Activation of protein kinase B/Akt in the periphery contributes to pain behavior induced by capsaicin in rats.

Protein kinase B (PKB/Akt) is a member of the second-messenger regulated subfamily of protein kinases. It is implicated in signaling downstream of growth factors, insulin receptor tyrosine kinases and phosphoinositide 3-kinase (PI3K). Current studies indicate that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and PI3K help mediate inflammatory hyperalgesia. However, little is known about the role of PKB/Akt in the nociceptive system. In this study, we investigated whether PKB/Akt in primary sensory neurons is activated after noxious stimulation and contributes to pain behavior induced in rats by capsaicin. We demonstrated that phospho-PKB/Akt (p-PKB/Akt) is increased in dorsal root ganglia (DRG) at 5 min after intradermal injection of capsaicin. p-PKB/Akt is distributed predominantly in small- and medium-sized DRG cells. After capsaicin injection, p-PKB/Akt (473) is colocalized with isotectin-B4 (IB4), tyrosine kinase A (TrkA), and calcitonin gene-related peptide (CGRP). Furthermore, most transient receptor potential vanilloid type 1 (TRPV1) positive DRG neurons double label for p-PKB/Akt. Behavioral experiments show that intradermal injection of a PI3K (upstream of PKB/Akt) inhibitor, wortmannin, dose-dependently inhibits the changes in exploratory behavior evoked by capsaicin injection. The PKB/Akt inhibitor, Akt inhibitor IV, has the same effect. The results suggest that the PKB/Akt signaling pathway in the periphery is activated by noxious stimulation and contributes to pain behavior.

The effect of a kainate GluR5 receptor antagonist on responses of spinothalamic tract neurons in a model of peripheral neuropathy in primates.

The responses of antidromically identified spinothalamic tract (STT) neurons to mechanical and thermal stimuli were compared in anesthetized normal and neuropathic monkeys before and after administration of a GluR5 kainate receptor antagonist (LY382884) into the spinal cord dorsal horn through a microdialysis fiber. Peripheral neuropathy was induced by tight ligation of the L7 spinal nerve 13-15 days prior to the experiment. STT neurons recorded in the animals with neuropathy showed increased responsiveness to weak mechanical stimuli and to heating and cooling of the skin compared to STT cells in normal animals. In both normal and the neuropathic monkeys the responses of the STT neurons to mechanical and thermal stimuli were attenuated by LY382884 application in a concentration-dependent manner. Intraspinal application of LY382884 in the neuropathic animals led to a potent reduction of those responses of the STT neurons that were aggravated by the peripheral neuropathy (weak mechanical, heat and innocuous cooling stimuli). These results suggest that kainate receptors are involved in synaptic activation of STT cells in the normal state and may also play an important role in pathological pain states such as peripheral neuropathy in primates. Kainate receptor antagonists could thus be useful for the treatment of certain forms of allodynia and hyperalgesia.

Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin.

In a recent study, we have demonstrated that the dorsal root reflex (DRR)-mediated acute cutaneous neurogenic inflammation following intradermal injection of capsaicin (CAP) is sympathetically dependent and subject to modulation by peripheral alpha(1)-adrenoceptors. Postganglionic sympathetic neurons contain not only adrenergic neurotransmitters, but also non-adrenergic substances, including neuropeptide Y (NPY). In this study, we examined if peripheral NPY receptors participate in the flare following CAP injection. Different NPY receptor subtypes were studied by using relatively specific agonists and antagonists for the Y(1) and Y(2) subtypes. Changes in cutaneous blood flow on the plantar surface of the foot were measured using a laser Doppler flowmeter. Following CAP injection, cutaneous flare spread more than 20 mm away from the site of CAP injection. Removal of the postganglionic sympathetic nerves by surgical sympathectomy reduced dramatically the CAP-evoked flare. If the foot of sympathectomized rats was pretreated with either NPY or Y(2) receptor agonists by intra-arterial injection, the spread of flare induced by CAP injection could be restored and prolonged. However, if the spinal cord was pretreated with a GABA(A) receptor antagonist, bicuculline, to prevent DRRs, NPY or an Y(2) receptor agonist no longer restored the CAP-evoked flare. A Y(1) receptor agonist did not affect the CAP-evoked flare in sympathectomized rats. In sympathetically intact rats, blockade of either peripheral NPY or Y(2) receptors with [D-Trp(32)]-NPY or BIIE0246 markedly reduced the flare induced by CAP injection, whereas blockade of peripheral Y(1) receptors by BIBP3226 did not obviously affect the flare. It is suggested that NPY is co-released with NE from the postganglionic sympathetic terminals to activate NPY Y(2) and alpha(1) receptors following CAP injection. Both substances are involved, at least in part, in modulation of the responses of CAP sensitive afferents thereby affecting their ability to evoke the release of inflammatory agents from primary afferents.

