Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays.

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as "background amplification". After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.

What can toxins tell us for drug discovery?

Toxins are of interest in drug design because the toxins provide three-dimensional templates for creating small molecular mimics with interesting pharmacological properties. Toxins are also useful in drug discovery because they can be used as pharmacological tools to uncover potential therapeutic targets. With their high potency and selectivity, toxins are often more useful in functional experiments than standard pharmacological agents. We have used two groups of neurotoxins, the dendrotoxins and the muscarinic toxins (MTs), to explore the involvement of subtypes of potassium ion channels and muscarinic receptors, respectively, in processes involved in cognition and the changes in neuronal properties with aging. From our current work, quantitative autoradiographic studies with radiolabelled dendrotoxins reveal widespread distribution of binding sites throughout rat brain sections, but few differences exist between young adult and aged rats. However, displacement studies with toxin K, which preferentially binds to the Kv1.1 subtype of cloned potassium channel, show the selective loss of such sites in regions of the hippocampus and septohippocampal pathway with aging. MTs have been tested for effects on performance of rats in memory paradigms. MT2, which activates m1 receptors, improves performance of rats in a step-down inhibitory avoidance test, whereas MT3, which blocks m4 receptors, decreases performance when given into the hippocampus. This is the first clear demonstration of a role for m4 muscarinic receptors in cognition.

Effect of early unilateral visual deprivation on cholinergic muscarinic receptors and acetylcholinesterase in chick optic lobe.

Chicks of 1-8 days after hatching were submitted to unilateral visual deprivation and, in both optic lobes, muscarinic receptors, labeled with L-[3H]quinuclidinyl benzylate [( 3H]L-QNB) and acetylcholinesterase (AChE) were assayed. Between 1 and 6 days the number of [3H]L-QNB binding sites was lower in the contralateral optic lobe; the AChE underwent a drastic reduction (-42% at 2 days); later on the differences became not significant. The pre- and postsynaptic localization of the two cholinergic markers and the possible influence of light on axonal transport are discussed.