Peripheral adenosine A2A receptors are involved in carrageenan-induced mechanical hyperalgesia in mice.

Here we studied the role of peripheral adenosine A(2A) receptors in mechanical hyperalgesia during inflammation using mice lacking the A(2A) receptors. Unilateral s.c. administration of the local inflammatory agent λ-carrageenan induced profound mechanical hyperalgesia 24 h after administration in the ipsilateral hind paw in wild-type mice. In homozygous mice lacking the A(2A) receptors, carrageenan-induced hyperalgesia was significantly reduced compared to wild type controls. The reduction in inflammatory hyperalgesia seen in A(2A) receptor knock-out mice was not associated with changes in paw edema. CGS 21680, a selective A(2A) receptor agonist, produced significantly more mechanical hyperalgesia in wild type females than in wild type males upon direct s.c. injection into the hindpaw whereas it had no effect upon systemic administration. The hyperalgesic effect of CGS 21680 was markedly reduced in the A(2A) knock-out mice of both sexes. Subcutaneous ZM-241,385, a selective A(2A) receptor antagonist, injected into the hindpaw reduced the mechanical hyperalgesia following carrageenan in female mice, but not in males. The results indicate that activation of peripheral adenosine A(2A) receptors during inflammation is associated with mechanical hyperalgesia, and that this effect is more prominent in females than in males.

Decreased inflammatory pain due to reduced carrageenan-induced inflammation in mice lacking adenosine A3 receptors.

Mice with a targeted disruption of adenosine A(3) receptor (A(3)AR) gene were assessed for their nociceptive threshold and for their localized inflammatory response following carrageenan injected into the hindpaw. Under basal conditions no difference was seen between A(3)AR knock-out (A(3)AR(-/-)) and wild-type (A(3)AR(+/+)) mice in nociceptive response to mechanical or heat stimuli. The antinociceptive response to the intrathecal adenosine analogue R-phenylisopropyl adenosine (R-PIA) was also unchanged in the A(3)AR(-/-) mice. In contrast, heat hyperalgesia, plasma extravasation and edema following carrageenan-induced inflammation in the hind paw were significantly reduced in A(3)AR(-/-) mice compared to the A(3)AR(+/+) controls. Thus, mice lacking A(3)AR had deficits in generating the localized inflammatory response to carrageenan, supporting a pro-inflammatory role of A(3)AR in peripheral tissues. However, no evidence for a role of A(3)AR in nociception and the antinociceptive effect of R-PIA was found.

Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor.

Caffeine is believed to act by blocking adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R), indicating that some A(1) receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A(1)R (A(1)R(-/-)). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A(1)R(+/+) mice, but A(1)R(-/-) mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A(1)R(-/-) mice. In A(1)R(+/-) mice the potency of adenosine was halved, as was the number of A(1)R. In A(1)R(-/-) mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A(1)Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.

Marked enhancement of anti-allodynic effect by combined intrathecal administration of the adenosine A1-receptor agonist R-phenylisopropyladenosine and morphine in a rat model of central pain.

BACKGROUND: There is often no satisfactory treatment for chronic pain after spinal cord injury. We have previously reported that intrathecal (i.t.) administration of the adenosine A1-receptor agonist R-phenylisopropyl-adenosine (R-PIA) or the opioid morphine has anti-allodynic effects in a model of presumed chronic central pain after photochemically induced spinal cord injury in rats. In the present study, we set out to investigate the possible interaction between i.t. R-PIA and morphine in spinally injured rats. METHODS: Sprague-Dawley rats displaying allodynia-like behaviors to mechanical and cold stimuli after photochemically induced spinal cord injury with minor motor deficits were used. R-PIA and morphine, either alone or in combination, were administered i.t. through an implanted catheter to lumbar spinal cord. RESULTS: Cumulative doses of R-PIA or morphine dose-dependently reduced the mechanical allodynia-like behavior, with a threshold of 1 nmol and 1.5 nmol, respectively. When co-administrated, R-PIA and morphine produced marked suppression of mechanical allodynia at doses of 5 pmol and 7.5 pmol, respectively. The effect of i.t. co-administration of R-PIA and morphine on cold allodynia was comparable to i.t. R-PIA alone. The combination of R-PIA and morphine did not increase adverse effects such as motor deficits in comparison to either drug alone. CONCLUSION: These results demonstrate a supra-additive interaction between the adenosine A1-receptor agonist R-PIA and morphine to reduce mechanical allodynia-like behavior in rats with chronic spinal cord injury. The combination of R-PIA and morphine administered spinally may be superior to R-PIA or morphine alone for treating such pain.