Increased phosphorylation of the GluR1 subunit of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor in rats following intradermal injection of capsaicin.

Ionotropic glutamate receptors are ligand-gated ion channels that help mediate rapid excitatory neurotransmission in the CNS. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are critical for synaptic plasticity in central nociceptive transmission. The current study was designed to investigate the role of the AMPA receptor subunit, GluR1, and its phosphorylated forms (at Ser-831 and Ser-845) in central sensitization in rat spinal cord. Western blots and immunohistochemistry were performed to examine the expression and localization of GluR1 and the phosphorylated forms of GluR1 (phospho-GluR1) at Ser-831 and Ser-845 with specific antibodies. Results showed that immunolabeling of GluR1 protein in rat spinal cord can be detected at 110 kD, and two phospho-GluR1 proteins were found at 106 kD. A significant upregulation of phospho-GluR1 both at Ser-831 and Ser-845 was found by 5 min after capsaicin treatment, and this increase lasted at least 60 min. Immunostaining showed that GluR1 and its phosphorylated forms were localized in the superficial laminae of dorsal horn and quantitative image analysis supported the immunoblotting results. Our findings are consistent with the suggestions that AMPA receptors show increased responsiveness because of their phosphorylation and that this may contribute to central sensitization following intradermal injection of capsaicin.

Fos expression in spinothalamic and postsynaptic dorsal column neurons following noxious visceral and cutaneous stimuli.

The spinothalamic tract (STT) has been classically viewed as the major ascending pathway for pain transmission while the dorsal column (DC) was thought to be involved primarily in signaling innocuous stimuli. Recent clinical studies have shown that limited midline myelotomy, which transects fibers in the DC, offers good pain relief in patients with visceral cancer pain. Experimental studies provided evidence that a DC lesion decreases the activation of thalamic neurons by visceral stimuli and suggested that this effect is due to transection of the axons of postsynaptic dorsal column (PSDC) neurons. In our study, Fos protein expression in retrogradely labeled STT and PSDC neurons in the lumbosacral enlargement in rats was used as an anatomical marker of enhanced activation to compare the role of these neurons in cutaneous and visceral pain. The noxious stimuli used were intradermal injection of capsaicin and distention of the ureter. Retrogradely labeled PSDC neurons were found in laminae III-IV and in the vicinity of the central canal. STT neurons were located in laminae I, III-VII and X. Ureter distention evoked Fos expression in PSDC and STT neurons located in all laminae in which retrogradely labeled cells were found, with the maximum in the L(2) spinal segment. The Fos-positive PSDC neurons represented a significantly higher percentage of the retrogradely labeled PSDC neurons (19.3+/-2.3% SEM) than of the STT Fos-positive neurons (13.2+/-1.5% SEM). Intradermal capsaicin injection also evoked Fos expression in both PSDC and STT neurons, but with no significant difference between these two, when expressed as a percentage of the retrogradely labeled cells (11.6+/-2.9% SEM, 10.8+/-1.1% SEM). These results show that both PSDC and STT neurons are activated by cutaneous and visceral noxious stimuli. Their particular role in transmission and modulation of painful stimuli needs to be investigated further.

Serotonergic neural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons after spinal hemisection in rat.

