Induction of a physiological memory in the cerebral cortex by stimulation of the nucleus basalis.

Auditory cortical receptive field plasticity produced during behavioral learning may be considered to constitute "physiological memory" because it has major characteristics of behavioral memory: associativity, specificity, rapid acquisition, and long-term retention. To investigate basal forebrain mechanisms in receptive field plasticity, we paired a tone with stimulation of the nucleus basalis, the main subcortical source of cortical acetylcholine, in the adult guinea pig. Nucleus basalis stimulation produced electroencephalogram desynchronization that was blocked by systemic and cortical atropine. Paired tone/nucleus basalis stimulation, but not unpaired stimulation, induced receptive field plasticity similar to that produced by behavioral learning. Thus paired activation of the nucleus basalis is sufficient to induce receptive field plasticity, possibly via cholinergic actions in the cortex.

Ca2+/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-d-aspartate receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.

Short-term synaptic enhancement and long-term potentiation in neocortex.

Repetitive stimuli reliably induce long-term potentiation (LTP) of synapses in the upper layers of the granular somatosensory cortex but not the agranular motor cortex of rats. Herein we examine, in these same cortical areas, short-term changes in synaptic strength that occur during the LTP induction period. theta-Burst stimulation produced a strong short-term enhancement of synapses in the granular area but only weak enhancement in the agranular area. The magnitude of enhancement during stimulation was strongly correlated with the magnitude of LTP subsequently expressed. Short-term enhancement was abolished by an antagonist of N-methyl-D-aspartate (NMDA) receptors but remained in the presence of a non-NMDA receptor antagonist. Inhibitory postsynaptic potentials of the granular and agranular areas displayed similar frequency sensitivity, but the frequency sensitivity of NMDA receptor-dependent excitatory postsynaptic potentials differed significantly between areas. We propose that pathway-specific differences in short-term enhancement are due to variations in the frequency dependence of NMDA currents; different capacities for short-term enhancement may explain why repetitive stimulation more readily induces LTP in the somatosensory cortex than in the motor cortex.

Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.

Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection.

During tumor progression, variants may arise that grow more vigorously. The fate of such variants depends upon the balance between aggressiveness of the variant and the strength of the host immunity. Although enhancing host immunity to cancer is a logical objective, eliminating host factors necessary for aggressive growth of the variant should also be considered. The present study illustrates this concept in the model of a spontaneously occurring, progressively growing variant of an ultraviolet light-induced tumor. The variant produces chemotactic factors that attract host leukocytes and is stimulated in vitro by defined growth factors that can be produced or induced by leukocytes. This study also shows that CD8+ T-cell immunity reduces the rate of tumor growth; however, the variant continues to grow and kills the host. Treatment with a monoclonal anti-granulocyte antibody that counteracts the infiltration of the tumor cell inoculum by non-T-cell leukocytes did not interfere with the CD8+ T-cell-mediated immune response but resulted in rejection of the tumor challenge, indicating a synergy between CD8+ T-cell-mediated immunity and the inhibition of paracrine stimulation.

Decreased brain reward produced by ethanol withdrawal.

Abstinence from chronic administration of various drugs of abuse such as ethanol, opiates, and psychostimulants results in withdrawal syndromes largely unique to each drug class. However, one symptom that appears common to these withdrawal syndromes in humans is a negative affective/motivational state. Prior work in rodents has shown that elevations in intracranial self-stimulation (ICSS) reward thresholds provide a quantitative index that serves as a model for the negative affective state during withdrawal from psychostimulants and opiates. The current study sought to determine whether ICSS threshold elevations also accompany abstinence from chronic ethanol exposure sufficient to induce physical dependence. Rats prepared with stimulating electrodes in the lateral hypothalamus were trained in a discrete-trial current-intensity ICSS threshold procedure; subsequently they were subjected to chronic ethanol administration in ethanol vapor chambers (average blood alcohol level of 197 mg/dl). A time-dependent elevation in ICSS thresholds was observed following removal from the ethanol, but not the control, chambers. Thresholds were significantly elevated for 48 hr after cessation of ethanol exposure, with peak elevations observed at 6-8 hr. Blood alcohol levels were directly correlated with the magnitude of peak threshold elevation. Ratings of traditional overt signs of withdrawal showed a similar time course of expression and resolution. The results suggest that decreased function of reward systems (elevations in reward thresholds) is a common element of withdrawal from chronic administration of several diverse classes of abused drugs.