Lewis B. Haberly - US grants

In recent years, basic level physiological and pharmacological studies have revealed a previously unsuspected diversity in inhibitory processes in cerebral cortex. Recent anatomical and immunocytochemical studies have revealed an equally diverse assortment of neurons that are good candidates for mediating these inhibitory events, but thus far there has been little progress in matching of the morphological substrate with physiological processes. Furthermore, there has been little analysis of the functional role of inhibitory processes either at the level of integrative processes in single neurons or at the level of information processing by systems of neurons. With these deficiencies in mind, the proposed experiments will examine inhibitory processes in the piriform cortex, a phylogenetically old part of the cerebral cortex that has a convenient segragation of different types of probable inhibitory interneurons at different depths and locations - a feature that will facilitate analysis of their physiological properties. In addition, there is a precise complementary lamination of fiber systems in piriform cortex - a feature that will facilitate study of the role of inhibition in integrative processes. Experimental approaches will include an analysis of inhibitory events and their interactions with excitatory events in pyramidal cells (principal cell type in cerebral cortex) by intracellular recording of potentials in brain slices maintained in vitro and by computation of the spatial and temporal distribution of the membrane currents that generate these potentials with the current source density technique in an in vivo preparation. The three populations of interneurons that have been postulated to mediate the different types of inhibitory processes observed in pyramidal cells will be studied with a combined physiological-morphological-immunocytochemical approach in the in vitro slice preparation. Direct analysis of synaptic effects mediated by these neurons on pyramidal cells will be carried out in cell pair intracellular stimulation-recording experiments.

The goal is to contribute to the understanding of neuronal interactions that underlie seizure activity in the cerebral cortex. Piriform cortex will be used for the proposed experiments because it is highly susceptible to epileptogenesis, may play an important role in temporal lobe epilepsy, and has structural, physiological, and pharmacological features that qualify it as a model system for study of cerebral cortex. The proposed experiments will focus on long-lasting "induction" processes that are believed to be involved in the development and progression of some forms of epilepsy, and on neuronal mechanisms that underlie spread of seizure activity from epileptic foci. Studies of long-lasting induction processes will be carried out by physiological analysis of the chances that underlie the development of epileptiform EPSPs in slices of piriform cortex subjected to bursting activity, and in slices from rats in which epilepsy has been "kindled" by repeated shock stimulation. Previous studies have localized the neurons in which the induced changes take place; in the proposed studies hypotheses concerning the identity of these changes will be tested using intracellular recording techniques. Studies of the mechanism of spread of epileptiform activity will be carried out on an anaesthetized rat preparation in which interictal- and ictal-like epileptiform activity spreads throughout the piriform cortex and adjacent cortical areas when a pharmacologically disinhibited focus is repetitively activated by 1 or 2 Hz shock stimulation. The hypothesis will be tested that repetitive bursting activity conducted from the focus by association axons induces a transient reduction in inhibitory processes in the surrounding cortex, thereby initiating a slow regenerative spread of epileptiform activity. Changes will be analyzed in each of the 4 inhibitory processes that have been identified. These studies will employ intracellular recording and current source-density analysis techniques in an anaesthetized rat preparation. Current source-density analysis gives a graphic picture of the sequence of neuronal events over depth and time via mathematical analysis of extracellularly recorded field potentials. To assist in interpretation of the physiological results, both local axon collateral systems and projection pathways that are believed to be involved in the induction and spread of seizure activity will be studied morphologically. Techniques to be used include intracellular injection of biocytin in slices maintained in vitro, and extracellular injection of Phaseolus vulgaris leucoagglutinin (PHA-L) in intact animals.

DESCRIPTION (Adapted from the Investigator's Abstract): In the past few years a series of fundamental-level advances have been made in our understanding of the olfactory system at the receptor level, and at the initial stage of central processing in the olfactory bulb. However, it is probably fair to say that none of the fundamental questions have been answered concerning how the olfactory cortex participates in further processing, despite the existence of a rather detailed 'circuit diagram' for piriform cortex (the largest subdivision of olfactory cortex), and the existence of considerable data on responses to odorants in piriform cortex recorded with a variety of techniques in several species. The comparative lack of progress and interest in olfactory cortex is surprising in view of the relative ease with which this highly laminated system can be studied, the strong arguments for its usefulness as a model to study questions of a general nature concerning cortical function, and the fact that operation of the olfactory bulb clearly cannot be understood in isolation from the cortex in view of the return projection it receives from the cortex that is an order of magnitude heavier than it's output projection to the cortex. The basic rationale for the proposed research program is that, although separate analyses of neuronal circuitry and odor responses in piriform cortex have failed to provide insights into mechanisms of its operation, a carefully designed combination of both approaches will provide a powerful analytical tool. The specific aims will test a series of predictions from the hypothesis that piriform cortex contains both spatial and ensemble representations of odor quality in different subdivisions, and that the ensemble representation enables powerful analysis, memory, and associative functions via"parallel-distributed" processes similar to those developed in studies of artificial networks. The experiments will use an integrated approach in which the recording of responses to odorants will be coordinate with anatomical analysis of the connections of single cells and functional groups of cells.

PR_tBEB.. The proposed experiments use an in vivo model for partial epilepsy of temporal lobe origin to test hypotheses regarding the spread of seizure into normally functioning cortex from areas with epileptogenic abnormalities. Studies in vitro have identified many processes that are potentially involved; determining which of these are actually involved and how they contribute will require studies in intact animals that build on findings from in vitro analysis. The experiments will be performed on rat piriform cortex (PC) where partial seizures that secondarily generalize can be readily recruited in behaving animals by activity emanating from a small disinhibited focus. This same process can be duplicated under urethane anesthesia, enabling analytical study in vivo. Electrographic seizures are recruited when normal PC and adjoining limbic cortex are subjected to several seconds of low rate (3-4 Hz) rhythmic excitatory volleys generated by interictal-like discharges in a distant disinhibited focus. A detailed working hypothesis has been developed for the cascade of processes that underlies this transformation. Features of the hypothesis include: (1) K* plays a causal role for seizure spread into normal cortex (despite evidence to the contrary for seizure initiation). (2) Mechanisms for recruitment of seizure in hippocampus differ from those in most other limbic areas including PC. These stem from the lack of an inwardly rectifying cr channel (CIC-2) that regulates somatic-region CI- in hippocampal pyramidal cells but not in PC and many other limbic areas, and glial-like inwardly-rectifying K[unreadable] channels that are weak or absent in hippocampal neurons but present in PC and other limbic areas. (3) Based on our findings from current source-density (CSD) analysis and transmembrane potential recordings in vivo, we propose that ephaptic-field transmission plays a central role in the generation of ictal activity by allowing high-rate, self-sustained discharges to occur after synaptic transmission is attenuated by depletion of docked synaptic vesicles and other factors. A key method is CSD analysis with a 22-site silicon probe that allows rapidly-evolving dendritic-region as well as somatic-region membrane currents to be visualized. Propagation of ephaptic-field driven discharges will be studied using new approaches for 2- and 3- dimensional CSD analysis. These experiments will provide essential information about the spread of epileptic activity into normal cortex, and clues concerning how this spread can be curtailed.