Hemisection of the rat spinal cord at thoracic level 13 provides a model of spinal cord injury that is characterized by chronic pain attributable to hyperexcitability of dorsal horn neurons. Presuming that this hyperexcitability can be explained in part by interruption of descending inhibitory modulation by serotonin, we hypothesized that intrathecal transplantation of RN46A-B14 serotonergic precursor cells, which secrete serotonin and brain-derived neurotrophic factor, would reduce this hyperexcitability by normalizing the responses of low-threshold mechanoreceptive, nociceptive-specific, and multireceptive dorsal horn neurons. Three groups (n=45 total) of 30-day-old male Sprague-Dawley rats underwent thoracic level 13 spinal hemisection, after which four weeks were allowed for development of allodynia and hyperalgesia. The three groups of animals received transplants of no cells, 10(6) RN46A-V1 (vector-only) or 10(6) RN46A-B14 cells at lumbar segments 2-3. Electrophysiological experiments were done two weeks later. Low-threshold mechanoreceptive, nociceptive-specific, and multireceptive cells (n=394 total) were isolated at depths of 1-300 and 301-1000 micro in the lumbar enlargement. Responses to innocuous and noxious peripheral stimuli were characterized, and analyses of population responses were performed. Compared with normal animals, dorsal horn neurons of all types in hemisected animals showed increased responsiveness to peripheral stimuli. This was true for neurons on both sides of the spinal cord. After hemisection, the proportion of neurons classified as multireceptive cells increased, and interspike intervals of spontaneous discharges became less uniform after hemisection. Transplantation of RN46A-B14 cells restored evoked responses to near-control levels, normalized background activity, and returned the proportion of multireceptive cells to the control level. Restoration of normal activity was reversed with methysergide.These electrophysiological results corroborate anatomical and behavioral studies showing the effectiveness of serotonergic neural precursors in correcting phenomena associated with chronic central pain following spinal cord injury, and provide mechanistic insights regarding mode of action.

Postsynaptic dorsal column neurons express NK1 receptors following colon inflammation.

Recent clinical and experimental studies have suggested that the dorsal column pathway and specifically postsynaptic dorsal column neurons play an important role in the transmission of visceral pain. In our study we have mapped the distribution of postsynaptic dorsal column neurons in thoracic, lumbar and sacral spinal cord segments. The presence of immunoreactivity for neurokinin 1 receptors on these postsynaptic dorsal column neurons was examined under control conditions and after colon inflammation. The largest number of postsynaptic dorsal column neurons was found in the lumbar enlargement. They were mostly located in laminae III-IV, but depending on the spinal segment, about 7-15% of them were in the deep medial dorsal horn and in the central canal area. Under control conditions none of the 1438 postsynaptic dorsal column neurons examined expressed neurokinin 1 receptors. However, after induction of colon inflammation about 1.4% of the 2015 postsynaptic dorsal column neurons observed in the experimental group showed immunoreactivity for neurokinin 1 receptors. These neurons were preferentially found in the lower thoracic and lumbosacral spinal segments where they represented about 3-4% of the total population of postsynaptic dorsal column neurons examined. The de novo expression of neurokinin1 receptors on postsynaptic dorsal column neurons after colon inflammation suggests that substance P released from visceral primary afferents under inflammatory conditions could help produce central sensitization of these neurons.

Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats.

Protein phosphorylation is a major mechanism for regulation of N-methyl-D-aspartate (NMDA) receptor function. The NMDA receptor 1 subunit (NR1) is phosphorylated by protein kinase A (PKA) on serine 890 and 897. We have recently reported that there is enhanced phosphorylation of NR1 on serine 897 in dorsal horn and spinothalamic tract (STT) neurons after intradermal injection of capsaicin (CAP) in rats [Zou et al. (2000) J. Neurosci. 20, 6989-6997]. Whether or not this phosphorylation, which develops during central sensitization following CAP injection, is mediated by PKA remains to be determined. In this study, western blots and immunofluorescence staining were employed to observe if pretreatment with a PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H89), blocks the enhanced phosphorylation of NR1 on serine 897 following injection of CAP into the glabrous skin of one hind paw of anesthetized rats. Western blots showed that pretreatment with H89 caused a decrease in CAP-induced phosphorylation of NR1 protein in spinal cord segments L(4)-S(1). In experiments using immunofluorescence staining, the numbers of phospho-NR1-like immunoreactive (p-NR1-LI) neurons seen after CAP injection were significantly decreased in the dorsal horn of the L(4)-L(5) segments on the side ipsilateral to the injection after PKA was inhibited. When STT cells were labeled by microinjection of the retrograde tracer, fluorogold, we found that the proportion of p-NR1-LI STT cells on the side ipsilateral to the injection in the superficial laminae of spinal cord segments L(4)-L(5) was markedly reduced when H89 was administered intrathecally before CAP injection. However, the proportion of p-NR1-LI STT cells in deep laminae was unchanged unless the PKC inhibitor, chelerythrine chloride, was co-administered with H89. Combined with our previous findings, the present results indicate that NR1 in spinal dorsal horn neurons, including the superficial dorsal horn STT cells, is phosphorylated following CAP injection and that this phosphorylation is due to the action of PKA. However, the phosphorylation of deep STT cells involves both PKA and PKC.