Intrathecal galanin alleviates allodynia-like behaviour in rats after partial peripheral nerve injury.

We have previously suggested that the neuropeptides galanin and galanin message-associated peptide (GMAP) may have an inhibitory role in spinal nociception. The present study examined the effects of intrathecal (i.t.) administration of these two peptides on allodynia-like behaviours in response to mechanical and cold stimulation in rats after photochemically induced ischaemic peripheral nerve injury. I.t. galanin significantly alleviated the mechanical- and cold-allodynia-like behaviours in nerve injured rats, and was not associated with motor impairment or sedation. I.t. GMAP relieved mechanical allodynia much less than galanin. I.t. M-35, a high-affinity galanin receptor antagonist, did not significantly alter the response of the rats to mechanical or cold stimulation. At 1 or 2 weeks postinjury, around 15% of dorsal root ganglion (DRG) neuron profiles showed galanin-like immunoreactivity. These profiles were mostly small sized. Although the number of galanin positive cells was thus increased in the DRG in the present model, the increase was substantially less than after complete sciatic nerve section, as previously shown. The present results showed that spinal administration of galanin inhibited some abnormal pain-like behaviours in rats after partial peripheral nerve injury. These results further support an inhibitory function for galanin in nociception. However, endogenous galanin may not play a significant role in suppressing nociceptive input after partial ischaemic peripheral nerve injury, as the upregulation of galanin is moderate.

Reduced anti-allodynic effect of the adenosine A1-receptor agonist R-phenylisopropyladenosine on repeated intrathecal administration and lack of cross-tolerance with morphine in a rat model of central pain.

UNLABELLED: We examined the development of tolerance to the antiallodynic effect of chronic intrathecal (IT) administration of the adenosine analog R-phenylisopropyladenosine (R-PIA) in a rat model of central pain after ischemic spinal cord injury. After 10 days of IT R-PIA treatment, the effect of IT morphine was also assessed to examine whether cross-tolerance between R-PIA and morphine was present. IT R-PIA completely alleviated allodynia-like behaviors to mechanical and cold stimuli in spinally injured rats. The anti-allodynic effect of R-PIA was maintained for 6-7 days with twice-daily administration and was reduced thereafter, particularly with respect to cold allodynia. IT morphine alleviated mechanical and cold allodynia in rats rendered tolerant to R-PIA to a degree comparable to that in R-PIA-naive (control) rats, which indicates that the anti-allodynic property of R-PIA is independent of the mechanisms by which morphine acts. The possibility of using agonists of adenosine receptors in treating refractory pain in patients with spinal cord injury is discussed. IMPLICATIONS: There is often no satisfactory treatment for chronic pain after spinal cord injury. Our study suggests such pain can be treated with a spinal injection of R-phenylisopropyladenosine in rats. Reduced effect to R-phenylisopropyladenosine was noted with repeated administrations. However, there was no cross-tolerance to morphine.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Intrathecal administration of the adenosine A1 receptor agonist R-phenylisopropyl adenosine reduces presumed pain behaviour in a rat model of central pain.

Effects of intrathecally (i.t.) administered R-phenylisopropyl adenosine (R-PIA), an adenosine A1 receptor agonist, on presumed pain behaviour were assessed in a rat model of chronic central pain. Spinal cord injury was induced photochemically via laser irradiation of the spinal cord after intravenous injection of erythrosin B in rats. The chronic allodynia-like behaviour that developed in some animals was studied. R-PIA (3 and 10 nmol), injected i.t. reduced the mechanical and cold allodynia-like symptoms as tested with von Frey filaments and ethyl-chloride spray, respectively. No side effects were observed. The effect of R-PIA was significant for up to 5 h and was reversed by theophylline, an adenosine receptor antagonist.

Nociceptive responses in interleukin-6-deficient mice to peripheral inflammation and peripheral nerve section.

