Craig E. Jahr - US grants

sensory feedback; neurotransmitters; neural transmission; afferent nerve; neural information processing; chemoreceptors; neuropharmacology; spinal nerves; ligands; membrane permeability; aspartate; glutamates; synapses; dorsal column; adenosine triphosphate; embryo /fetus; tissue /cell culture; immunochemistry; histochemistry /cytochemistry; electrophysiology;

Patch clamp recording will be used to study the properties of single channel and whole cell events activated by glutamate (the probable neurotransmitter mediating fast synaptic excitation) and to compare these to the properties of fast synaptic excitation in cultures of hippocampal and spinal cord neurons. Conditions have been found in which a phenomenon similar to long term potentiation can be induced between two neurons in dissociated cultures of hippocampus. this presents the first opportunity where the molecular mechanisms which produce long term potentiation can be examined in both pre- and postsynaptic neurons simultaneously using dual whole cell recordings. In addition, a new but simple technique of patch pipette perfusion will permit intracellular injection of compounds and enzyme systems thought to modulate or mediate synaptic plasticity. Material can be independently injected into either neuron of synaptically coupled pairs after a period of recording in control conditions thus limiting the action of drugs and enzymes to either pre- or postsynaptic elements. This will permit detailed examination of the loci of actions of drugs which previously could only be applied globally. Three protein kinase systems will be investigated for effects on synaptic efficacy and plasticity. Calcium/calmodulin type II protein kinase, protein kinase C and protein kinase A have all been implicated as factors which control or modulate synaptic plasticity. Intracellular injections of both pre- and postsynaptic neurons with the activated kinases and co-factors as well as their activators and inhibitors will be used to identify the loci of action of the kinases. The effects of these systems on single channel events will also be studied using cell-attached and excised patches in order to determine their mechanisms of action at the molecular level. The information sought by the proposed research will increase our understanding of the molecular mechanisms of chemically mediated synaptic transmission and synaptic plasticity. Since long term potentiation is one of the best cellular models for long term information storage in the central nervous system, determining its basic mechanism could aid our understanding of learning and memory and therefore deficits in these functions in pathological states.

The objective of the proposed work is the understanding of the mechanisms that control the kinetics and plasticity of glutamate mediated excitatory synaptic neurotransmission in the mammalian CNS. The glutamate receptors directly gating ion channels and those coupled to G-proteins will be studied using whole cell and single channel patch clamp techniques in primary cultures of hippocampal neurons. Glutamate agonists and antagonists will be applied externally to cells by fast perfusion techniques. Controlled internal dialysis involving patch pipette perfusion and dual whole cell recording will be utilized to pharmacologically manipulate intracellular second messenger systems. The fast non-NMDA and slow NMDA receptor components of the excitatory postsynaptic current will be examined and compared to responses induced by glutamate analogues to determine if channel kinetics, receptor desensitization, transmitter diffusion and uptake or some combination of these factors can account for the differences in their time courses. The cellular mechanisms that regulate the efficacy of excitatory synaptic transmission will be studied by investigating the influence of activation of the G-protein coupled glutamate receptor and other second messenger systems on both currents induced synaptically and by exogenous glutamate ligands. Synaptic transmission through non-NMDA receptor gated ion channels is likely to represent the majority of fast cell to cell communication in the CNS, and both the NMDA component and the G-protein coupled receptor are involved in intracellular signalling and plasticity. The glutamate receptor system is capable of rapid up-and down-regulation and has been implicated in both developmental and adult plasticity. Over activation can result in epileptiform behavior and excitotoxicity. A detailed knowledge of the way in which the activation of kinetics of glutamate receptor subtypes and their biochemical regulation govern the behavior of glutamate synapses is therefore fundamental to the understanding of the CNS in both normal and abnormal states.