Differential effects of muscimol upon the firing frequency of large and small amplitude antidromic dorsal root action potentials in rat spinal cord in vitro.

The effects of bath applied muscimol upon spontaneous and evoked antidromic activity recorded from lumbar dorsal roots was investigated in hemisected, isolated preparations of rat spinal cord. In magnesium free medium containing 0.1 microM 4-aminopyridine, bursts of high amplitude (up to 1 mV), dorsal root reflexes were recorded. These were blocked by low concentrations of muscimol (2-5 microM). Higher concentrations (5-20 microM) of muscimol caused a concentration-dependent increase in the frequency of small amplitude (<200 microV) spontaneous dorsal root action potentials. The possibility that the large and small amplitude extracellular action potentials reflect activity in large and small diameter dorsal root axons, and that these respond in different ways to the GABA(A) agonist muscimol, is discussed.

Cerebellar stimulation modulates the intensity of a visceral nociceptive reflex in the rat.

The cerebellum modulates different nociceptive phenomena and influences visceral functions. This study shows cerebellar modulation of an abdominal reflex elicited by a visceral noxious stimulus (colorectal distension, CRD). The intensity of the reflex was measured by electromyographic (EMG) recording from the rectus abdominus muscle, and the cerebellar cortex (vermis, lobule VIII), the fastigial nucleus, or the dentate nucleus was stimulated using D, L-homocysteic acid (0.1 M, 1 micro l). To release the fastigial nucleus from inhibition by the Purkinje cells, bicuculline (GABA(A) receptor antagonist, 100 micro M, 1 micro l) was used. Stimulation of the cerebellar cortex enhanced, whereas stimulation or disinhibition of the fastigial nucleus decreased, the responses to CRD measured by EMG. Stimulation of the dentate nucleus did not have an obvious effect on the intensity of the reflex. These results are in agreement with the hypothesis that the cerebellum modulates visceral nociceptive functions, whereby the cerebellar cortex and the fastigial nucleus, respectively, play a pro-nociceptive and an anti-nociceptive role.

Stimulation in the rat fastigial nucleus enhances the responses of neurons in the dorsal column nuclei to innocuous stimuli.

The cerebellum was recently proposed to play a role in cognition and sensation in addition to motor phenomena. We have shown that the cerebellum is involved in the processing of sensory nociceptive information. In this study, the activity of neurons in the dorsal column nuclei (DCN) was tested following stimulation in the rat fastigial nucleus. The results showed an enhancement of the extracellularly recorded responses of DCN neurons to somatic non-noxious stimuli following injection of D,L-homocysteic acid (0.1 M, 1 microl) into the area of the fastigial nucleus. We conclude that the cerebellum influences the processing of non-noxious somatosensory information at the level of the DCN, an important relay and a center for the processing of fine tactile and vibratory information. This observation is not yet supported by clinical data.

NMDA or non-NMDA receptor antagonists attenuate increased Fos expression in spinal dorsal horn GABAergic neurons after intradermal injection of capsaicin in rats.

GABAergic neurons play an important role in the generation of primary afferent depolarization, which results in presynaptic inhibition and, if large enough, triggers dorsal root reflexes. Recent electrophysiological studies by our group have suggested that increased excitation of spinal GABAergic neurons by activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors following intradermal injection of capsaicin results in the generation of DRRs that contribute to neurogenic inflammation. The present study was to determine if changes in the expression of Fos protein occur in GABAergic neurons in the lumbosacral spinal cord following injection of capsaicin into the glabrous skin of one hind paw of anesthetized rats and if pretreatment with an NMDA receptor antagonist, D-(-)-2-amino-7-phosphonoheptanoic acid (AP7) or a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocks Fos expression in these neurons. The experiments used western blots and immunofluorescence double labeling staining following capsaicin or vehicle injection. Western blots showed that Fos protein was increased on the ipsilateral side in spinal cord tissue 0.5 h after capsaicin injection. Pretreatment with AP7 or CNQX caused a decrease in capsaicin-induced Fos expression. Immunofluorescence double labeling showed that the proportion of Fos-positive GABAergic neuronal profiles was significantly increased following capsaicin injection (48.8+/-4.8%) compared to the vehicle injection (23.8+/-5.1%) in superficial laminae on the ipsilateral side in lumbosacral spinal cord (P<0.05). However, when the spinal cord was pretreated with AP7 (5 microg) or CNQX (0.2 microg), only 9.1+/-0.6% or 7.1+/-0.8% of GABA-immunoreactive neuronal profiles were stained for Fos following capsaicin injection. The blockade of the capsaicin-evoked Fos staining was dose-dependent. These findings suggest that GABAergic neurons take part in dorsal horn circuits that modulate nociceptive information and that the function of GABAergic neurons following capsaicin injection is partially mediated by NMDA and non-NMDA receptors.

