Karen L. Smith

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.

Model neural prostheses with integrated microfluidics: a potential intervention strategy for controlling reactive cell and tissue responses

Model silicon intracortical probes with microfluidic channels were fabricated and tested to examine the feasibility of using diffusion-mediated delivery to deliver therapeutic agents into the volume of tissue exhibiting reactive responses to implanted devices. Three-dimensional probe structures with microfluidic channels were fabricated using surface micromachining and deep reactive ion etching (DRIE) techniques. In vitro functional tests of devices were performed using fluorescence microscopy to record the transient release of Texas Red labeled transferrin (TR-transferrin) and dextran (TR-dextran) from the microchannels into 1% w/v agarose gel. In vivo performance was characterized by inserting devices loaded with TR-transferrin into the premotor cortex of adult male rats. Brain sections were imaged using confocal microscopy. Diffusion of TR-transferrin into the extracellular space and uptake by cells up to 400 /spl mu/m from the implantation site was observed in brain slices taken 1 h postinsertion. The reactive tissue volume, as indicated by the presence of phosphorylated mitogen-activated protein kinases (MAPKs), was characterized using immunohistochemistry and confocal microscopy. The reactive tissue volume extended 600, 800, and 400 /spl mu/m radially from the implantation site at 1 h, 24 h, and 6 weeks following insertion, respectively. These results indicate that diffusion-mediated delivery can be part of an effective intervention strategy for the treatment of reactive tissue responses around chronically implanted intracortical probes.

Tetanus toxin-induced seizures in infant rats and their effects on hippocampal excitability in adulthood

A new experimental model of developmental epilepsy is reported. Behavioral and EEG features of seizures produced by unilateral intrahippocampal injection of tetanus toxin in postnatal day 9-11 rats, are described. Within 24-72 h of tetanus toxin injection, rat pups developed frequent and often prolonged seizures which included combinations of repetitive wet dog shakes, and wild running-jumping seizures. Intrahippocampal and cortical surface EEG recordings showed that coincident with these behaviors, electrographic seizures occurred not only in the injected hippocampus, but also in the contralateral hippocampus and bilaterally in the neocortex. Analysis of the interictal EEG revealed multiple independent spike foci. One week following tetanus toxin injection, the number of seizures markedly decreased; however, interictal spiking persisted. After injection rats were allowed to mature some were observed to have unprovoked behavioral seizures and/or epileptiform EEG activity. Mature animals were also studied using in vitro slice techniques. Recordings from hippocampal slices demonstrated spontaneous epileptiform burst discharges in the majority of rats which had tetanus toxin induced seizures as infants. These events occurred in area CA3 and consisted of interictal spikes and intracellularly recorded paroxysmal depolarization shifts (PDSs). On rarer occasions, electrographic seizures were recorded. The use of the tetanus toxin model in developing rats may facilitate a better understanding of the unique features of epileptogenesis in the developing brain and the consequences early-life seizures have on brain maturation and the genesis of epileptic conditions in later life.

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.

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.

Developmental Neuroplasticity and Epilepsy

Results from numerous experimental and clinical studies suggest that the immature nervous system is unusually susceptible to seizures during periods of postnatal life. In rats and mice, this period is postnatal weeks 2 and 3. Similar to that in human neonates, the first week of life in rodents is marked by poorly synchronized neuronal discharging. However, by postnatal week 2, a dramatic transition occurs, and animals demonstrate a marked increase in epileptogenicity reflected in increases in rates of neocortical focal epileptogenesis, amygdala and hippocampal kindling, and susceptibility to convulsant drugs (1). This critical period of enhanced seizure susceptibility has also been demonstrated in in vitro models, which permit more detailed examination of the underlying processes. For instance, when in vitro slices are taken from the hippocampus of 2and 3-week-old rats and exposed to convulsant drugs, such as GABA,-receptor antagonists, they generate prolonged electrographic seizures. However, under identical recording conditions, slices taken from rats <5 days of age do not produce synchronized events, and slices from adult rats produce only brief interictal discharges (2). A late onset of GABA-mediated synaptic inhibition could conceivably play a role in enhanced seizure susceptibility. However, this seems unlikely, given that GABA,-receptor antagonists are such potent convulsants in early life. On the contrary, GABA,-mediated synaptic inhibition appears to be all important in preventing seizures. Electrophysiologic, pharmacologic, neurochemical, and anatomic results all suggest that excitatory amino acid synaptic transmission is enhanced during postnatal weeks 2 and 3 and could contribute to seizure generation (3). This is particularly true in hippocampal area CA3, where recurrent excitatory synapses between mutually excitatory pyramidal cells play an important role in seizure generation. Computer reconstruction of recurrent axon arbors from single CA3 pyramidal cells have shown that few axonal branches are present before the critical period of seizure susceptibility. However, by postnatal days 9 and 10, an exuberant outgrowth has taken place. Half of these axonal branches are pruned by adulthood. Thus, there is good correlation between developmental remodeling of excitatory connectivity and changes in seizure susceptibility (4). In general, remodeling of neuronal connectivity during brain development is dependent on neuronal activity (5). The correlated or synchronous activation of synapses is thought to lead to their selection and maintenance into adulthood. Inactive synapses or synapses activated out of phase with other more effective inputs to cells are thought to be selected against and pruned. Thus, investigators have questioned what the effects of recurring seizures are on the formation of neuronal networks. To address this question, the tetanus toxin model was first developed (6). A unilateral intrahippocampal injection of tetanus toxin on day 10 produces brief but recurrent seizures that decrease in frequency after 1 week. As adults, these rats are chronically epileptic, as demonstrated by video-EEG recordings and recordings from CA3 pyramidal cells in vitro slices (6,7). Results of slice experiments also suggest that hyperexcitable CA3 recurrent excitatory networks play a key role in seizures in these rats (8). To determine the anatomic substrates for the epileptiform discharges, individual CA3 pyramidal cells were labeled intracellularly with biocytin (9). Several outcomes were possible. Most often, speculation has been that early-life seizures “freeze” developing networks into immature patterns of connectivity. For instance, during hippocampal development, when repeated seizures occur, the synchronous discharging of pyramidal cells might prevent axonal remodeling that normally takes place in area CA3. Thus, the excess in axonal branching that is present in early life could be maintained into adulthood. However, other outcomes are possible. The growth of recurrent axon arbors that take place during week 2 could be promoted by seizure discharges. Sprouting of recurrent excitatory collaterals could produce profuse innervation by local networks. A third outcome would follow from results of studies of the neuromuscu-