Excitatory synaptic transmission in many pathways of the vertebrate CNS is due to presynaptic release of an acidic amino acid, probably L-glutamate, that binds to postsynaptic receptors directly linked to ion channels. In addition to these tightly coupled receptor/channels, other classes of glutamate receptors are apparently coupled to their effectors by indirect mechanisms. L-glutamate and quisqualate stimulate IP3 turnover in both Xenopus oocytes injected with brain mRNA and in hippocampal neurons. At presynaptic receptors activated specifically by L-2-amino-4- phosphonobutyrate (L-APB), L-glutamate inhibits excitatory transmission, probably by decreasing voltage-dependent calcium currents (see Preliminary Results). Finally, in the retina, ON bipolar cells are hyperpolarized by L-glutamate released by photoreceptors by acting at a receptor specifically activated by L-APB. Dialysis of ON bipolar cells results in the loss of this response (see Results). The proposed research will attempt to determine the mechanisms by which the effects of L-glutamate at APB receptors are produced in selected pathways in the brain and retina. Electrophysiological techniques which measure synaptic activity in populations of neurons and at single synapses as well as molecular processes of single ion channels will be used. The actions of L-APB on synaptic transmission will be characterized in neuronal cultures derived from hippocampus and entorhinal cortex and in the hippocampal slice. The molecular pathways altered by this receptor will be determined by directly monitoring its effects on voltage-dependent currents and by attempting to mimic its effects with exogenous compounds that alter second messenger systems and the functioning of GTP binding proteins. The only neuron in the CNS that has clearly been shown to have a direct electrical response to L-APB is the ON bipolar cell of the neural retina. This neuron is hyperpolarized by both L-glutamate and L-APB, the end effect of which is a decrease in transmitter release. When these neurons are dialyzed by whole cell patch pipets, the response to the agonists "wash out" suggesting that the response is mediated by indirect coupling of the receptor to the conductance. The conductance changes induced by glutamate and L-APB will be studied in slices of retina using whole cell and single channel recording to determine the intracellular mechanisms which couple this receptor to the hyperpolarization.

DESCRIPTION: (adapted from Applicant's Abstract) The purpose of this proposal is to study the kinetic parameters that determine the probability of opening (Po) of NMDA channels at synaptic and non-synaptic sites in conditions that favor phosphorylation or dephosphorylation to determine the rage over which modulation is possible. Because the Po is generally found to be the channel property affected by modulatory events (changes in single channel conductance and channel number are only rarely reported), the means of altering Po and the range over which Po can change are vital pieces of information in understanding the consequences of activating modulatory mechanisms. Estimates in the literature of NMDA channel Po range from 0.003 to 0.3; that is, if an NMDA channel were to remain in a state from which it could open (bound by glutamate and glycine) for 1 second, it would spend a total of either 3 milliseconds or 300 milliseconds in the open state, respectively. Recent reports suggest that the Po is increased about 10- fold by excising an outside-out patch and that both non-synaptic and synaptic receptors in undisturbed cells have similar Po's. The evidence for altering Po by patch detachment appears good, but the estimates of synaptic receptor Po are not straightforward and, again, estimates vary 10-fold. If the variability is real but due to different experimental conditions, it implies that alterations in synaptic strength as high as 10-fold can be achieved by normal physiological mechanisms. The proposed experiments will assess the Po of NMDA channels in a variety of experimental conditions. NMDA receptor responses will be evoked by synaptic stimulation and rapid agonist applications in hippocampal neurons in culture and in slice preparations. Responses will be recorded with whole cell, cell-attached and detached patch clamp techniques.

DESCRIPTION (provided by applicant): A basic tenet in neuroscience is that the synapse is the functional unit in the brain, and therefore in mentation. Each synapse acts relatively independently of its neighbors in that it primarily responds to its own released transmitter and either not at all or much less so to that of its neighbors. Exocytosis of synaptic vesicles is generally assumed to occur only at ultrastructurally defined presynaptic active zones. If this is true, receptors not located within the synaptic cleft must be activated by transmitter that diffuses out of the cleft, or not be activated at all. However, AMPA receptor-mediated quantal events resulting from climbing fiber release are recorded in Bergmann glial cells in the cerebellar cortex. These quantal events are not coincident with quanta recorded in neighboring Purkinje cells which receive input from the same climbing fiber and therefore it appears that exocytosis can occur from ectopic climbing fiber release sites located directly across from Bergmann glial membranes. If ectopic release is a general phenomenon, heterosynaptic and neural-glial interactions are more likely to result from this form of cross-talk rather than from the low concentrations of transmitter achieved by spillover from release within synaptic clefts. Using electrophysiological, optical, and pharmacological approaches, we will determine the characteristics of ectopic release including its calcium dependency, which types of voltage dependent calcium channels provide the necessary calcium influx, release probability, and the sensitivity of ectopic release to presynaptic receptor activation, all in comparison to release at conventional active zones. In addition, we will determine the advantages of high concentration transients of transmitter in the extrasynaptic space including differential calcium permeability of AMPA receptors and effects on motility of Bergmann glial processes.

