3. Axons are remodeled with maturation, and nearly half of the branches are eliminated. Postnatal weeks 2 and 3 also coincide with a "critical" period of development, when CA3 networks have a marked propensity to generate electrographic seizures. In an attempt to prevent axonal remodeling, local circuit activity was blocked unilaterally in dorsal hippocampus by continuous infusion of tetrodotoxin (TTX). Field potential recordings from behaving animals were dramatically altered when TTX infusion was initiated at the beginning of the critical period, week 2, but not later in life. Spontaneous, synchronized spikes and electrographic seizures with behavioral accompaniments were observed after 4 weeks of TTX infusion and persisted into adulthood. When recordings were made during TTX infusion, synchronized spiking was recorded in ventral hippocampus as early as 2 weeks after infusate introduction. At this same time, extracellular field recordings from in vitro slices demonstrated spontaneous network-driven "mini-bursts" arising from ventral hippocampal slices. These were abolished by glutamate receptor antagonists. Whole-cell recordings from CA3 neurons revealed bursts of excitatory synaptic potentials coincident with the network bursts recorded extracellularly. Thus, local assemblies of mutually excitatory CA3 pyramidal cells are hyperexcitable in these rats. Whether alterations in developmental axonal remodeling mediate these effects awaits further studies.

Novel hippocampal interneuronal subtypes identified using transgenic mice that express green fluorescent protein in GABAergic interneurons.

The chief inhibitory neurons of the mammalian brain, GABAergic neurons, are comprised of a myriad of diverse neuronal subtypes. To facilitate the study of these neurons, transgenic mice were generated that express enhanced green fluorescent protein (EGFP) in subpopulations of GABAergic neurons. In one of the resulting transgenic lines, called GIN (GFP-expressing Inhibitory Neurons), EGFP was found to be expressed in a subpopulation of somatostatin-containing GABAergic interneurons in the hippocampus and neocortex. In both live and fixed brain preparations from these mice, detailed microanatomical features of EGFP-expressing interneurons were readily observed. In stratum oriens of the hippocampus, EGFP-expressing interneurons were comprised almost exclusively of oriens/alveus interneurons with lacunosum-moleculare axon arborization (O-LM cells). In the neocortex, the somata of EGFP-expressing interneurons were largely restricted to layers II-IV and upper layer V. In hippocampal area CA1, two previously uncharacterized subtypes of interneurons were identified using the GIN mice: stratum pyramidale interneurons with lacunosum-moleculare axon arborization (P-LM cells) and stratum radiatum interneurons with lacunosum-moleculare axon arborization (R-LM cells). These newly identified interneuronal subtypes appeared to be closely related to O-LM cell, as they selectively innervate stratum lacunosum-moleculare. Whole-cell patch-clamp recordings revealed that these cells were fast-spiking and showed virtually no spike frequency accommodation. The microanatomical features of these cells suggest that they function primarily as "input-biasing" neurons, in that synaptic volleys in stratum radiatum would lead to their activation, which in turn would result in selective suppression of excitatory input from the entorhinal cortex onto CA1 pyramidal cells.

A role for L-type calcium channels in the maturation of parvalbumin-containing hippocampal interneurons.

While inhibitory interneurons are well recognized to play critical roles in the brain, relatively little is know about the molecular events that regulate their growth and differentiation. Calcium ions are thought to be important in neuronal development and L-type voltage gated Ca(+2) channels have been implicated in activity-dependent mechanisms of early-life. However, few studies have examined the role of these channels in the maturation of interneurons. The studies reported here were conducted in hippocampal slice cultures and indicate that the L-type Ca(+2) channel agonists and antagonists accelerate and suppress respectively the growth of parvalbumin-containing interneurons. The effects of channel blockade were reversible suggesting they are not the result of interneuronal cell death. Results from immunoblotting showed that these drugs have similar effects on the expression of the GABA synthetic enzymes, glutamic acid decarboxylase65, glutamic acid decarboxylase67 and the vesicular GABA transporter. This suggests that L-type Ca(+2) channels regulate not only parvalbumin expression but also interneuron development. These effects are likely mediated by actions on the interneurons themselves since the alpha subunits of L-type channels, voltage-gated calcium channel subunit 1.2 and voltage-gated calcium channel subunit 1.3 were found to be highly expressed in neonatal mouse hippocampus and co-localized with parvalbumin in interneurons. Results also showed that while these interneurons can contain either subunit, voltage-gated calcium channel subunit 1.3 was more widely expressed. Taken together results suggest that an important subset of developing interneurons expresses L-type Ca(+2) channels alpha subunits, voltage-gated calcium channel subunit 1.2 and especially voltage-gated calcium channel subunit 1.3 and that these channels likely regulate the development of these interneurons in an activity-dependent manner.

