Franco A. Taverna

Enhanced LTP in Mice Deficient in the AMPA Receptor GluR2

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

α5GABAA Receptors Mediate the Amnestic But Not Sedative-Hypnotic Effects of the General Anesthetic Etomidate

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the α5 subunit (α5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by α5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not α5 null mutant (α5−/−) mice. In addition, etomidate impaired memory performance of WT but not α5−/− mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The α5−/− mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and α5−/− mice. Deletion of the α5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

Endogenous Zn2+ is required for the induction of long-term potentiation at rat hippocampal mossy fiber-CA3 synapses

The functional role of the abundant Zn2+ found in some hippocampal synapses has been an enigma. We show here, using N‐[6‐methoxy‐8‐quinolyl]‐P‐toluenesulfonamide (TSQ) staining, that chelatable‐Zn2+ can be removed from hippocampal synaptic boutons using dietary depletion or with Zn2+ chelators. A chronic dietary deficiency of bouton Zn2+ resulted in the impairment of long‐term potentiation (LTP) at mossy fiber‐CA3 synapses. The averaged normalized fEPSP slope 30 min after tetanus was 209 ± 28% of baseline value in control (mean ± SEM, n = 10), and 118 ± 12% in Zn2+‐deficient rats (mean ± SEM, n = 12, P < 0.01). In the deficient rats with Zn2+ supplements, mossy fiber LTP returned to normal levels. The acute depletion of bouton Zn2+ in the hippocampal slice with membrane‐permeable Zn2+ chelators, dithizone, or diethyldithiocarbamic acid (DEDTC) blocked the induction of mossy fiber LTP. The mean amplitudes of EPSCs after tetanus were 194 ± 22% of baseline value in control (n = 5), compared to 108 ± 14% in dithizone (n = 6) and 101 ± 12% in DEDTC (n = 5). The averaged value of LTP, at the associational commisural fiber‐CA3 synapses, was 193 ± 20% in the control (n = 6), compared to 182 ± 21% (n = 6, P > 0.1) in the presence of dithizone. The blockade of mossy fiber LTP by dithizone was reversible after washout. In addition, normal LTP could be induced by tetanus if exogenous Zn2+ was applied immediately following dithizone. Our results indicate that the endogenous Zn2+ is specifically required for LTP induction at the mossy fiber input into CA3 neurons. Synapse 38:187–197, 2000.

Sensitivity of AMPA receptors to pentobarbital

The inhibitory effects of pentobarbital on various AMPA receptors expressed (GluR1, GluR3, GluR1/3, GluR1/2, and GluR2/3) in Xenopus oocytes were examined. Combinations of AMPA receptor subunits that included GluR2 demonstrated a much higher sensitivity to blockade by this barbiturate and the apparent co-operativity of the interaction of pentobarbital with the receptor was reduced. This evidence demonstrates that the GluR2 subunit alters the structure of AMPA receptors in such a way as to facilitate any interaction with this barbiturate.

Expression of functional metabotropic and ionotropic glutamate receptors in baculovirus-infected insect cells

An ionotropic glutamate receptor of the kainate subtype (GluR6) and a G-protein coupled metabotropic glutamate receptor (mGluR1 alpha) were expressed and studied in two insect cell lines: sf9 cells from Spodoptera frugiperda and MG1 cells from Trichoplusia ni. Application of kainate to GluR6-infected MG1 cells produced kainate-activated currents. Glutamate activation of mGluR1 alpha in MG1- and sf9-infected cells caused rapid, transient increases in intracellular calcium levels. This effect was more pronounced in MG1 cells compared to sf9 cells. These results indicate that functional glutamate receptors can be expressed in the baculovirus system, and that MG1 cells may have several advantages over the widely used sf9 cells for studying the functional properties of receptors and channels.