The cutaneous nociceptive response threshold to mechanical and thermal stimulation, the development of hyperalgesia and plasma extravasation after subcutaneous injection of carrageenan and the development of autotomy behaviour after nerve section were assessed in interleukin-6-deficient (IL-6-/-) and age-matched wild-type (IL-6+/+) mice. IL-6-/- mice had significantly lower response threshold to both mechanical and thermal stimulation in comparison to IL-6+/+ controls. Both IL-6-/- and IL-6+/+ mice developed hyperalgesia to mechanical and thermal stimulation after localized carrageenan injection, but the magnitude of the hyperalgesia was less in the IL-6-/- than in the IL-6+/+ controls. IL-6-/- mice also exhibited less plasma extravasation after carrageenan injection. No difference was noted between males and females in basal nociception and inflammatory hyperalgesia. However, female IL-6-/- mice exhibited autotomy behaviour, a sign of neuropathic pain, significantly more frequently and after a shorter interval following peripheral nerve injury than male IL-6-/- or male and female IL-6+/+ mice. It is suggested that IL-6-/- mice exhibited numerous changes in nociceptive responses compared to controls, some of which are sex related. The mechanisms of these changes in relation to null-mutation of the IL-6 gene and the influence of genetic background are discussed. 1997 Academic Press Limited.

Intrathecal pituitary adenylate cyclase activating polypeptide facilitates the spinal nociceptive flexor reflex in the rat.

We have examined the effects of intrathecal (i.t.) pituitary adenylate cyclase activating polypeptide on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanaesthetized rats. The flexor reflex was elicited by electrical stimulation applied subcutaneously to the sural nerve innervation area and recorded as electromyogram activity from ipsilateral hamstring muscles. Pituitary adenylate cyclase activating polypeptide(l-27) was administered over a wide dose range (10 ng to 10 mu g) and elicited a dose-dependent facilitation of the flexor reflex and did not depress the reflex at any dose. Furthermore, pituitary adenylate cyclase activating polypeptide did not inhibit the facilitation of the flexor reflex induced by repetitive stimulation of C-fibres. It is concluded that pituitary adenylate cyclase activating polypeptide had an excitatory effect on spinal cord function which may indicate a role for this peptide in nociceptive transmission and modulation. Moreover, in contrast to previous studies, we found no evidence suggesting that pituitary adenylate cyclase activating polypeptide exerts antinociceptive action at spinal level.

Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study.

The effect of axotomy (3, 10 and 21 days) on the expression of some neuronal markers was analysed in dorsal root ganglia and spinal cord of guinea-pigs using immunohistochemistry. Three weeks following injury, substance P-like immunoreactivity (-LI) was slightly reduced in the DRGs of the ipsilateral side, whereas a marked increase in neuropeptide Y(NPY)-LI could be detected ipsilaterally and a smaller increase contralaterally. NPY-LI was mainly expressed in small, but also some medium-sized and large neuron profiles after axotomy. Galanin-LI showed a moderate bilateral increase. No significant changes could be observed in DRGs for calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- or nitric oxide synthase-LIs. In the ventral horn CGRP-LI was slightly increased bilaterally in motoneurons, most pronounced on the injured side. Autotomy behaviour was seen in seven of the nine animals in the twenty-one day group. The present results demonstrate that also in guinea-pigs several peptides undergo distinct changes in their expression after peripheral nerve injury. However, in contrast to rats and monkeys, galanin-LI is only moderately increased in guinea-pigs. Neuropeptide Y showed a dramatic increase mainly in small neurons, in contrast to the upregulation in large neurons in the rat. Thus, distinct species differences exist with regard to the cellular response to nerve injury.

Increased levels of GMAP, VIP and nitric oxide synthase, and their mRNAs, in lumbar dorsal root ganglia of the rat following systemic resiniferatoxin treatment.