DESCRIPTION (provided by applicant): Cell-to-cell transmission of information in the CNS is thought to occur at individual synapses, isolated from their neighbors. However, phenomena such as transmitter spillover and pooling between synapses have suggested that point-to-point synaptic communication between neurons is not the only method of information transfer in the CNS. Whether spillover and pooling of transmitter occurs depends on several variables including the amount of transmitter released per synapse per presynaptic action potential. If clearance mechanisms are overwhelm by release, transmitter will spread away from synapses. At some synapses, manipulations that increase the probability of vesicular release result in the release of multiple vesicles from individual synapses following single presynaptic action potentials. This large bolus of transmitter can overwhelm local transporters and result in spillover. At other synapses, however, multivesicular release has not been found regardless of the release probability suggesting that there is a mechanism that prevents release of more than a single vesicle. Transmitter spillover and pooling are at least in part responsible for activation of extrasynaptic receptors including metabotropic receptors which in turn are required for several types of short and long term plasticity. The main objectives of this proposal are to determine the extent to which plasticity depends on transmitter spillover and the degree to which spillover depends on multivesicular release. This will be accomplished using a combination of electrophysiological and optical techniques in the study of three different synapses each with distinct characteristics. The plan is to use the attributes of each of these synapses to probe, first, the generality of multivesicular release and, second, the multiplicity of its consequences. All CNS functions including sensory processing, motor behavior, consciousness, and memory depend on intact synaptic transmission. Knowledge of the fundamental molecular and cellular properties of transmission is required for the development of rational interventions in pathological states.

[unreadable] DESCRIPTION (provided by applicant): Astrocytes throughout the CNS provide an array of services for their neighboring neurons including removing transmitters following release and providing metabolic sustenance. The realization that astrocytes are not merely caretakers but are also intimately involved in the main purpose of the CNS, processing information, is recent. Although the responses of astrocytes cannot match the speed of neurons, they can alter the interactions between neurons by isolating or opening pathways of communication between adjacent synapses. Additionally, astrocytes communicate between themselves and, bi-directionally, with neurons by responding to alterations in ionic gradients, released neurotransmitters, and by releasing neuroactive substances. The aim of the present research is to determine the ability of, and mechanisms by which, astrocytes alter the isolation of synapses from neighboring synapses by the extension and retraction of astrocytic lamellipodial processes. These objectives will be addressed using a combination of 2 photon microscopy to directly observe process elaboration, electrophysiology to measure synaptic strength, pharmacological manipulation of receptor activity, 2 photon photolysis of caged glutamate to rapidly activate receptors in dendritic spine-sized volumes, flash photolysis of intracellular caged calcium, and expression of normally absence glutamate- gated ion channels to alter calcium influx. These studies will uncover strategies to control the necessary but potentially damaging effects of releasing glutamate into the extracellular space. Disruption of glutamate clearance followed by excitotoxic damage has been implicated in a variety of neurological pathologies. In addition, in schizophrenia and major depression, glial cell number is subnormal suggesting diminished trophic support of neurons that likely contributes to the reduced neuronal size, density of dendritic spines, and levels of synaptic proteins, as well as the abnormalities observed in fMRI in these conditions. Understanding astrocyte plasticity and its role in synaptic function promises unique approaches to managing the abnormalities in transmission in mental disease. [unreadable] [unreadable] [unreadable]

NMDA receptors (NMDARs) are one of two major types of ion channels located in the postsynaptic membrane at the vast majority of excitatory synapses in the CNS. They not only mediate excitation but, because of their permeability to calcium, raise intracellular calcium concentrations and thus trigger a variety of intracellular pathways leading to short and long term changes in synaptic strength. They also contribute to neuronal death in pathological conditions. Recent studies suggest that NMDARs are located not only at postsynaptic sites but also at presynaptic release sites and can modify the likelihood of vesicular release. As these receptors are important in both physiological and pathological conditions, determining where they are located will help define their effects. We will test whether NMDARs are expressed presynaptically with a combination of calcium imaging using two photon microscopy and electrophysiology to measure pre- and postsynaptic responses to global and local applications of NMDAR agonists and antagonists. We have chosen two types of neurons for this study: cerebellar stellate cells and neocortical layer 5 pyramidal cells. It is clear that NMDAR activation alters transmitter release from these two cell types but it is unclear if the same mechanisms are involved. Whether these receptors alter release by directly increasing intracellular calcium at release sites or rather by depolarizing release sites is not clear. If the former is true, NMDARs would have to be located very close to sites of release because calcium does not diffuse far intracellularly. If the regulation of release by NMDARs is secondary to their depolarizing influence, however, they need not be located near release sites because depolarizations can spread electrotonically for hundreds of micrometers. These two mechanisms are not mutually exclusive and could be mutually reinforcing. NMDAR-mediated depolarization by itself may have multiple effects on vesicular release by 1) directly activating voltage-sensitive calcium channels (VSCCs) and thereby increasing release by elevating baseline calcium concentrations, 2) inactivating axonal Kv1-type potassium channels leading to a broadening of action potentials, enhanced activation of VSCCs and thus greater release, 3) a calcium-independent, though uncharacterized, regulation of the release process and 4) inactivation of voltage-dependent sodium channels and/or VSCCs and therefore a blockade of action potential propagation to release zones and decreased calcium influx leading to decreased neurotransmitter release. We will determine whether NMDARs are located at presynaptic release zones and whether somatic depolarizations alter release by one or a combination of these mechanisms.

