Interval timing in genetically modified mice: a simple paradigm.

We describe a behavioral screen for the quantitative study of interval timing and interval memory in mice. Mice learn to switch from a short-latency feeding station to a long-latency station when the short latency has passed without a feeding. The psychometric function is the cumulative distribution of switch latencies. Its median measures timing accuracy and its interquartile interval measures timing precision. Next, using this behavioral paradigm, we have examined mice with a gene knockout of the receptor for gastrin-releasing peptide that show enhanced (i.e. prolonged) freezing in fear conditioning. We have tested the hypothesis that the mutants freeze longer because they are more uncertain than wild types about when to expect the electric shock. The knockouts however show normal accuracy and precision in timing, so we have rejected this alternative hypothesis. Last, we conduct the pharmacological validation of our behavioral screen using d-amphetamine and methamphetamine. We suggest including the analysis of interval timing and temporal memory in tests of genetically modified mice for learning and memory and argue that our paradigm allows this to be done simply and efficiently.

Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS.

The hyperpolarization-activated cation current (termed I(h), I(q), or I(f)) was recently shown to be encoded by a new family of genes, named HCN for hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective. When expressed in heterologous cells, each HCN isoform generates channels with distinct activation kinetics, mirroring the range of biophysical properties of native I(h) currents recorded in different classes of neurons. To determine whether the functional diversity of I(h) currents is attributable to different patterns of HCN gene expression, we determined the mRNA distribution across different regions of the mouse CNS of the three mouse HCN genes that are prominently expressed there (mHCN1, 2 and 4). We observe distinct patterns of distribution for each of the three genes. Whereas mHCN2 shows a widespread expression throughout the CNS, the expression of mHCN1 and mHCN4 is more limited, and generally complementary. mHCN1 is primarily expressed within neurons of the neocortex, hippocampus, and cerebellar cortex, but also in selected nuclei of the brainstem. mHCN4 is most highly expressed within neurons of the medial habenula, thalamus, and olfactory bulb, but also in distinct neuronal populations of the basal ganglia. Based on a comparison of mRNA expression with an electrophysiological characterization of native I(h) currents in hippocampal and thalamic neurons, our data support the idea that the functional heterogeneity of I(h) channels is attributable, in part, to differential isoform expression. Moreover, in some neurons, specific functional roles can be proposed for I(h) channels with defined subunit composition.

B2 RNA and 7SK RNA, RNA polymerase III transcripts, have a cap-like structure at their 5' end.

We found that hydrolysates of poly(A)+ RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xppp5'G, where x has a non-nucleotide structure. About half of all B2 RNAs contained this group at the 5' end. Previously, Epstein et al. (1) detected a similar structure at the 5' end of small nuclear U6 RNA. Later, Singh and Reddy (2) showed methyl to be the blocking group in the component x of U6 RNA. Besides B2 RNA, we found 5' ends containing methyl groups in 7SK RNA.

The most abundant nascent poly(A) + RNAs are transcribed by RNA polymerase III in murine tumor cells.

Twelve to twenty percent of newly synthesized poly(A) + RNA is transcribed by RNA polymerase III in Ehrlich ascites carcinoma and P3O1 plasmocytoma mouse tumors. Most of this RNA designated as pol IIIpoly(A) + RNA has a size of 160 to 800 nucleotides with a maximum of distribution of ca. 300 nucleotides. Pol IIIpoly(A) + RNA fraction consists of two major classes of molecules corresponding to previously described B1 RNA and B2 RNA with the ratio of 1:4 to 2:3. All B2 RNAs present in poly(A) + fraction contain a long poly(A) segments at the 3' ends. Thus, RNA polymerase III transcripts can be polyadenylated. Several transcripts that hybridize with B2 probe were also observed in poly(A)- RNA. The major components consist of 180, 160, 120 and 95 nucleotides. The 180-nucleotide B2 RNA seems to be a primary transcript from B2 repeat. We suggest that other B2 RNAs are transcribed from truncated copies of B2 element.