Using in situ hybridization, the expression of mRNA encoding galanin, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and nitric oxide synthase (NOS), respectively, was studied in lumbar dorsal root ganglia of rats given a single s.c. dose of 300 micrograms kg-1 resiniferatoxin (RTX), an ultrapotent capsaicin analogue. In control animals, 10% of the DRG neurones were positive for galanin mRNA, whereas no message for VIP, NPY or NOS could be detected. One week after RTX treatment, a markedly increased number (approximately 30%) of the neurones expressed galanin mRNA. Simultaneously, VIP and NOS mRNA became detectable in 6-8% of the neurones. The number of galanin-positive neurones declined after 2 weeks and returned to control levels by 8 weeks. The increase in number of VIP-, or NOS-positive neurones persisted up to 4 weeks after RTX treatment and declined thereafter. Also, there was a small increase in NPY mRNA-positive neurones. In parallel immunohistochemical experiments, similar increases were observed for galanin message-associated protein (GMAP)-, VIP- and NOS-like immunoreactivities. Our findings suggest that RTX can cause changes (messenger plasticity) in galanin, VIP and NOS expression in capsaicin-sensitive sensory neurones of the rat, similar to those described following axotomy.

New high affinity peptide antagonists to the spinal galanin receptor.

1. The role of endogenous galanin in somatosensory processing has been studied with galanin receptor antagonists. The new galanin receptor ligands C7, M32, M38 and M40 bind with high affinity (Kd in nanomolar range) to spinal cord galanin receptors and possess oxidative stability as compared to earlier generations of peptide ligands. These peptides have been examined in the spinal flexor reflex model where exogenous galanin exhibited biphasic excitatory and inhibitory effects. 2. Intrathecal administration of C7 [galanin(1-13)-spantide] and M32 [galanin (1-13)-neuropeptide Y(25-36) amide] blocked facilitation of the nociceptive flexor reflex induced by 30 pmol intrathecal galanin in decerebrate, spinalized rats in a dose-dependent manner, thus behaving as antagonists of the galanin receptor. In contrast, M38[galanin(1-13)-(Ala-Leu)3-Ala amide] and M40 [galanin(1-13)-Pro-Pro-(Ala-Leu)2-Ala amide], exhibited only weak antagonism at high doses in this model. Moreover, lower doses of M40 potentiated galanin-induced reflex facilitation. C7 was neurotoxic at high doses in the rat spinal cord. 3. M32 and C7 were potent antagonists of galanin receptors in rat spinal cord, in correlation with their in vitro binding characteristics. In contrast, M38 and M40, despite their high in vitro affinity, exhibited only very weak antagonism. Moreover, M40 may also behave as a partial agonist. 4. Previous studies have shown that the galanin receptor may be heterogeneous. The discrepancy between in vitro binding and in vivo antagonistic potency of M38 and M40 may also suggest the presence of different galanin receptor subtypes within the rat spinal cord. However, other explanations for the discrepancy, such as differences in metabolic stability, diffusion rates and penetration to the site of action are also possible.

Effect of administration of high dose intrathecal clonidine or morphine prior to sciatic nerve section on c-Fos expression in rat lumbar spinal cord.

The effects of moderate and high intrathecal doses of clonidine, an alpha 2 adrenoceptor agonist, or a high dose of morphine on sciatic nerve section-induced expression of c-Fos-like immunoreactivity was studied in laminae I and II of the dorsal horn and laminae VIII and IX of the ventral horn of rat lumbar spinal cord. c-Fos-like immunoreactivity was examined by immunohistochemistry in normal rats (group 1), rats implanted with an intrathecal catheter with its tip on the lumbar spinal cord (group 2), injected with 10 micrograms (group 3) or 50 micrograms (group 4) clonidine intrathecally 3 h before being killed. In other groups, saline, 10 or 50 micrograms clonidine or 30 micrograms morphine was injected 1 h before unilateral nerve section, and the expression of c-Fos-like immunoreactivity was examined 2 h after axotomy. Few labeled neurons were found in normal controls. The intrathecal catheter itself caused a significant increase in bilateral c-Fos-like immunoreactivity in spinal dorsal and ventral horn compared to normals. The level of c-Fos-like immunoreactivity after 10 or 50 micrograms intrathecal clonidine was similar as in the intrathecal catheter group. Sciatic nerve section caused a significant ipsilateral increase in c-Fos-like immunoreactivity in the dorsal horn compared to the intact side in rats injected with saline. Pretreatment with 10 or 10 micrograms clonidine did not reduce sciatic nerve section-induced expression of c-Fos-like immunoreactivity, but instead caused a significant bilateral increase in c-Fos-like immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of intrathecal galanin message-associated peptide (GMAP) on the flexor reflex in rats.