Extracellular levels of glutamate are controlled with great precision both temporally and spatially, achieving efficient and selective synaptic excitation and preventing neuronal death by excitotoxicity. On one hand, glutamate concentrations in the synaptic cleft must rise rapidly to millimolar concentrations to ensure activation of postsynaptic ionotropic receptors. On the other hand, the average extracellular concentration of glutamate must be maintained at sub-micromolar levels to prevent cell death. These requirements are met by the explosive exocytotic release of glutamate and the high capacity and high affinity glutamate uptake system provided by the family of Na-dependent glutamate transporters. When transporter function is compromised experimentally or by metabolic crises such as ischemia, elevated tonic levels of glutamate and slowed clearance around synaptic release sites can result in seizures, enhanced spreading damage from ischemic insults and neuronal and organismal death. The objective of this proposal is to determine how much glutamate escapes from the synaptic cleft following release, how far from the release site glutamate reaches concentrations sufficient to activate receptors, how rapidly the uptake system sequesters glutamate, and how these processes are affected by physiological alterations in the amount of glutamate released including multivesicular release. We will use whole cell and outside-out patch clamp recordings in conjunction with two photon laser scanning microscopy and glutamate uncaging in acute slices of rat cerebellum and hippocampus to address the consequences of ionotropic and metabotropic glutamate receptor activation inside and outside of the synaptic cleft. Three dissimilar synapses will be studied to compare how their unique morphologies and expression patterns of receptors and transports affect the actions of glutamate following synaptic release.

DESCRIPTION (provided by applicant): The purpose of this postdoctoral program is to train Ph.D.'s and M.D.'s for careers in neuroscience research. The program is highly focused in neuronal signaling, encompassing many of the most exciting areas of modern neuroscience, which rely on techniques of cellular and molecular neuroscience. Studies on neuronal signaling pathways have lead to new insights in understanding neurological diseases such as Huntington's, Alzheimer's, Parkinson's, epilepsy and stroke. A common theme in these diseases is often defects in signaling pathways. The focus of this program was chosen because of its impact in normal physiology and pathological diseases and the expertise of the training faculty. The sixteen members of the training faculty were selected because of their scientific expertise and their substantial records of successful training of postdoctoral fellows. All of the mentors are either members of the Vollum Institute (VI) or closely affiliated centers at Oregon Health and Science University. The concentration of outstanding faculty in a strongly collaborative environment offers a highly focused and interactive environment for postdoctoral training. In addition to laboratory research training, postdoctoral trainees will participate in numerous other activities designed to enhance their career development. Included among these are a course on The Neurobiology of Disease, the Vollum Seminar series (outside speakers), Vollum noon seminar series (short talks by postdoctoral fellows), scientific writing course, and a series of career development workshops which include such topics as grant writing, setting up a laboratory, job interviewing, etc. Each trainee will have a three member career development committee that will advise the trainee for two years and also work with the trainee when he/she initiates a job search. Trainees will meet bimonthly as a group with mentors and present updates on their research.

DESCRIPTION (provided by applicant): The likelihood of transmitter release from presynaptic release sites is regulated by several factors including the frequency of action potentials and the activity of presynaptic receptors. In addition to these mechanisms, subthreshold depolarizations of the somatodendritic compartment recently have been shown to alter action potential-driven release at distant locations of the axon. This form of regulation of release is analog in nature, i.e., though it alters the probability of release to an action potential, its potential to alter reease requires neither action potentials nor local receptor activation. Rather, these subthreshold depolarizations passive spread through the axon and are reported to affect release by calcium-dependent and calcium-independent mechanisms. Analog signaling adds another dimension to the control of neuronal circuit function which depends not only on the history of action potential frequency but also on the subthreshold changes in membrane potential that preceded a given action potential. Presynaptic receptors can also regulate release probability and alter the axonal membrane potential. The changes in axonal membrane potential can passively propagate antidromically and alter the excitability of the axon initial segment, a function which until recently was thought to be the exclusive domain of synapses on the somatodendritic membrane. The objective of this proposal is to determine the mechanisms underlying orthodromic and antidromic analog signaling and to determine if these mechanisms are used generally in the CNS. Orthodromic signaling will be studied in three dissimilar neurons, cerebellar molecular layer interneurons, dentate gyrus granule cells and cortical layer 5 pyramidal cells. The proposed mechanisms for orthodromic signaling in these three cells types are contradictory but include both calcium-dependent and calcium-independent processes. Antidromic signaling has only been demonstrated in one neuronal type, cerebellar granule cells. Though a number of axonal receptor types may affect initial segment excitability, NMDA receptors are particularly interesting candidates because of their use-dependence. Abundant evidence indicates that cortical layer 4 spiny stellate neurons in both visual and barrel cortex express presynaptic NMDA receptors that alter release properties and are required for the induction of long term depression. We will use two photon laser scanning microscopy, two photon laser uncaging and electrophysiology to determine the mechanisms of orthodromic analog signaling and the extent of presynaptic NMDA receptor expression, the physiological conditions necessary for their activation and their effects on the excitability of the axon initial segments of the layer 4 spiny stellate neurons.