Systematic review of prognostic factors for mortality in dogs with immune-mediated hemolytic anemia.

BACKGROUND: Treatment of dogs with primary immune-mediated hemolytic anemia (IMHA) is difficult and frequently unrewarding. Prognostic factors have been evaluated in a number of previous studies, and identification of such factors would be beneficial to enable selection of appropriate therapeutic regimens and supportive care. OBJECTIVES: The aim of the current study was to undertake a critical appraisal of the risk of bias in evidence relating to prognostic indicators for mortality in dogs with IMHA. ANIMALS: Three hundred and eighty client-owned dogs with spontaneous primary idiopathic IMHA reported in 6 previous studies. METHODS: A systematic review was conducted to evaluate evidence relating to prognostic factors for mortality in dogs with primary IMHA. Search tools were employed to identify articles and a validated appraisal tool was used to assess the quality of individual studies by considering inclusion and exclusion criteria, measurement of prognostic, outcome and confounding variables, and statistical methods. RESULTS: Few studies evaluated prognostic indicators for IMHA in dogs, and all of these suffered from methodologic flaws in at least 1 major area. Fifteen different variables were identified as prognostic indicators, with 2 variables identified by >1 study. CONCLUSIONS AND CLINICAL IMPORTANCE: There are few pieces of high-quality evidence available to enable estimation of prognosis for dogs presenting with primary IMHA.

GABAergic neurons that pioneer hippocampal area CA1 of the mouse: morphologic features and multiple fates.

Dramatic changes occur in the expression of glutamic acid decarboxylase (GAD67) immunoreactivity in mouse hippocampus during postnatal development. Most striking is the presence of a dense population of immunopositive cells in stratum radiatum and stratum oriens in area CA1 during the first postnatal week. Between days 5 and 10, these cells disappear and the GAD67 immunoreactivity begins to resemble that of adulthood. These neurons are considered pioneer cells, and studies were undertaken to determine their fate. Between days 5 and 50, area CA1 doubles in size; however, the loss of cells expressing GAD67 mRNA cannot be explained solely by dilution resulting from hippocampal growth. In stratum radiatum, cell loss is particularly dramatic. Although between days 5 and 15, many cells seem to migrate from stratum radiatum to its border with stratum lacunosum-moleculare, both fate maps of pioneer cells labeled with bromodeoxyuridine (BrdU) on embryonic day 13 (E13) and in situ DNA end-labeling studies suggest that some cells die by means of programmed cell death. However, not all pioneer cells die, because many cells labeled with BrdU on E13 are present in adulthood and express markers for and have anatomic features of hippocampal interneurons. In conclusion, events that underlie the age-dependent disappearance of gamma-aminobutyric acid (GABA) -ergic pioneer cells are complex and cannot be completely explained by dilution in an expanding neuropile. Although some GABAergic pioneer cells likely undergo programmed cell death during the first postnatal weeks, others relocate within hippocampal laminae and terminally differentiate into the interneurons of adulthood.

Axonal remodeling during postnatal maturation of CA3 hippocampal pyramidal neurons.

