Structural basis for the coiled-coil architecture of human CtIP.

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.

Baclofen and the Release of Neuropeptides in the Cat Spinal Cord.

The antibody microprobe technique was used to study the effect of baclofen on the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of pentobarbitone-anaesthetized spinalized cats. Both peptides were released in the region of the substantia gelatinosa during ipsilateral noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of baclofen suppressed the excitation of lumbar dorsal horn neurons, but did not produce detectable alterations of the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial grey matter dorsal to these neurons. The results suggest that the antinociceptive action of baclofen does not involve a reduction of the intraspinal release of substance P or calcitonin gene-related peptide from the central terminals of nociceptive sensory fibres.

Periaqueductal grey stimulation: an association between selective inhibition of dorsal horn neurones and changes in peripheral circulation.

In barbiturate-anaesthetized and paralysed cats, dorsal horn neurones were studied during electrical stimulation of the periaqueductal grey matter (PAG) and the midbrain ventral tegmentum (VT). Responses to impulses in unmyelinated primary afferents were selectively inhibited by stimulation in the PAG, whereas stimulation in the VT non-selectively reduced both these responses and those to innocuous cutaneous stimuli. Stimulation in the PAG but not the VT produced changes in peripheral circulation. This was observed as a rise in the levels of carbon dioxide in expired air, a rise in muscle temperature in the hind limb and a fall in skin temperature of the pinna or glabrous skin. The combination of suppression of spinal transmission of impulses related to pain and an increase in perfusion of muscles may be a mechanism appropriate to coping with a potentially injurious environment.

Modulation of cutaneous nociceptor activity by electrical stimulation in the brain stem does not inhibit the nociceptive excitation of dorsal horn neurons.

In anesthetized cats, recordings were obtained from single lumbar dorsal horn neurons and from primary afferent fibers of the posterior tibial nerve excited by controlled noxious radiant heating of glabrous hindpaw skin. Electrical stimulation in four brain stem regions (periaqueductal gray and lateral reticular formation in the midbrain, raphe and reticular formation in the medulla) during noxious skin heating markedly reduced the nociceptive excitation of the dorsal horn neurons. In contrast, such brain stem stimulation had small and variable effects upon the noxious heat-evoked activity in the primary afferent fibers; both increases and decreases were observed. The brain stimulation also produced transient changes in blood pressure, suggesting that circulatory effects may underlie the mechanism of nociceptor modulation. It is concluded that brain stem stimulation can modulate cutaneous nociceptor activity, but that this modulatory effect on nociceptor inflow is too small and inconsistent to explain the marked descending inhibition of the nociceptive excitation of dorsal horn neurons.

Inhibition of nociceptive responses of lumbar dorsal horn neurones by remote noxious afferent stimulation in the cat.

In cats anaesthetized with nitrous oxide and sodium pentobarbital, multireceptive lumbar dorsal horn neurones excited by controlled noxious radiant heating of glabrous hind paw skin were recorded by extracellular microelectrodes. These noxious heat responses were inhibited by concomitant noxious stimulation of the ipsilateral forepaw or pinna, or repetitive electrical stimulation of the ipsilateral forelimb deep radial nerve. Similar extents of inhibition were produced by noxious peripheral stimulation and by deep radial nerve stimulation in repetitive trains at intensities sufficient to excite small myelinated fibres or unmyelinated fibres. A greater inhibitory effect was produced by continuous repetitive high-intensity stimulation of the deep radial nerve. With a constant frequency (5 Hz) of continuous deep radial nerve stimulation, graded increases in stimulation intensity revealed the threshold for inhibition in the small myelinated fibre range, and an additional increment of the inhibitory effect when unmyelinated fibres were also activated. When suprathreshold for unmyelinated fibres, the efficacy of continuous deep radial nerve stimulation increased with graded increases in stimulation frequency, with a threshold frequency for inhibition between 0.5 and 1 Hz and maximal effect at 5 Hz. Two nociceptive-specific neurones studied were also inhibited by deep radial nerve stimulation. The results indicate that 'diffuse noxious inhibitory controls' (DNIC) occur in the cat and can be activated by remote electrical or natural noxious stimulation.

Release of sensory neuropeptides in the spinal cord: studies with calcitonin gene-related peptide and galanin.

In anaesthetized cats, antibody microprobes were used to investigate the release of immunoreactive calcitonin gene-related peptide and galanin in the lower lumbar spinal cord. In the absence of applied stimulation, a basal release of both peptides was detected at the level of the substantia gelatinosa. This release of calcitonin gene-related peptide was not altered by innocuous thermal cutaneous stimulation nor by electrical stimulation of low-threshold myelinated primary afferent fibres, but was increased by noxious thermal or noxious mechanical cutaneous stimuli and by electrical stimulation of unmyelinated primary afferents. A simultaneous release of both calcitonin gene-related peptide and substance P was detected in the substantia gelatinosa region by the use of pairs of microprobes. In contrast, none of the peripheral stimulation procedures increased intraspinal galanin release. The results suggest that the spinal transmission of nociceptive information may involve the simultaneous release and action of several neuropeptides within the superficial layers of the dorsal horn.

