We review many of the recent findings concerning mechanisms and pathways for pain and its modulation, emphasizing sensitization and the modulation of nociceptors and of dorsal horn nociceptive neurons. We describe the organization of several ascending nociceptive pathways, including the spinothalamic, spinomesencephalic, spinoreticular, spinolimbic, spinocervical, and postsynaptic dorsal column pathways in some detail and discuss nociceptive processing in the thalamus and cerebral cortex. Structures involved in the descending analgesia systems, including the periaqueductal gray, locus ceruleus, and parabrachial area, nucleus raphe magnus, reticular formation, anterior pretectal nucleus, thalamus and cerebral cortex, and several components of the limbic system are described and the pathways and neurotransmitters utilized are mentioned. Finally, we speculate on possible fruitful lines of research that might lead to improvements in therapy for pain.

Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. We disrupted Gapds expression by gene targeting to selectively block sperm glycolysis and assess its relative importance for in vivo sperm function. Gapds ؊/؊ males were infertile and had profound defects in sperm motility, exhibiting sluggish movement without forward progression. Although mitochondrial oxygen consumption was unchanged, sperm from Gapds ؊/؊ mice had ATP levels that were only 10.4% of those in sperm from WT mice. These results imply that most of the energy required for sperm motility is generated by glycolysis rather than oxidative phosphorylation. Furthermore, the critical role of glycolysis in sperm and its dependence on this sperm-specific enzyme suggest that GAPDS is a potential contraceptive target, and that mutations or environmental agents that disrupt its activity could lead to male infertility. glycolysis ͉ gene targeting ͉ infertility S perm motility is essential for normal fertilization, and asthenozoospermia, or low sperm motility, is common in infertile men. In a recent study of 1,085 sperm samples from infertile men, 81% had defects in motility, and 19% had asthenozoospermia without other defects in sperm number or morphology (1). Motility is generated by the extremely long flagellum that comprises Ͼ90% of the length of a mammalian sperm. This process requires substantial ATP to support coordinated movement of the central axoneme and surrounding flagellar structures (2). ATP is hydrolyzed by dynein ATPases, which function as force-generating molecular motors along the axoneme. Although quiescent in the epididymis, mammalian sperm display vigorous forward movement, termed activated or progressive motility, immediately upon ejaculation or collection into physiological medium. The motility waveform changes in the female reproductive tract, with increases in both the amplitude and asymmetry of flagellar bending. These changes result in a whiplash-like motion, termed hyperactivated motility, which facilitates sperm transport in the oviduct and penetration of the zona pellucida surrounding the oocyte (3).Potential sources of ATP to support sperm motility are compartmentalized in distinct regions along the length of the flagellum. Oxidative phosphorylation is confined to the proximal segment of the flagellum where the mitochondria are localized (middle piece). In contrast, glycolysis appears to be restricted to the principal piece, which is distal to the middle piece and is the longest segment of the sperm flagellum (4-8). Several glycolytic enzymes in mammalian sperm are distinct from the isozymes present in somatic tissues. Thr...

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Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

A-kinase anchoring proteins (AKAPs) tether cyclic AMP-dependent protein kinases and thereby localize phosphorylation of target proteins and initiation of signal-transduction processes triggered by cyclic AMP. AKAPs can also be scaffolds for kinases and phosphatases and form macromolecular complexes with other proteins involved in signal transduction. Akap4 is transcribed only in the postmeiotic phase of spermatogenesis and encodes the most abundant protein in the fibrous sheath, a novel cytoskeletal structure present in the principal piece of the sperm flagellum. Previous studies indicated that cyclic AMP-dependent signaling processes are important in the regulation of sperm motility, and gene targeting was used here to test the hypothesis that AKAP4 is a scaffold for protein complexes involved in regulating flagellar function. Sperm numbers were not reduced in male mice lacking AKAP4, but sperm failed to show progressive motility and male mice were infertile. The fibrous sheath anlagen formed, but the definitive fibrous sheath did not develop, the flagellum was shortened, and proteins usually associated with the fibrous sheath were absent or substantially reduced in amount. However, the other cytoskeletal components of the flagellum were present and appeared fully developed. We conclude that AKAP4 is a scaffold protein required for the organization and integrity of the fibrous sheath and that effective sperm motility is lost in the absence of AKAP4 because signal transduction and glycolytic enzymes fail to become associated with the fibrous sheath.