Anatomical substrates were investigated for local circuit hyperexcitability that occurs in the CA3 subfield of the rat hippocampus during postnatal week 2. A transient excess of excitatory local circuit connectivity was hypothesized to underlie this hyperexcitability. To test this hypothesis, recurrent excitatory axon arbors from single biocytin-filled CA3 pyramidal cells were reconstructed. Arbors were analyzed in segments of area CA3 comparable in size to in vitro minislice preparations, which were shown to reproduce the developmental hyperexcitability seen in intact slices during postnatal week 2. Segments were then adjusted for hippocampal growth, based on age-dependent changes in neuron density in stratum pyramidale. Axon arbors were found to be short and possessed very few branches during the first postnatal week. By the second postnatal week, arbors had undergone dramatic growth and were much longer and more complex in their branching patterns. By adulthood, a significant decrease in all measures of arbor length and complexity was observed. Following growth adjustment, measures of axon length and varicosity number during week 2 were not significantly different from that of adulthood. However, the number of axon branches decreased by 50%. These results suggest that, during early postnatal life, there is exuberant outgrowth of local CA3 recurrent axons, and with maturation these recurrent collaterals are remodeled. Short-ranging, profusely branched axons appear to be replaced by longer-ranging arbors that possess fewer branches. Maturational changes in the dendritic location rather than the number of early-formed recurrent excitatory synapses may explain developmental hyperexcitability of the hippocampal CA3 subfield.

Treatment of infantile spasms with high-dose ACTH: efficacy and plasma levels of ACTH and cortisol.

Fifteen children with infantile spasms and a hypsarrhythmic EEG defined by EEG-videotelemetry monitoring received a regimen of high-dose (150 IU/m2/d) ACTH for their seizures. We carried out an endocrinologic evaluation before and after initiation of the ACTH and conducted a time course study of plasma ACTH and cortisol levels after ACTH dosing. Spasms were controlled and the EEG normalized in 14 of the 15 children. Prior to starting ACTH therapy all the patients had normal prolactin, insulin, cortisol, and ACTH levels in plasma and normal thyroid function. Although the pattern of rise of ACTH levels in plasma after ACTH dosing was similar in all the children, there was great individual variation in the absolute concentrations. However, both the pattern of rise and absolute level of cortisol in plasma after ACTH was highly predictable in all patients. Plasma cortisol rose rapidly within 1 hour of ACTH administration and continued a slower rise for 12 to 24 hours after the ACTH dose. High-dose ACTH therapy seems quite effective in infantile spasms, perhaps because of a sustained high level of plasma cortisol. This sustained plateau of cortisol may be more effective in controlling infantile spasms than the pulse effect expected with oral steroids or lower doses of ACTH.

A role for sodium and chloride in kainic acid-induced beading of inhibitory interneuron dendrites.

Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors. In experiments reported here, transient (1h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5mM kainic acid, more than half of the immunopositive apical dendrites in area CA(1) had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl-D-aspartate receptor antagonists, the L-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading.Thus, we suggest that the movement of Na(+) and Cl(-), rather than Ca(2+), into cells underlies the focal swellings of interneuron dendrites in hippocampus.

Expression of calretinin in diverse neuronal populations during development of rat hippocampus.