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.

Release of immunoreactive somatostatin in the spinal dorsal horn of the cat.

Antibody microprobes were used to investigate the possible release of immunoreactive somatostatin (irSS) within the lumbar spinal cord of anaesthetized cats. A basal release of irSS was detected in the region of the substantia gelatinosa of the dorsal horn. By comparison with in vitro standards the concentration of SS detected in this region was estimated at 10(-7) M. This release of irSS was not significantly altered by electrical stimulation of large myelinated primary afferent fibres but was increased when unmyelinated afferents were additionally stimulated. Release of irSS was also detected at the spinal cord surface. The results support a role for somatostatin in nociceptive transmission in the spinal cord.

Analysis of antibody microprobe autoradiographs by computerized image processing.

A computerized digitizing method to analyze the autoradiographs derived from the antibody microprobe technique is described. The method enables the averaging of groups of microprobes that have been placed in similar locations in the central nervous system during the same physiological stimulation. Control and experimental groups of microprobes can be compared, and a level of significance for the release of a neuropeptide obtained. In addition estimates can be made of the concentration of peptide present in regions of release.

The preparation and use of antibody microprobes.

A new method of detecting release of neuropeptides in the central nervous system is described. Glass micropipettes are treated with gamma-aminopropyltriethoxysilane resulting in a fine outer coating of a siloxane polymer containing free amino groups. Glutaraldehyde is then used to covalently couple protein A which in turn binds antibodies to a particular peptide. Following use in the central nervous system, microprobes are incubated in a radiolabelled form of the peptide being studied and release is detected on autoradiographs as localized zones of inhibition of binding of the labelled peptide. The spatial resolution of the method is at least 100 micron. Necessary tests of the validity of the technique are also described.

Inhibition of spinal nociceptive transmission accompanies cardiovascular changes from stimulation in diencephalic 'defence' regions of cats.

In anaesthetized cats, diencephalic regions were electrically stimulated while recording evoked responses from lumbar dorsal horn neurones and cardiovascular parameters. Neuronal responses to impulses evoked electrically in unmyelinated primary afferents were inhibited by stimulation in many diencephalic regions. Responses to non-noxious cutaneous stimulation (hair deflection) were inhibited at relatively few sites. Indirect circulatory measurements showed that this selective spinal inhibition was accompanied by increases in cardiac output and muscle blood flow but reduced cutaneous perfusion. This association between selective inhibition of nociceptive spinal neuronal responses and a cardiovascular response pattern associated with the defence reaction supports the proposal that analgesia could be an important component of defensive behaviour.

Diffuse noxious inhibitory controls of lumbar spinal neurons involve a supraspinal loop in the cat.

In anesthetized cats, lumbar dorsal horn neurons were excited by brief noxious radiant heating of glabrous hindpaw skin. These nociceptive responses were inhibited by concomitant repetitive electrical stimulation of the ipsilateral deep radial nerve. Noxious heat responses were linearly correlated with skin temperature during heating. The slope of this stimulus-response function was decreased, and the response threshold increased, by deep radial nerve stimulation. Microinjection of lidocaine into the medullary raphe attenuated the inhibition induced by deep radial nerve stimulation. The results indicate that in the cat, 'diffuse noxious inhibitory controls' (DNIC) involve medial medullary regions.

Calcitonin: brainstem microinjection but not systemic administration inhibits spinal nociceptive transmission in the cat.

In anaesthetized cats, nociceptive responses of lumbar dorsal horn neurons were studied during administration of salmon calcitonin (sCT). Systemic sCT administration (4-95 IU/kg i.v.) produced no change in neuronal responses produced by noxious skin heating or by impulses evoked electrically in afferent C-fibres. Responses to skin heating were reduced during electrical stimulation in the brainstem, but the efficacy of this descending inhibition was not altered by systemic sCT administration. In contrast, noxious heat responses were clearly reduced by microinjection of sCT into the mesencephalic periaqueductal grey or the medullary raphe regions. These results suggest that calcitonin or a related peptide could act at specific brainstem sites to inhibit the spinal transmission of nociceptive information.

Dynorphin A: in vivo release in the spinal cord of the cat.