Nitric oxide synthase in spinal cord central sensitization following intradermal injection of capsaicin.

Nitric oxide (NO) is believed to be an important messenger molecule in signal transduction pathways that enhance nociceptive transmission in the central nervous system (CNS). The role of nitric oxide synthase (NOS) I and II, which synthesize NO, in central sensitization induced by an intradermal capsaicin injection was investigated. To elucidate whether changes in NOS I and NOS II activities caused by capsaicin injection contribute to behavioral changes, responses to von Frey filaments with two different innocuous bending forces applied on the rat foot were tested. The allodynic responses induced by capsaicin injection in the foot were partially reversed by the administration of either the selective NOS I inhibitor, 7-nitroindazole (7-NINA), or the selective NOS II inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT). To confirm changes at the level of single nociceptive neurons, extracellular recordings were made from rat dorsal horn neurons. The electrophysiological results showed that increased responses to noxious and innocuous stimuli caused by capsaicin injection were blocked by either 7-NINA or AMT delivered through a microdialysis fiber inserted through the dorsal horn. Finally, the expression of both NOS I and NOS II in the spinal cord as demonstrated by Western blots was increased by 20 min following intradermal capsaicin injection in the rat foot. These results suggest that both NOS I and NOS II are upregulated following intradermal capsaicin injection and that both cause NO release that contributes to the secondary hyperalgesia and allodynia following this noxious chemical stimulus.

Nociceptive visceral stimulation modulates the activity of cerebellar Purkinje cells.

The cerebellum is a system with various input and output functions that influence motor, sensory, cognitive, and other processes. In a previous study, we showed that cerebellar cortical stimulation increases spinal neuronal responses to visceral noxious stimulation by colorectal distension (CRD). However, the neuronal network underlying the cerebellar modulation of nociceptive phenomena is largely unknown. Purkinje cells of the cerebellar cortex receive ascending and descending inputs and exert a major inhibitory control over neurons in the underlying cerebellar nuclei that constitute the cerebellar output. Therefore, in this study, we tested the effect of CRD and other somatic stimuli on the firing rate of Purkinje cells using in vivo extracellular recording techniques. The results suggest that Purkinje cells respond to nociceptive visceral and somatic stimulation in the form of early and delayed changes in activity. Based on these and previous findings, we propose a negative feedback circuitry involving the cerebellum for the modulation of peripheral nociceptive events.

GABA(A) and 5-HT(3) receptors are involved in dorsal root reflexes: possible role in periaqueductal gray descending inhibition.

The dorsal root reflex (DRR) is a measure of the central excitability of presynaptic inhibitory circuits in the spinal cord. Activation of the periaqueductal gray (PAG), a center for descending inhibition of spinal cord nociceptive transmission, induces release of variety of neurotransmitters in the spinal cord, including GABA and serotonin (5-HT). GABA has been shown to be involved in generation of DRRs. In this study, pharmacological agents that influence DRRs and their possible mechanisms were investigated. DRRs were recorded in anesthetized rats from filaments teased from the cut central stump of the left L(4) or L(5) dorsal root, using a monopolar recording electrode. Stimulating electrodes were placed either on the left sciatic nerve or transcutaneously in the left foot. Animals were paralyzed and maintained by artificial ventilation. Drugs were applied topically to the spinal cord. A total of 64 units were recorded in 34 Sprague-Dawley rats. Peripheral receptive fields were found for nine of these units. In these units, DRRs were evoked by brush, pressure, and pinch stimuli. Nine units were tested for an effect of electrical stimulation in the periaqueductal gray on the DRRs. In eight cases, DRR responses were enhanced following PAG stimulation. The background activity was 4.2 +/- 1.9 spikes/s (mean +/- SE; range: 0-97.7; n = 57). The responses to agents applied to the spinal cord were (in spikes/s): artificial cerebrospinal fluid, 7.1 +/- 3.6 (range: 0-86.9; n = 25); 0.1 mM GABA, 16.8 +/- 8.7 (range: 0-191.0; n = 22); 1.0 mM GABA, 116.0 +/- 26.5 (range: 0.05-1001.2; n = 50); and 1.0 mM phenylbiguanide (PBG), 68.1 +/- 25.3 (range: 0-1,073.0; n = 49). Bicuculline (0.5 mM, n = 27) and ondansetron (1.0 mM, n = 10) blocked the GABA and PBG effects, respectively (P < 0.05). Significant cross blockade was also observed. It is concluded that GABA(A) receptors are likely to play a key role in the generation of DRRs, but that 5-HT(3) receptors may also contribute. DRRs can be modulated by supraspinal mechanisms through descending systems.