The prenatal and postnatal expression of calretinin was studied in hippocampus of the rat using immunohistochemical procedures. Calretinin was detected as early as embryonic day 15 in the primordial hippocampus where calretinin-containing neurons and fibres were localized to the primitive plexiform layer. Upon emergence of the hippocampal plate (the prospective stratum pyramidale), large numbers of immunopositive multipolar cells were observed in the marginal zone. Fewer cells with fusiform cell bodies were observed bordering the hippocampal plate and subplate. During the perinatal period (embryonic day 20 to postnatal day 0), large numbers of immunoreactive pyramidal-like neurons were observed at the margin of the hippocampal plate with the subplate. At this same time, many calretinin-containing neurons with irregularly shaped dendrites were observed in stratum radiatum. Soon after birth (postnatal day 3), the calretinin immunoreactivity of both these later cell types rapidly declined and a new population of calretinin-immunopositive cells emerged, the Cajal-Retzius cells of stratum lacunosum-moleculare and the dentate gyrus. The Cajal-Retzius cells rapidly matured but disappeared by the second postnatal week. During the second postnatal week, calretinin interneurons of the adult hippocampal formation began to appear. Their immunoreactivity increased by postnatal day 15, when the number of calretinin-immunopositive interneurons in area CA1 and stratum radiatum of CA3 exceeded that of the adult. At this time, the soma and proximal dendrites of many calretinin interneurons were found to contact each other. The frequency of such cellular appositions decreased in adulthood. The results presented here show that calretinin immunohistochemistry can be very useful in recording the development of subpopulations of hippocampal neurons that are present during distinct embryonic and postnatal periods. Although some neuronal types may exist only briefly during hippocampal development, others appear to express calretinin transiently during restricted phases of neuronal differentiation. Surprisingly, this includes some hippocampal pyramidal cells. However, even as the adult pattern of immunostaining emerges in week 2, morphological refinement of interneurons continues to take place, which eventually leads to the population of calretinin-containing interneurons of the mature hippocampus.

Long-term depression of perforant path excitatory postsynaptic potentials following synchronous network bursting in area CA3 of immature hippocampus.

Various forms of synaptic long-term potentiation and depression have been studied in detail in hippocampus and neocortex. Each are produced by specific patterns of synaptic activation and rely on electrical stimulation of afferents for their induction. Few studies have explored the ability of activity produced by cortical networks themselves to generate similar long-term changes in synaptic efficacy. Experiments have shown that periods of synchronized network bursting in both slices and dissociated cultures of hippocampus can lead to persistent network discharges. Conversely, one study has reported that network discharging can disrupt the induction of long-term potentiation in hippocampus. Whether long-term depression can be induced by synchronous network discharging is unknown. In experiments reported here, we examined the long-term effects of synchronized activity within hippocampal CA3 networks on synaptic potentials produced by the converging perforant path. Prior to induction of network bursting, slices were incubated in a perfusate containing picrotoxin and elevated (4 mM) Ca2+ and Mg2+. To induce network discharges, the concentration of both divalent cations were reduced to normal levels (1.5 mM). Following 20 min of network bursting, perforant path synapses, that did not participate in network discharging, underwent a 30% non-decrementing long-term depression. At the same time, synchronized network discharges, that were absent prior to induction, persisted upon return to preinduction conditions. The antagonist of the N-methyl-D-aspartate receptor, D(-)2-amino-5-phosphonovalerate, blocked both long-term depression of perforant path excitatory postsynaptic potentials and persistent network discharging. Results suggest that activity generated by hippocampal networks is able to produce long-term depression of non-coincidentally active synapses.

Postnatal development of GABA-mediated synaptic inhibition in rat hippocampus.

Developmental alterations in GABAergic synaptic transmission were examined physiologically and biochemically in hippocampus of rats from 3 days of age to adulthood. Neither antidromic nor orthodromic stimulation could elicit identifiable inhibitory postsynaptic potentials in CA1 neurons in slices from rats 5 or 6 days of age. In contrast, at this age these stimuli result in large inhibitory postsynaptic potentials in CA3 pyramidal cells. In the latter cells orthodromic stimulation produced a brief monosynaptic excitatory postsynaptic potential which was followed by a large prolonged biphasic hyperpolarization. These signals were strikingly similar to those recorded in 1-month-old rats. In addition, large recurrent inhibitory postsynaptic potentials were produced by antidromic stimulation. By postnatal day 9 similar inhibitory postsynaptic potentials could be elicited in a majority of neurons of the CA1 subfield. As in mature pyramidal cells, application of GABA antagonists, such as bicuculline, selectively eliminated the antidromic inhibitory postsynaptic potential and the first component of the biphasic inhibitory postsynaptic potential generated by stimulation of stratum radiatum. In the CA3 subfield, this blockade of GABA receptors resulted in prolonged afterdischarges in slices from immature but not month-old rats. Measurements of the equilibrium potential and the conductance of antidromic inhibitory postsynaptic potentials in CA3 neurons were very similar when made during the first postnatal week and at 1 month of age. While on days 10-11 the equilibrium potential was very similar to measurements made at these other ages, the conductance was 3-4 times greater. The activity of glutamate decarboxylase, the synthetic enzyme for GABA, was very low at 3 days in hippocampus, and increased until 30 days of age at which time adult values were obtained. By comparison, hippocampal GABA levels were high early in postnatal life. Glutamate decarboxylase activities in microdissected CA3 and CA1 subfields were similar in immature hippocampus. These results demonstrate dramatic differences in the ontogenesis of functional GABAergic inhibitory synaptic transmission in the CA1 and CA3 subfields of rat hippocampus. The late development of GABA-mediated synaptic inhibition in the CA1 subfield could play a role in the susceptibility of immature hippocampus to seizures. However, the large GABA-mediated inhibitory postsynaptic potentials present in the CA3 subfield at the same age have a critical role in dampening neuronal excitability.(ABSTRACT TRUNCATED AT 400 WORDS)

Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy.

Studies were undertaken to examine the effects recurrent early-life seizures have on the ability of rats to acquire spatial memories in adulthood. A minute quantity of tetanus toxin was injected unilaterally into the hippocampus on postnatal day 10. Within 48 h, rats developed recurrent seizures that persisted for 1 week. Between postnatal days 57 and 61, rats were trained in a Morris water maze. Toxin-injected rats were markedly deficient in learning this task. While these rats showed gradual improvement in escape latencies over 20 trials, their performance always lagged behind that of controls. Poor performance could not be explained by motor impairments or motivational difficulties since swimming speed was similar for the groups. Only eight of 16 toxin-injected animals showed focal interictal spikes in the hippocampus during electroencephalographic recordings. This suggests that learning deficiencies and chronic epilepsy may be independent products of recurrent early-life seizures. A quantitative analysis of hippocampus revealed a significant decrease in neuronal density in stratum pyramidale of experimental rats. However, the differences were largely explained by a concomitant increase in the area of stratum pyramidale. Studies of glial fibrillary acidic protein expression and spread of horseradish peroxidase-conjugated tetanus toxin in the hippocampus suggest that the dispersion of cell bodies in stratum pyramidale can neither be explained by a reactive gliosis nor the direct action of the toxin itself. Taken together, we suggest that recurrent seizures beginning in early life can lead to a significant deficiency in spatial learning without ongoing hippocampal synchronized network discharging or a substantial loss of hippocampal pyramidal cells.

Anatomical properties of fast spiking cells that initiate synchronized population discharges in immature hippocampus.

Minislices of the CA3 hippocampal subfield were prepared from 10- to 15-day-old rats and exposed to penicillin, a GABAA receptor antagonist. Synchronized population discharges occurred spontaneously but could also be entrained by action potentials in single, fast spiking cells. This was unexpected, since fast spiking cells in the hippocampus are normally thought to be inhibitory interneurons. Experiments were thus undertaken to determine the anatomical identity of these cells. Biocytin injections showed that these cells had the anatomical feature of inhibitory interneurons. Two classes of cells were identified: basket cells (including cells with pyramidal or multipolar dendritic arbors) and bistratified cells. Basket cells had characteristic dense axonal arbors in the stratum pyramidale. They also possessed wide ranging axons in strata radiatum and oriens. The axons of bistratified cells avoided the cell body layer and produced a web-like plexus of axons in strata radiatum and oriens. In the majority of minislices, dye coupling was also observed. Interneurons were preferentially dye-coupled to other interneurons. We speculate that, in early life, hippocampal interneurons may have dualistic synaptic properties. Normally, they inhibit nearby pyramidal cells; however, when GABAA receptors are suppressed a secondary excitatory property of these cells is uncovered.

A chronic focal epilepsy with mossy fiber sprouting follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infant rats.