We have examined the effects of intrathecal (i.t.) galanin message-associated peptide (GMAP), the C-terminal flanking peptide in the galanin (GAL) precursor protein, which is produced in equimolar quantities with galanin and which is upregulated upon axotomy, on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats. I.t. GMAP elicited a moderate facilitation of the flexor reflex. No depression of baseline flexor reflex was observed with any dose of GMAP. The facilitation of the flexor reflex induced by conditioning stimulation (CS) of cutaneous C-afferents was dose-dependently blocked by GMAP. The reflex facilitatory effect of exogenously applied substance P (SP), one of the endogenous modulators of reflex hyperexicitability following C-fiber CS, was only blocked by GMAP at a relatively high dose. I.t. GMAP did not antagonize the reflex facilitatory effect of vasoactive intestinal peptide and did not potentiate the reflex depressive effect of i.t. morphine or clonidine. Finally, 1 micrograms i.t. GMAP did not influence spinal cord blood flow whereas 10 micrograms GMAP induced a transient decrease in spinal cord blood flow in some experiments. The ability of GMAP to block the increase in spinal cord excitability following repetitive C-fiber stimulation may be through a presynaptic action. Although some of the effects of GMAP were similar to galanin, distinct differences were found, particularly in interaction with other excitatory and inhibitory agents. It is possible that GMAP exerts its action in the spinal cord through its own specific receptor. GMAP may act similarly to GAL in some, but not all pharmacological functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral axotomy increases the expression of galanin message-associated peptide (GMAP) in dorsal root ganglion cells and alters the effects of intrathecal GMAP on the flexor reflex in the rat.

We have previously reported that galanin message-associated peptide (GMAP), a fragment of galanin precursor protein, occurs in a limited number of dorsal root ganglion (DRG) cells in rats with intact sciatic nerves. In the present study, the localization of GMAP in dorsal root ganglia, dorsal roots and dorsal horn was analyzed immunohistochemically and compared between rats with intact and sectioned sciatic nerves. Furthermore, the effects of intrathecal (i.t.) GMAP on the flexor reflex in rats with intact and sectioned nerves were examined. In rats with intact sciatic nerves, i.t. GMAP elicited a moderate facilitation of the flexor reflex. The facilitation of the flexor reflex induced by conditioning stimulation (CS) of cutaneous C-fibers was strongly blocked by GMAP. GMAP also selectively antagonized the reflex facilitatory effect of i.t. substance P (SP), but not i.t. vasoactive intestinal peptide (VIP). Unilateral sciatic nerve section induced an upregulation of GMAP in the ipsilateral dorsal root ganglia 2 weeks after axotomy. The effect of GMAP on the baseline reflex was similar in normal and axotomized rats, but the blocking effect of GMAP on C-fiber CS-induced facilitation was significantly reduced after axotomy. GMAP did not antagonize the reflex facilitatory effect of SP after axotomy, whereas an antagonism on VIP-induced facilitation was observed. The possible role of GMAP in spinal transmission and comparison with the effects of galanin are discussed.

The effects of pretreatment with tachykinin antagonists and galanin on the development of spinal cord hyperexcitability following sciatic nerve section in the rat.

The effects of acute section of the sciatic nerve on the excitability of the flexor reflex was examined in decerebrate, spinalized, unanaesthetized rats. In control experiments without drugs, the excitability of the flexor reflex was dramatically increased in two phases following axotomy. An early intense, brief reflex hyperexcitability was followed by a less intense, prolonged period of facilitation. The selective NK1 tachykinin receptor antagonist CP-96,345 injected intrathecally at lower (1.2-2.4 nmol) and higher (12 nmol) doses blocked both components of spinal sensitization. The selective NK2 tachykinin receptor antagonist Men 10376 at a dose of 2.4 nmol also reduced both response components, as did the same dose of the inhibitory neuropeptide galanin. Thus, antagonists of excitatory neuropeptides released during and after nerve section, such as substance P and neurokinin A, can block the spinal response to peripheral nerve injury. Furthermore, the inhibitory neuropeptide galanin also reduced spinal cord sensitization.

Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy.

