A major part of our effort is to understand the ionic mechanisms that regulate the activity of local circuit GABAergic interneurons and how these mechanisms impact hippocampal function using patch clamp, immunohistochemical and biochemical techniques from wild type and transgenic animals. Our work over the past year has focused on differential mechanisms of synaptic transmission within the hippocampal formation and the role of novel voltage gated potassium channels expressed in inhibitory neurons within the developing hippocampus 1. DIFFERENTIAL MECHANISMS OF TRANSMISSION AT THREE TYPES OF MOSSY FIBER, AMPA RECEPTOR CONTAINING SYNAPSES.The mossy fiber axons of dentate gyrus granule cells provide a common afferent pathway to both inhibitory neurons and principal cells of the CA3 hippocampus. These axons however do not behave as a single compartment but are specialized depending on the nature of the target neuron being innervated. Mossy fiber, glutamatergic synapses on single st. lucidum interneurons comprise two main subtypes; either containing calcium-permeable or calcium-impermeable AMPA receptors. In contrast mossy fiber synapses onto CA3 pyramidal cells occur exclusively at calcium impermeable AMPA receptor synapses. We have studied the differential mechanisms of both short and long term plasticity that exist at mossy fiber synapses onto interneurons versus principal cells. Transmission at principal cell synapses showed marked frequency dependent facilitation and NMDA-independent forms of LTP. The fidelity of transmission was unaltered at these synapses following induction of long term plasticity. In contrast mossy fiber synapses onto str. lucidum were either stable i.e. showing no long term plasticity or demonstrated a novel form of long term depression (iLTD) only at calcium permeable AMPA receptor containing synapses. Induction of iLTD altered the fidelity of transmission onto these synapses consistent with a mechanism involving changes in release probability. These experiments demonstrate that the properties of mossy fiber synaptic transmission are target specific. Furthermore protocols that result in potentiation of transmission onto principal neurons results in depression of transmission onto specific inhibitory neuron synapses. How this alteration in excitatory:inhibitory balance within the CA3 network is currently under study.2. PKA PHOSPHORYLATION OF KV3.2 POTASSIUM CHANNELS SETS THE UPPER LIMIT OF FIRING FREQUENCY IN DENTATE GYRUS INTERNEURONS. The voltage-dependent potassium channel subunit Kv3.2 activates rapidly at high thresholds, shows little time-dependent inactivation and is negatively modulated by PKA phosphorylation. This biophysical profile suggests that channels containing Kv3.2 may play a role in action potential repolarization. To determine the role played by Kv3.2 within neurons of the mammalian hippocampus we combined, immunohistochemical, biochemical and electrophysiological techniques from both wildtype and animals containing a deletion of the Kv3.2 gene. Within the mammalian hippocampus Kv3.2 is expressed in all parvalbumin- and approximately 30% of somatostatin-containing hippocampal local circuit inhibitory interneurons, but is absent from calbindin, and calretinin- containing interneurons as well as pyramidal neurons of all subfields. SDS-PAGE and Western blotting techniques were used to determine Kv3.2 expression in wild type murine brains ranging in age from 0 to 51 postnatal days. Using crude membrane homogenates, the Kv3.2 protein was not detected until P7. At P7, a single band with a Mr of ~100 kDa increased in staining intensity until P21, at which point it reached a plateau and was maintained until P51. Currents through Kv3.2 containing channels determine the upper limit of fast spiking in dentate gyrus interneurons. Activators of PKA reduced sustained outward potassium currents through Kv3.2 containing channels and modulated the maximum firing frequency in WT interneurons but not in interneurons from Kv3.2 knockout animals. In the absence of glutamatergic synaptic transmission spontaneous high frequency oscillations were observed in the CA3 pyramidal cell layer that required intact GABA-A receptor function, confirming a role for inhibitory interneurons in high frequency oscillations. Oscillations were negatively modulated by PKA activation in WT but not KO hippocampus. These datademonstrate thatKv 32 sets the upper limit for firing frequency in parvalbumin-containing interneurons, which in turn paces high frequency oscillations within the CA3 hippocampus.3. NEURONAL BASIS FOR HIPPOCAMPAL EPILEPTOGENESIS IN A MURINE MODEL OF HUMAN LISSENCEPHALY.A number of genetic mutations that disrupt the normal development of the human cerebral cortex have been described. Classical or Type I lissencephaly, defines a subgroup of human neuronal migration disorders characterized by generalized agyria/pachygyria, four abnormal cortical layers, enlarged ventricles, generalized neuronal heterotopias and often defects in the corpus callosum. In the U.S.A. ~1 in 40,000 infants are born annually with Type I lissencephaly, including isolated lissencephaly sequence (ILS) and Miller-Dieker syndrome (MDS). These infants present with severe cognitive and motor impairments and often die from seizures in the first years of life. The defective gene, termed PAFAH1B1 a.k.a. LIS1, was identified from patient samples with informative deletions of 17p13.3 and was subsequently confirmed when dominant point mutations and a hemizygous intragenic deletion of the LIS1 gene were found in ILS patients. The LIS1 gene encodes a brain-specific, 45 kDa noncatalytic subunit of platelet- activating factor acetylhydrolase 1b (PAFAH1b), an enzyme that inactivates PAF. Seizures are universal in humans with Type I lissencephaly. Precisely how a deficiency of LIS1 protein disrupts normal brain development and precipitates seizures is not known although the hippocampus may be a potential focus. We now demonstrate severe neuronal dysplasia and heterotopia throughout the granule cell and pyramidal cell layers of both human lissencephalic hippocampus and mice containing an heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birthdating analysis using bromodeoxyuridine revealed that pyramidal neurons in Lis1+/- murine hippocampus are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice are stunted and possess fewer dendritic branches, while dentate granule cells were hypertrophic and formed spiny basilar dendrites from which the principal axon emerged. Both somatostatin- and parvalbumin-containing inhibitory neurons were heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission are severely disrupted, revealing hyperexcitability at Schaffer- collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission was less than wildtype. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K+]o model of epilepsy. In Lis1+/- hippocampus intense interictal bursting was observed on elevation of extracellular potassium to 6.5mM, a condition which resulted in only minimal bursting in wildtype. These anatomicaland physiological hippocampal defects provide a neuronal basis for seizures associated with lissencephaly.
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