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, EGFPexpressing 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.

Excitotoxicity, resulting from the excessive release of glutamate, is thought to contribute to a variety of neurological disorders, including epilepsy. Excitotoxic damage to dendrites, i.e., dendrotoxicity, is often characterized by the formation of large dendritic swellings, or "beads." Here, we show that hippocampal interneurons that express the neuropeptide somatostatin are highly vulnerable to the excitotoxic effects of the ionotropic glutamate receptor agonist kainate. Brief, focal iontophoretic application of kainate rapidly induced bead formation in dendrites of somatostatinergic interneurons that express green fluorescent protein (GFP) from mice of the transgenic line GIN (GFP-expressing inhibitory neurons). Surprisingly, beads often did not form at the site of kainate application or even in the dendritic segment to which kainate was applied; instead, dendritic beading occurred more distally, often encompassing all branches distal to the application site. We have termed this phenomena, "distally directed dendrotoxicity." Distally directed beading was induced regardless of the branch order of the site of application and was found to be dependent on activation of voltage-gated sodium channels. Subsequent to induction, distally directed beading would reverse in most cells; in other cells, however, beading irreversibly invaded proximal dendritic segments and gradually encompassed the entire dendritic tree. These results demonstrate that distal dendritic segments are highly vulnerable to excitotoxic injury and imply that excessive excitatory activity originating in one synaptic pathway can impact synapses at more distal dendritic segments of the same neuron. The discovery of this phenomenon will likely be important in understanding interneuronal dysfunction following excitotoxic injury.

The effects recurring seizures have on the developing brain are an important area of debate because many forms of human epilepsy arise in early life when the central nervous system is undergoing dramatic developmental changes. To examine effects on glutamatergic synaptogenesis, epileptiform activity was induced by chronic treatment with GABAa receptor antagonists in slice cultures made from infant rat hippocampus. Experiments in control cultures showed that molecular markers for glutamatergic and GABAergic synapses recapitulated developmental milestones reported previously in vivo. Following a 1-week treatment with bicuculline, the intensity of epileptiform activity that could be induced in cultures was greatly diminished, suggesting induction of an adaptive response. In keeping with this notion, immunoblotting revealed the expression of NMDA and AMPA receptor subunits was dramatically reduced along with the scaffolding proteins, PSD95 and Homer. These effects could not be attributed to neuronal cell death, were reversible, and were not observed in slices taken from older animals. Co-treating slices with APV or TTX abolished the effects of bicuculline suggesting that effects were dependent on NMDA receptors and neuronal activity. Neurophysiological recordings supported the biochemical findings and demonstrated decreases in both the amplitude and frequency of NMDA and AMPA receptor-mediated miniature EPSCs (mEPSCs). Taken together these results suggest that neuronal network hyperexcitability interferes with the normal maturation of glutamatergic synapses, which could have implications for cognitive deficits commonly associated with the severe epilepsies of early childhood.

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.

Neuronal activity is thought to play an important role in refining patterns of synaptic connectivity during development and in the molecular maturation of synapses. In experiments reported here, a 2-week infusion of tetrodotoxin (TTX) into rat hippocampus beginning on postnatal day 12 produced abnormal synchronized network discharges in in vitro slices. Discharges recorded upon TTX washout were called 'minibursts', owing to their small amplitude. They were routinely recorded in area CA3 and abolished by CNQX, an AMPA receptor antagonist. Because recurrent excitatory axon collaterals remodel and glutamate receptor subunit composition changes after postnatal day 12, experiments examined possible TTX-induced alterations in recurrent excitation that could be responsible for network hyperexcitability. In biocytin-labelled pyramidal cells, recurrent axon arbors were neither longer nor more highly branched in the TTX infusion site compared with saline-infused controls. However, varicosity size and density were increased. Whereas most varicosities contained synaptophysin and synaptic vesicles, many were not adjacent to postsynaptic specializations, and thus failed to form anatomically identifiable synapses. An increased pattern of excitatory connectivity does not appear to explain network hyperexcitability. Quantitative immunoblots also indicated that presynaptic markers were unaltered in the TTX infusion site. However, the postsynaptic AMPA and NMDA receptor subunits, GluR1, NR1 and NR2B, were increased. In electrophysiological studies EPSPs recorded in slices from TTX-infused hippocampus had an enhanced sensitivity to the NR2B containing NMDA receptor antagonist, ifenprodil. Thus, increases in subunit protein result in alterations in the composition of synaptic NMDA receptors. Postsynaptic changes are likely to be the major contributors to the hippocampal network hyperexcitability and should enhance both excitatory synaptic efficacy and plasticity.

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 synchronized 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 hippocampal slice cultures to explore the effects that recurring seizure-like activity had on the developing hippocampus. We found that developing networks adapted rapidly to recurring synchronized 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 synchronized network activity. Our results showed that hippocampal pyramidal cells and their networks adapted rapidly to intense synchronized activity and that calcineurin played an important role in the underlying processes.

Objective: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries.We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms. Methods: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1, acts through the IGF-1 receptor to abolish spasms. Results: Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3) IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals. Interpretation: Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder.