Timothy J. Teyler - US grants

The proposed research project is designed to investigate the mechanism and distribution of action of delta-9-tetrahydrocannabinol (THC - a major psychoactive constituent of marihuana) in the rodent brain. THC is a potent excitatory neuromodulator of hippocampal activity in picomolar concentrations and appears to act at membrane-active estrogen receptors in hippocampus. We propose to examine this hypothesis and determine which other brain areas display this neuromodulation. The role of membrane-acting estrogen receptors will be studied using estrogen antagonists to see if they can also block the THC effect in hippocampal slice preparations. This hypothesis will also be tested by castrating male rats and priming them with heterotypic or homotypic gonadal steroids, a treatment that alters the cellular response to applied estrogen. If the membrane-acting estrogen receptor is involved, we expect to see a THC effect only in the group primed with the heterotypic steroid. Brain localization will be determined electrophysiologically following an autoradiography uptake study to identify regions concentrating the labeled THC. This data will also allow us to evaluate the nature of the estrogen receptor hypothesis by comparing the distribution of THC neuromodulation to the known distribution of estrogen cytosol receptors. Since an estrogen receptor appears to mediate the THC effect, we shall examine the nature of the THC neuromodulation in hippocampus of female rats tested in various stages of the estrous cycle. Other localization studies will attempt to determine if cholinergic systems are preferentially affected by THC and will examine the cellular locus of THC neuromodulation. The demonstrated hippocampal THC neuromodulation may be related to the human effects of THC intoxication on learning, memory and emotionality. The role of THC in cortical regions may similarly be related to the cognitive and perceptual alterations noted in human experience with this drug.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus have been a fruitful model for studying the neuronal processes believed to underlie learning and memory. The discovery of the NMDA receptor/Ca2+ - channel as part of the mechanism of LTP has provided a solid foundation for further studies into additional substrates of synaptic plasticity. In this proposal we wish to study a form of LTP that does not utilize NMDA receptors. Namely, LTP mediated by non-NMDA calcium channel activation. Hippocampal LTP can be induced by brief exposure to elevated calcium concentrations if extracellular potassium concentration is elevated (^[K+]e). This appears to reflect the involvement of a voltage-dependent mechanism which enhances postsynaptic calcium influx, because, in intracellular experiments, depolarizing current injection can substitute for elevated [K+]e. A small increase in [K+]e is sufficient to produce the required depolarization. Tetanic stimulation produces rapid and large increases in [K+]e in the synaptic layer of hippocampus. These results suggest that the post-synaptic depolarization required to activate NMDA receptor-gated ion channels in LTP might arise in part from release of potassium into the extracellular space. The voltage-dependent calcium influx appears to be unaffected by the NMDA antagonist APV, but is abolished by specific dihydropyridine calcium channel blockers. Thus, our preliminary results indicate that postsynaptic depolarization can promote a non-NMDA, voltage-dependent post-synaptic calcium influx, leading to a long-term Ca2+ -induced potentiation. Furthermore, we have new evidence that the non-NMDA receptor-mediated calcium influx can be activated during tetanus-induced LTP in normal media, as well. Thus, tetanus-induced LTP appears to have two calcium-dependent components - one mediated by Ca2+ influx through NMDA receptor-mediated channels, and another mediated by Ca2+ influx through voltage sensitive calcium channels. The two components of LTP appear to be induced by different patterns of afferent activity and appear to have somewhat different properties. We propose to study this phenomena. We wish to determine the magnitude, spatial distribution, clearance kinetics, and source of the ^[K+]e associated with tetanus-induced hippocampal LTP. We wish to confirm the identity and study the role of voltage-dependent calcium channels in LTP. We wish to confirm the identity and study the role of voltage-dependent calcium channels in LTP. Finally, since the effect of tetanus-induced ^[K+]e can be expected to depolarize adjacent membranes, we wish to investigate the role of this mechanism in associative LTP. These studies will be done on CA1 cells of hippocampal slices using intracellular and field potential recordings. Ion-sensing microelectrodes will be used to measure [K+]e. Pharmacological agents will be used to manipulate the two components of LTP.

DESCRIPTION (Adapted from applicant's abstract): This proposal is the second resubmission of one reviewed in June 1998. The conceptual framework guiding the proposed research is that the biophysical properties of different LTP mechanisms should be reflected in different aspects of learning and memory. The PI has made important contributions toward understanding these, as well as the cellular and biological properties of LTP, including the recent discovery of an NMDA receptor independent form of LTP in the Schaffer collateral-CA1 synapse (Grover & Teyler, Nature, 1990). The PI now proposes to investigate the links between learning and memory and the cellular and synaptic mechanisms of LTP.The PI discovered a form of long-term potentiation (LTP) that is independent of NMDA receptors. This form of LTP is induced by 200 Hz stimulation through voltage dependent calcium channels, and contributes to the LTP observed in typical in vitro and in vivo experiments (i.e. 100 Hz stimulation). In contrast to NMDA receptor-dependent LTP, which is induced quickly and decays, voltage-dependent calcium channel LTP (vdccLTP) can be induced in the presence of NMDA receptor antagonists, builds up slowly (~20 minutes) and decays slowly (no change > 10 h in vitro). The two patterns of stimulation are differentially sensitive to different enzymatic poisons: Serine/threonine kinase poison H-7 inhibits PKC, PKA, PKG, and CamKII, and prevents NMDA-dependent LTP induction (25Hz tetanus); tyrosine kinase poisons (e.g. genistein and lavendustin A) did not significantly attenuate 25Hz LTP induction, but prevented the vdccLTP (200Hz stim) from lasting more than 80 minutes. The proposed research will explore the relationship between learning and memory and the mechanisms underlying these two forms of plasticity. Specifically, the PI will test the effects of drugs that selectively block either voltage-dependent or NMDA receptor dependent LTP on memory performance in the radial maze.To test the hypothesis that vdccLTP is required for long-term memory retention, whereas NMDA receptor-dependent LTP is required for the acquisition of shorter-term reference memory, rats will be trained and tested in the 4/8 task on a radial maze. In this task, food is placed in 4 of the 8 arms of the maze at the start of each trial without replacement, and the task for the rat is to enter each of these baited arms once to get the food most efficiently. Entries into arms that never have food define reference memory errors, and repeated entries into arms with food within a trial define working memory errors (episodic memory). Hippocampal lesions impair acquisition of both working and reference memory in this task, but lesions given after training impair only the working memory component. Two major unanswered questions remain: 1. How does the within-trial working memory persist when NMDA receptors are blocked? 2. How and where in the brain is the long-term reference memory stored? The proposal focuses on the 2nd of these questions. NMDA receptor antagonists and voltage-dependent calcium channel LTP blockers will be given, alone and in combination, to behaving rats. In separate experiments, the drugs will be given systemically and into the hippocampus directly. Dose/response curves for the effects of the drugs on LTP induction will be tested in vivo. The hypothesis is that voltage-dependent LTP is required for long-term memory retention, whereas NMDA receptor dependent LTP is required for the shorter-term acquisition of reference memory. The drugs will also be given in combination.

DESCRIPTION (provided by applicant): We have discovered that the the fast repetitive presentation of a visual checkerboard (a photic "tetanus") leads to a persistent enhancement of early components of the visual evoked potential in normal humans. This lasting enhancement of the visual evoked potential is thought to be a form of human long-term potentiation (LTP) - the neuronal process underlying learning and memory. The potentiated response is largest in the hemisphere contralateral to the tetanized visual hemifield, and is limited to one component of the visual evoked response. Exposure to a photic tetanus also results in improved performance in a behavioral task. This study will be the first to investigate non-invasively the parameters and possible mechanisms of a form of LTP in humans. The goal of this proposal is to: 1) determine the optimal stimulus parameters of this potentiation, and 2) determine if this potentiated response obeys the established rules and cellular mechanisms of LTP and long-term depression (LTD) and 3) study its behavioral relevance. The study involves three research sites. The overall design, coordination, and animal studies will be by Dr. Teyler, at the University of Idaho, the co-discoverer of "human LTP". The optimal stimulus parameters and "rules testing" will be by Drs. Kirk and Hamm at the University of Auckland, New Zealand. The Yale site, led by Drs. Cavus and Krystal, will test whether the potentiation of this visual evoked response in humans is dependent on NMDA receptor activity. This study will provide us for the first time with tools to assess cortical plasticity noninvasively in humans. Such ability could have future applications in better understanding LTP and in the assessment of disorders that are thought to involve impairment in the cortical plasticity, such as Alzheimer's disease, Depression and Schizophrenia.