Defective place cell activity in nociceptin receptor knockout mice with elevated NMDA receptor-dependent long-term potentiation

There is growing evidence that NMDA receptor‐dependent long‐term potentiation (LTP) in the hippocampus mediates the synaptic plasticity that underlies spatial learning and memory. LTP deficiencies correlate well with spatial memory deficits and LTP enhancements may improve spatial memory. In addition, LTP deficiencies are associated with abnormal place cells as expected from the spatial mapping hypothesis of hippocampal function. In contrast, nothing is known on how enhanced NMDA receptor‐dependent LTP affects place cells. To address this question we recorded place cells from mice lacking the nociceptin receptor (NOP1/ORL1/OP4) that have enhanced hippocampal LTP. We found that the enhanced LTP was mediated by NMDA receptors, did not require L‐type calcium channels, and occurred only when high frequency tetanizing stimulus trains were used. Place cells in nociceptin receptor knockout mice were abnormal in several ways: they were less stable, had noisier positional firing patterns, larger firing fields and higher discharge rates inside and outside the firing fields. Our results suggest that excessive LTP can cause subnormal hippocampal place cell function. The effects of LTP enhancement on place cell function may therefore also depend on molecular details of synaptic plasticity, including the relationship between stimulus frequency and synaptic strength, and not merely on the magnitude of synaptic strength increases. The data have important clinical implications on development of strategies to improve cognitive function.

Impairment in long-term retention but not short-term performance on a water maze reversal task following hippocampal or mediodorsal striatal N-methyl-D-aspartate receptor blockade.

Male Long-Evans rats were injected with 32 ng/mul of the N-methyl-D-aspartate (NMDA) receptor antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) or vehicle and trained to locate a hidden platform in a different location (reversal training) than used on the initial 4 days of training. Rats treated with vehicle or CPP into the dorsal hippocampus, basolateral amygdala, or mediodorsal striatum had similar latencies to locate the platform on the reversal day. Rats infused with CPP into the dorsal hippocampus or mediodorsal striatum failed to search preferentially in the novel location during a 24-hr, drug-free retention test, whereas all other groups searched preferentially in this location. Therefore, blocking dorsal hippocampal or mediodorsal striatal NMDA receptors selectively blocked long-term spatial retention without producing short-term performance deficits.

Properties of a recombinant kainate receptor expressed in baculovirus-infected insect cells

GluR6 is a glutamate receptor of the kainate subtype that is expressed in the mammalian central nervous system. The cDNA coding for the rat brain receptor was subcloned into a baculovirus expression vector and the purified recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The pharmacological profile and the status of several post-translational modifications of the GluR6 protein were analyzed. Saturation analyses of [3H]kainic acid binding demonstrated that GluR6 expressed in Sf9 cell membranes bound [3H]kainic acid at a single high affinity site with a dissociation constant of 12 nM. Competition studies indicated the inhibitory potencies of various excitatory amino acids, including the potent neurotoxin domoic acid, were comparable to those observed in mammalian brain tissue. Immunoblots of infected Sf9 cell membranes using an anti-GluR6 antibody revealed two immunoreactive bands. Enzymatic deglycosylation indicated that the higher molecular weight form corresponded to the glycosylated receptor whereas the lower molecular weight form corresponded to the unglycosylated protein. The phosphorylation of GluR6 by cyclic AMP-dependent and cyclic GMP-dependent protein kinase was examined in partially purified preparations of the receptor. GluR6 was phosphorylated by cyclic AMP but not by cyclic GMP-dependent protein kinase in vitro. These results indicate that GluR6 expressed in Sf9 cells has similar pharmacological properties and is processed post-translationally in a manner similar to GluR6 expressed in mammalian cells and tissues. The ease of production and the high levels of expression in baculovirus-infected insect cells relative to other expression systems should facilitate further biochemical and pharmacological characterization of this receptor.

Textbook Use in the Sciences and Its Relation to Course Performance

There are limited studies with conflicting results examining textbook use and student performance at the university level. To learn more, we surveyed instructors and over one thousand students in 12 undergraduate introductory science courses. The majority (77%) of the students reported reading the textbook either “often” (>75% of the assigned reading) or “sometimes” (25%–75% of the assigned reading). Those who read “often” had better final marks those who read “sometimes,” but surprisingly, those who reported “rarely” reading the textbook did as well as those who read “often.” Perceptions of the usefulness of the textbook were generally more favorable in courses in which some marks were based solely on the readings. We conclude that there appears to be different types of learners: some may need to read the textbook “often” to do well, while others do not.