Studies were conducted to characterize a chronic epileptic condition that follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infancy. Wistar rat pups received a single injection of tetanus toxin in the right CA3 region on postnatal day 10. Animals were monitored for epileptiform activity by video electroencephalographic or visual observation during the following three to five days. Repeat evaluation six months later demonstrated interictal discharges in 79% (11 of 14) and electrographic seizures in 42% (six of 14) of adult rats with tetanus toxin-induced seizures in infancy. Five of the animals had interictal activity which occurred focally in either the left (n = 2) or right (n = 3) hippocampus. One animal had focal interictal activity independently in these regions and in the left and right cortical regions. The remaining five animals had interictal activity in the hippocampus and synchronously in the ipsilateral cortex or the contralateral hippocampus. Electrographic seizures were focal (nine of 14) or bilateral (five of 14) in onset. The behaviors that accompanied these seizures were quite variable. Clonic face and forelimb movements were observed in some animals. However, a significant portion of rats had electrographic seizures with no associated behavioral change. Timm staining was performed on hippocampal sections from experimental and control animals. There was a significantly greater Timm score (aberrant Timm granules) in the inner molecular layer of the dentate gyrus in tetanus toxin-treated rats than in control rats. Our findings suggest that intrahippocampal tetanus toxin injection in infant rats results in a chronic focal epilepsy that persists for at least six months and is associated with aberrant mossy fiber sprouting in the dentate gyrus. The model described here contributes significantly to the evidence for chronic effects of recurrent seizures in early life, and provides a model for investigation of the molecular and cellular events that contribute to the development of chronic epilepsy.

Fluorescence in situ hybridization method for co-localization of mRNA and GEP.

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.

Age-dependent alterations in the operations of hippocampal neural networks.

Results from numerous studies suggest that the functioning of rat hippocampal neural networks during the second postnatal week of life differs distinctly from that in the mature brain. During this critical period, network behavior might be considered hyperexcitable. Spontaneous network-driven bursts of synaptic potentials, which have not been reported in mature hippocampus, are commonly observed. While these events could be attributable to a late onset of GABAergic synaptic transmission, results suggest that this is not the case. In the immature hippocampus orthodromic stimulation leads to prolonged depolarizations and often repetitive synchronized discharging of the entire CA3 population. These events are in many ways reproduced by application of drugs that suppress GABAergic synaptic transmission. The synchronized discharging of the CA3 population is blocked by excitatory amino acid antagonists. This finding, coupled with our growing understanding of the role that recurrent excitation plays in CA3 network functioning, has led to the hypothesis that the differences in network behavior early in life may be largely attributable to an overabundance of local-circuit recurrent excitatory synapses. With maturation, axon collaterals and attendant synapses would regress to achieve an adult complement. Results from dual intracellular recordings as well as anatomical studies of individual CA3 pyramidal cells support this hypothesis. Unique properties of the NMDA receptor at these recurrent excitatory synapses early in life may also promote network excitability. The participation of extracellular Ca2+ in the voltage dependency of the NMDA receptor-linked iontophore could also contribute to synapse consolidation during maturation and thus in the establishment of network connectivity.

Localizing sites of intradendritic electrophysiological recordings by confocal light microscopy.

Studies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recordings were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio-Rad 600 confocal attachment on an Olympus BH-2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images. Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system.

Platelet-activating factor produces neuronal growth cone collapse.

It is generally believed that neuronal growth cone collapsing factors are proteins that interact with membrane-bound receptors. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) - a phospholipid autocoid, also interacts with a membrane-bound neuronal receptor which is similar in nature to collapsing factor receptors. We report that PAF and the nonhydrolyzable PAF agonist, methyl carbamyl PAF (1-O-hexadecyl-2N-methylcarbamyl-sn-glycero-3-phosphocholine, mc-PAF), evoke a dose-dependent neuronal growth cone collapse. This collapse is specifically attenuated by the PAF receptor antagonist BN-52021. These data point to a PAF receptor-mediated growth cone collapse. Therefore, PAF must be added to the list of collapsing factors which potentially guide axons to their proper targets in the developing nervous system.