The antibody microprobe technique was used to study the release of immunoreactive dynorphin A within the lower lumbar spinal cord of anaesthetised cats. A basal release was observed in the dorsal horn, centered in the region of lamina I, but was abolished by spinal cord transection at the thoracolumbar junction. Release of dynorphin A in the lamina I region was evoked by high-frequency electrical stimulation of unmyelinated primary afferent fibres, whereas stimulation of myelinated (including A delta) afferents was ineffective. Evidence was also obtained for release in laminae V-VI and at the spinal cord surface. These results suggest that in the lumbar spinal cord of the cat, dynorphin A is released in the superficial dorsal horn by impulses in descending pathways and in somatic unmyelinated primary afferent fibres.

Lateral reticular regions and the descending control of dorsal horn neurones of the cat: selective inhibition by electrical stimulation.

In barbiturate-anaesthetized cats, brainstem sites were electrically stimulated while studying the synaptic responses of lumbar dorsal horn neurones. The excitation of these neurones by impulses in unmyelinated primary afferents was selectively inhibited by stimulation of the ventrolateral medulla in the region of the caudal lateral reticular nucleus. The significance of this inhibition was heightened by the finding that stimulus currents producing inhibition from this area were less effective in the raphé region and not effective at intervening or dorsal sites. Bilateral lesions of the inhibition-producing ventrolateral sites reduced tonic descending inhibition of the responses of dorsal horn neurones to impulses in C fibres. The lateral reticular regions of the medulla may thus exert a considerable control over the transmission of nociceptive information in the cat spinal cord.

The effects of lesions of medullary midline and lateral reticular areas on inhibition in the dorsal horn produced by periaqueductal grey stimulation in the cat.

In barbiturate-anaesthetized cats, the excitation of lumbar dorsal horn neurones by impulses in unmyelinated primary afferent fibres was inhibited by electrical stimulation in the periaqueductal grey matter. This inhibition was slightly reduced by extensive electrocoagulation of the medullary midline and para-medial areas including the raphé, but significantly reduced by small bilateral lesions in the region of the caudal lateral reticular nuclei. When the lateral lesions were made subsequent to midline coagulation, the inhibition from periaqueductal grey stimulation was abolished. An important component of spinal inhibition from periaqueductal grey stimulation appears to relay in lateral reticular areas of the medulla.

Differing distributions of receptors for morphine and Met5-enkephalinamide in the dorsal horn of the cat.

In the dorsal horn of barbiturate-anaesthetized cats evidence was obtained for a different distribution of enkephalin-preferring and morphine-preferring receptors. When ejected electrophoretically from micropipettes, morphine reduced the nociceptive responses of neurones of laminae IV and V when administered in the substantia gelatinosa but not at more ventral sites including sites near cell bodies. By contrast, Met5-enkephalinamide reduced nociceptive responses at all of these sites of administration often with increased potency near cell bodies. It is proposed that morphine-preferring receptors are located mainly near the terminals of primary afferent fibres whereas those for enkephalin are present on these fibres and also on the dendrites and somata of dorsal horn neurones. These dendritic receptors may be located adjacent to sites of termination of nociceptive afferents, a mechanism which would inhibit nociceptive but not other inputs to these neurones.

Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat.

In barbiturate-anaesthetized spinal cats, antibody microprobes were used to examine immunoreactive substance P (irSP) release at sites within the spinal cord following cutaneous stimuli. A basal level of irSP release was detected in the region of the substantia gelatinosa of the lumbar spinal cord. No increase in this irSP release was produced by non-noxious thermal or mechanical cutaneous stimulation. Noxious thermal, mechanical or chemical cutaneous stimuli all increased release of irSP in the region of the substantia gelatinosa and in the overlying pia mater. The results support a role for SP in the transmission of information from nociceptors to spinal neurones.

Somatostatin: evidence for a role in thermal nociception.

In barbiturate-anaesthetized spinalized cats, antibody microprobes were used to investigate the release of immunoreactive somatostatin (irSS) in the lumbar dorsal horn in response to cutaneous stimuli. In the absence of applied stimulation, a significant basal release of irSS was present in the region of the substantia gelatinosa. Such release was not increased by innocuous or noxious cutaneous mechanical stimuli nor by innocuous thermal stimuli, but was increased by noxious thermal stimulation. The magnitude of this noxious heat-evoked release was estimated by comparing in vivo microprobes with those used to detect known concentrations of somatostatin in vitro. Pairs of microprobes were used to detect simultaneous release of both irSS and immunoreactive substance P in the substantia gelatinosa. The results support the putative role of somatostatin in the spinal transmission of thermal nociceptive information.

Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes.

Using a new method, the antibody microprobe technique, the release of immunoreactive substance P (SPiR) in the dorsal horn in response to noxious heating of the skin, was studied in barbiturate anaesthetized spinal cats. Release of SPiR was not produced by immersing the ipsilateral hind paw in water at 37 degrees C. With water at 50 and 52 degrees C, however release was consistently detected in the region of the substantia gelatinosa. These results directly show a central release of SPiR with excitation of nociceptors by heat.