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I-IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae I-II. [125I]PYY binding was most strongly attenuated by NPY13-36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13-36 completely abolished PYY binding, which was also reduced by [Leu31,Pro34] NPY. However, the increase in PYY binding seen in laminae I-IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31,Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I-IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons.

In this study the actions of NGF in regulating peptide expression were examined in vivo in adult rat primary sensory neurons. The hypothesis that NGF might tonically inhibit expression of some peptides was tested specifically. In situ hybridization and immunohistochemistry were used to detect presence or absence of alpha-CGRP, beta-CGRP, SP, SOM, VIP, CCK, NPY, and GAL as well as their mRNAs. In neurons in normal lumbar DRG alpha-CGRP, beta-CGRP, SP, and SOM are abundantly and heterogeneously expressed whereas few neurons have detectable VIP, CCK, NPY, or GAL. Two weeks following sciatic nerve transection, concentrations of alpha-CGRP, beta-CGRP, SP, and SOM plus their mRNAs have decreased to background in all but a few neurons. In contrast, VIP, CCK, NPY, and GAL are now synthesized in many neurons. Delayed intrathecal infusion of NGF (125 ng/microliter/hr) for 7 d, starting 2 weeks after injury counteracted the decrease in expression of alpha-CGRP, beta-CGRP and SP expression, but not SOM. This lack of influence of NGF on SOM is consistent with the absence of high-affinity NGF receptors and trk mRNA in SOM-positive neurons. Delayed infusion of NGF also reduced the number of neurons expressing VIP, CCK, NPY, and GAL after injury by approximately one-half in each subpopulation. Therefore, we suggest that NGF suppresses expression of these four peptides but only if the neurons also have NGF receptors. The results show that NGF can regulate peptide expression differentially and may also be part of the signal that allows reversion to normal of responses to injury as axons regenerate.

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord--IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/peptide histidine isoleucine.

Using immunofluorescence histochemistry and pre- and post-embedding immunoelectron microscopy the rat lumbar dorsal horn was analysed in normal rats and 14 days after unilateral transection of the sciatic nerve. A marked increase in neuropeptide Y-like immunoreactivity was observed in the ipsilateral, superficial dorsal horn, especially in laminae III and IV, of the lumbar 4-5 spinal cord segments after peripheral axotomy. In the ipsilateral lamina II two types of neuropeptide Y-immunoreactive, presumably primary afferent terminals could be identified at the ultrastructural level. The first type contained many large dense-core vesicles (100-155 nm in diameter), whereas a second, more common type had only a few and smaller large dense-core vesicles (80-100 nm in diameter), plus synaptic vesicles of varying diameter (50-85 nm), large empty vesicles and tubular structures. Only occasionally were neuropeptide Y-positive terminals in lamina II involved in the formation of axonal labyrinths. In the ipsilateral lamina III, the number of neuropeptide Y-positive nerve terminals markedly increased after axotomy, with a moderate increase in lamina IV. These neuropeptide Y-positive terminals were morphologically similar to the second type of neuropeptide Y-positive terminal in lamina II, i.e. contained many synaptic vesicles (45-50 nm in diameter), a few small large dense-core vesicles (80-100 nm in diameter), electron-dense granular matrix and a few tubular structures. Fusion of synaptic vesicles with the plasma membrane was often observed at these synapses. These terminals frequently formed glomeruli but were not involved in axonal labyrinths. With regard to local neurons, neuropeptide Y-like immunoreactivity was observed in many dendrite-like profiles mostly making synaptic contacts with neuropeptide Y-negative dendrites and only rarely contacting the central terminal of the glomeruli. Neuropeptide Y-positive nerve endings were mainly seen in lamina I and the outer third of lamina II. After peripheral axotomy the number of vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive terminals was increased in laminae I and II. They contained many large dense-core vesicles (100-120 nm in diameter), and some of them were positive for vasoactive intestinal polypeptide/peptide histidine isoleucine. Morphologically, the terminals were characterized by a granular matrix, tubular structures, empty vesicles, reduction in synaptic vesicles and absence of postsynaptic densities. Vasoactive intestinal polypeptide/peptide histidine isoleucine-like immunoreactivities were often found in association with labyrinth formation.(ABSTRACT TRUNCATED AT 400 WORDS)