There is now a wealth of experimental evidence showing that in the spinal cord inhibition is brought about by two quite distinct synaptic mechanisms (Frank & Fuortes, 1957;Eccles, 1961a Eccles, , b, 1962bEccles, Magni & Willis, 1962; Eccles, Kostyuk & Schmidt, 1962b, c; Eccles, Schmidt & Willis, 1963a; Schmidt & Willis, 1963a, b). One, post-synaptic inhibition, is effected by a specific synaptic action on the post-synaptic membrane, which counteracts the depolarization produced by excitatory impulses; the other, presynaptic inhibition, is effected by a specific synaptic action that depolarizes the synaptic terminals of primary afferent fibres and so reduces the amount of excitatory transmitter substance liberated therefrom.Although the transmitter substance mediating post-synaptic inhibition has not been identified, there is general agreement that a distinctive pharmacology is related to the operation of this substance. Depression of all types of post-synaptic inhibitory action is produced by some convulsant drugs (strychnine, brucein, thebaine and 5,7-diphenyl-1,3-diazadamantan-6-ol), which, on analogy with the pharmacological blockage of cholinergic excitatory synapses, is assumed to arise because of competitive occupation of the receptor sites for post-synaptic inhibition (Bradley, Easton

1. The contribution of activity in spinothalamic tract (STT) neurons to the pain and neurogenic hyperalgesia produced by an intradermal injection of 100 micrograms of capsaicin was investigated. Electrophysiological responses of identified STT neurons recorded in anesthetized monkeys were compared with psychophysical measurements of pain and hyperalgesia obtained in humans using identical stimuli. 2. Magnitude estimates of pain in humans were obtained after an injection of capsaicin or the vehicle. Capsaicin produced immediate burning pain that was most intense within 15 s after injection and then declined over the next 10-30 min. The vehicle produced no pain. 3. Cutaneous hyperalgesia to gentle stroking (allodynia) and also hyperalgesia to punctate stimulation developed in a wide area surrounding the capsaicin injection. Within this area, magnitude estimates of pain produced by a punctate stimulus (von Frey type with force of 225 mN) increased over preinjection values by an average of sixfold at test sites, 1, 2, and 3 cm away from the injection site. At the capsaicin injection site, magnitude estimates of pain in response to punctate simulation typically remained the same or were decreased. 4. After capsaicin, but not vehicle, the mean heat pain thresholds were lowered from approximately 45 degrees C before injection to 34 degrees C after, but only in the immediate vicinity of the injection site. At a site located 2 cm away, the thresholds were not significantly altered. Similarly, magnitude estimates of pain produced by suprathreshold heat stimuli were increased after capsaicin only at the injection site. 5. STT neurons were classified as high-threshold (HT) or wide-dynamic-range (WDR) cells according to responses evoked by graded cutaneous mechanical stimulation. An intradermal injection of capsaicin excited 4 of 7 HT cells and 10 of 12 WDR cells. The discharge rates of STT neurons correlated in time course with the magnitude estimates of pain in humans. The correlation was considerably better for WDR than for HT neurons, suggesting a predominant contribution of WDR neurons to the pain from capsaicin. 6. Capsaicin significantly increased the responses of HT neurons (9-fold) and the responses of WDR neurons (2-fold) to stroking the skin within the receptive field. Similar increases in responses to a standard punctate stimulus were observed at test sites, 1, 2, and 3 cm away from the injection site. After injection of vehicle, the responses to punctate stimulation increased by a mean of only 1.2- and 1.4-fold for HT and WDR neurons, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.