Cerebellar cortical stimulation increases spinal visceral nociceptive responses.

The role of the cerebellum in modulating nociceptive phenomena is unclear. In this study, we focus on the effects of cerebellar cortical stimulation on the responses of midline neurons of the lumbosacral spinal cord to graded nonnoxious and noxious visceral (colorectal distension) as well as somatic (brush, pressure, pinch) stimuli. Extracellular recording was used for the isolation and recording of spinal nociceptive neurons, while electrical current pulses and chemical injection of D, L-homocysteic acid were used to stimulate the cortex of the posterior cerebellar vermis. Cerebellar cortical stimulation increased the responses of all isolated cells to colorectal distension, whereas the effect on the responses to somatic stimuli was variable. These findings indicate that the posterior cerebellar vermis may exert a pro-nociceptive effect on spinal visceroceptive neurons.

Projections from the marginal zone and deep dorsal horn to the ventrobasal nuclei of the primate thalamus.

It has been concluded recently that if a projection from the marginal zone to the ventral posterior lateral (VPL) nucleus exists, it is sparse. Given the importance of the marginal zone in nociception, this conclusion has raised doubts about the significance of the role of the ventrobasal complex in nociception. We have reexamined this projection using injections of the retrograde tracer, cholera toxin subunit B, into one side of the lateral thalamus in macaque monkeys. The injections were confined to the ventrobasal complex (with minimal spread to adjacent nuclei that do not receive spinal projections) in two animals. Many retrogradely labeled neurons were found in lamina I (as well as in lamina V) of the contralateral spinal and medullary dorsal horn. The results are consistent with the view that neurons in the marginal zone contribute prominently to the spinothalamic and trigeminothalamic projections to the VPL and ventral posterior medial (VPM) nuclei. This pathway is likely to be important for the sensory-discriminative processing of nociceptive information with respect to the location and intensity of painful stimuli.

Changes in dorsal horn neuronal responses in an experimental wrist contracture model.

Joint contracture, a major complication after casting, usually makes the therapeutic outcome worse by causing a limited range of motion and related pain. We developed rat models of wrist contracture with fracture of the radius (group A) and wrist contracture without fracture (group B), and investigated whether contracture and fracture changed the characteristics of cervical dorsal horn neuronal responses and the behavior of the animals. After 4 weeks of immobilization, both groups showed wrist contracture and disuse tendencies in the treated forelimb. In an electrophysiological study, the responses of 403 cervical dorsal horn neurons to mechanical stimuli were examined. In normal (control) animals, the neurons had the following distribution: 63% were low-threshold (LT); 15% were high-threshold (HT); and 22% were wide-dynamic-range (WDR). In group A, the distribution of the neurons changed to 51% LT, 16% HT, and 33% WDR. Similar changes were observed for group B. Responses during wrist movement were also examined. Forty-one percent of cells in the control group were responsive to the movements, whereas the number of neurons responding to motion stimulus in both groups A and B was increased, to 77%. The changed population of WDR and LT neurons responding to wrist movement suggests that the characteristics of dorsal horn neurons may undergo plastic changes after contracture.

Haploinsufficiency of protamine-1 or -2 causes infertility in mice.

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.

The role of the dorsal column pathway in visceral nociception.

Neurosurgeons have successfully used punctate midline myelotomy to relieve visceral cancer pain in human patients. Animal experiments demonstrate a visceral nociceptive pathway in the posterior column that is more effective than the spinothalamic tract in activating thalamic neurons, eliciting behavioral responses and triggering increases in regional cerebral blood flow. This visceral nociceptive pathway involves postsynaptic dorsal column neurons in the central, visceral processing region of the spinal cord. Axons from the sacral cord ascend near the midline and from the thoracic cord at the junction of the gracile and cuneate fasciculi.