? DESCRIPTION (provided by applicant): NMDA receptors are involved in myriad aspects of CNS function including development, learning and memory but also neurological and psychiatric disorders. Understanding the intricacies of NMDA receptor properties, including permeability and gating, and how different subunit compositions can alter these properties, is critical in developing therapeutic interventions in CNS disorders. To be activated, NMDA receptors require the binding of two glycine and two glutamate molecules (or their equivalents). Several studies indicate that receptors bound by a single glutamate and two glycines do not open, though being singly-bound may facilitate subsequent opening upon a second exposure to glutamate. There are several ways in which NMDA receptors can become singly-bound by glutamate, including exposure to the low levels of ambient glutamate, spillover of glutamate from neighboring synapses and as synaptic receptors sequentially unbind their two glutamates following vesicular release. We propose to study the effects of NMDA receptors bound by two glycines and one glutamate on subsequent activation by a second glutamate binding event. As traditional methods of applying glutamate result in both singly and doubly-bound receptors, the effects of singly-bound receptors are difficult to study directly. Using two recently developed techniques that 1) force expression of NMDA receptors composed of two GluN1 subunits and two different GluN2 subunits and 2) covalently binding only one of the GluN2 subunits to a photoswitchable glutamate ligand, most, if not all, receptors can be forced into the singly-bound state and thus allow the study of a pure population of receptors. Using fast applications of glutamate to outside-out patches (saturating glycine), we will test whether singly-bound NMDA receptors can desensitize, whether the opening of a population of singly-bound receptors is facilitated following succeeding pulses of glutamate and whether the two glutamate binding sites exhibit cooperative binding. In addition, the glutamate unbinding rate from singly-bound receptors will be determined. This is an important parameter as its value determines the period during which a second pulse of glutamate can interact with singly-bound receptors. Together, determination of these properties of NMDA receptors will lead to better predictions about the timing and importance of activation of extrasynaptic receptors and synaptic receptors at neighboring synapses by spillover, as well as how synaptic NMDA receptors just across the synaptic cleft from active release sites respond to repetitive release events.

Long term synaptic plasticity is an experience-dependent increase or decrease in the strength of synaptic transmission that can last for at least an hour, if not days or longer. The mechanisms underlying long term potentiation and depression of synaptic strength depend on the particular region of the CNS and the type of synapse within the region. Both potentiation and depression are thought to provide information storage in the CNS, i.e., underlie learning and memory. At many types of synapses, the glutamate-gated ion channel NMDA receptor is involved in the induction process, if not the expression mechanism. While the location of these NMDA receptors at the best studied model synapse, that between Schaffer collaterals and CA1 pyramidal neurons in the hippocampus, is almost certainly the postsynaptic membrane, there are other synapses in which it appears that the important NMDA receptors are in the presynaptic membrane. This is particular evident for the induction of long term depression in the somatosensory cortex at the synapse between the layer 4 spiny stellate neuron, the neuron that receives the largest direct input from the thalamus, and the layer 2/3 pyramidal neuron. We have found, however, that this may not be the case but rather that the required NMDA receptors are postsynaptic, in the layer 2/3 dendrites. The objective of this proposal is to reassess the mechanism(s) of expression of long term depression, i.e., is the decrease in synaptic strength the result of a decrease in presynaptic release of glutamate or a decrease in the postsynaptic sensitivity to glutamate, for example, a decrease in receptor number. We will use whole cell recording in conjunction with 2 photon laser scanning microscopy and 2 photon glutamate uncaging in acute slices of mouse somatosensory cortex to address this issue.