Even in the absence of explicit stimulation, rats emit patterns of rhythmic whisking movements. Because of their stereotyped nature and their persistence after sensory denervation and cortical ablation, whisking movements have been assumed to reflect the output of a central pattern generator (CPG). However, identification of a movement pattern as the product of a CPG requires evidence that its generation, patterning, and coordination are independent of sensory input. To provide such evidence, we used optoelectronic instrumentation to obtain high-resolution records of the movement trajectories of individual whiskers in rats whose heads were fixed to isolate their exploratory whisking from exafferent inputs. Unconditioned whisking patterns were quantitatively characterized by a biometric analysis of the kinematics, rhythmicity, and coordination of bilaterally homologous vibrissa movements. Unilateral and bilateral sectioning of the infraorbital nerve, which innervates the whiskers, was then performed to block reafferent inputs generated by the animal's own whisking movements. Unilateral sectioning of the nerve has no effect on whisking kinematics but is followed by a significant but relatively transient bilateral increase in whisking frequency. However, bilateral deafferentation, when performed in a single-stage procedure, does not disrupt the generation, patterning, or bilateral coordination of whisking patterns in the rat. These findings provide strong behavioral evidence for a whisking CPG and are discussed in relation to its possible location and properties.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular (domains A, B, C, D, E, and F) nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins and itself using NAD(+) as a substrate. Early studies suggested that domain D is likely an interface for protein-protein interaction between PARP-1 and its targets and is also the primary region for automodification. However, determination of the modification sites has been complicated by the heterogeneous nature of the poly(ADP-ribose) polymer. Here we report a strategy to identify the modification sites on domain D using the PARP-1 E988Q mutant, which only catalyzes mono(ADP-ribosyl)ation. Trypsin digestion of the modified domain D followed by LC-MS/MS analysis led to the identification of three ADP-ribosylation sites in domain D (D387, E488, and E491). Our data also show, in contrast to early reports, that automodification of PARP-1 is not limited to domain D but occurs beyond this region. In addition, domain D is not essential for PARP-1 activity since PARP-1 mutant having domain D deleted is still catalytically active. Two synthetic peptides with amino acid sequences derived from the ADP-ribosylation sites of domain D were also demonstrated to act as PARP-1 substrates. The methodology and the results reported herein will facilitate future studies of PARP-1 catalysis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins using NAD (+) as a substrate. In this work, NMR methods were used to determine the solution structure of human PARP-1 protein. Domain C was found to contain a zinc-binding motif of three antiparallel beta-strands with four conserved cysteines positioned to coordinate the metal ligand, in addition to a helical region. The zinc-binding motif is structurally reminiscent of the "zinc-ribbon" fold, but with a novel spacing between the conserved cysteines (CX2CX12CX 9C). Domain C alone does not appear to bind to DNA. Interestingly, domain C is essential for PARP-1 activity, since a mixture containing nicked DNA and the PARP-1 ABDEF domains has only basal enzymatic activity, while the addition of domain C to the mixture initiated NAD (+) hydrolysis and the formation of poly(ADP-ribose), as detected by an NMR-based assay and autoradiography. The structural model for domain C in solution provides an important framework for further studies aimed at improving our understanding of how the various domains within the complex PARP-1 enzyme play their respective roles in regulating the enzyme activity when cells are under conditions of genotoxic stress.

The rat's mystacial vibrissae are active during exploratory and discriminative behaviors, with individual vibrissae serving as elements in a receptive array scanned across object surfaces. To facilitate neurobehavioral analysis of this sensorimotor system, we have developed an experimental paradigm that confines vibrissa movements to a defined physical location, makes possible on-line monitoring of "whisking" activity, and brings such activity under associative control using operant conditioning procedures. Rats were secured, and movements of an identified bilaterally homologous pair of vibrissae (right and left gamma straddlers) were detected by laser-based photodetectors. Subjects were maintained on a water deprivation schedule, and whisker movements were monitored during adaptation to the test situation and after the clipping of other vibrissae on both sides of the snout. Rats were reinforced with water delivery for emitting vibrissa movements in the presence of a conditioned stimulus (tone) whose presentation was made contingent upon a prior period of nonwhisking. The rate and temporal distribution of vibrissa movements were brought under experimental control by means of interval and ratio reinforcement schedules. Although the procedures provide minimal information about the kinematics or topography of conditioned vibrissa movements, they permit the investigator to manipulate response parameters normally under the voluntary control of the animal in a preparation amenable to neurophysiological analysis.