The relationship of bone-tumor-induced spinal cord astrocyte activation and aromatase expression to mechanical hyperalgesia and cold hypersensitivity in intact female and ovariectomized mice.

Recently, our group established a relationship between tumor-induced spinal cord astrocyte activation and aromatase expression and the development of bone tumor nociception in male mice. As an extension of this work, we now report on the association of tumor-induced mechanical hyperalgesia and cold hypersensitivity to changes in spinal cord dorsal horn GFAP and aromatase expression in intact (INT) female mice and the effect of ovariectomy on these parameters. Implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in INT animals, while ovariectomized (OVX) females had significantly less mechanical hyperalgesia. Cold hypersensitivity was apparent by post-implantation day 7 in INT and OVX females compared to their saline-injected controls and increased throughout the experiment. The decrease in mechanical hyperalgesia in OVX females was mirrored by significant decreases in spinal astrocyte activity in laminae I-II, III-IV, V-VI and X and aromatase expression in laminae V-VI and X in the dorsal horn of tumor-bearing animals. Administration of the aromatase inhibitor letrozole reduced tumor-induced hyperalgesia in INT females only suggesting that the tumor-induced increase in aromatase expression and its associated increase in spinal estrogen play a role in the development of bone tumor-induced hyperalgesia. Finally, intrathecal (i.t.) administration of 17ß-estradiol caused a significant increase in tumor-induced hyperalgesia in INT tumor-bearing females. Since i.t. 17ß-estradiol increases tumor pain and ovariectomy significantly decreases tumor pain, as well as spinal aromatase, estrogen may play a critical role in the spinal cord response to the changing tumor environment and the development of tumor-induced nociception.

Role of peripheral sigma-1 receptors in ischaemic pain: Potential interactions with ASIC and P2X receptors.

BACKGROUND: The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. METHODS: We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naïve rats. RESULTS: Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0-3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3-6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naïve rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αßmeATP. CONCLUSION: Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain.

Colocalization of aromatase in spinal cord astrocytes: differences in expression and relationship to mechanical and thermal hyperalgesia in murine models of a painful and a non-painful bone tumor.

While spinal cord astrocytes play a key role in the generation of cancer pain, there have been no studies that have examined the relationship of tumor-induced astrocyte activation and aromatase expression during the development of cancer pain. Here, we examined tumor-induced mechanical hyperalgesia and cold allodynia, and changes in Glial fibrillary acid protein (GFAP) and aromatase expression in murine models of painful and non-painful bone cancer. We demonstrate that implantation of fibrosarcoma cells, but not melanoma cells, produces robust mechanical hyperalgesia and cold allodynia in tumor-bearing mice compared to saline-injected controls. Secondly, this increase in mechanical hyperalgesia and cold allodynia is mirrored by significant increases in both spinal astrocyte activity and aromatase expression in the dorsal horn of fibrosarcoma-bearing mice. Importantly, we show that aromatase is only found within a subset of astrocytes and not in neurons in the lumbar spinal cord. Finally, administration of an aromatase inhibitor reduced tumor-induced hyperalgesia in fibrosarcoma-bearing animals. We conclude that a painful fibrosarcoma tumor induces a significant increase in spinal astrocyte activation and aromatase expression and that the up-regulation of aromatase plays a role in the development of bone tumor-induced hyperalgesia. Since spinal aromatase is also upregulated, but to a lesser extent, in non-painful melanoma bone tumors, it may also be neuroprotective and responsive to the changing tumor environment.

σ1 receptors activate astrocytes via p38 MAPK phosphorylation leading to the development of mechanical allodynia in a mouse model of neuropathic pain.

BACKGROUND AND PURPOSE: Spinal astrocytes have emerged as important mechanistic contributors to the genesis of mechanical allodynia (MA) in neuropathic pain. We recently demonstrated that the spinal sigma non-opioid intracellular receptor 1 (σ1 receptor) modulates p38 MAPK phosphorylation (p-p38), which plays a critical role in the induction of MA in neuropathic rats. However, the histological and physiological relationships among σ1, p-p38 and astrocyte activation is unclear. EXPERIMENTAL APPROACH: We investigated: (i) the precise location of σ1 receptors and p-p38 in spinal dorsal horn; (ii) whether the inhibition of σ1 receptors or p38 modulates chronic constriction injury (CCI)-induced astrocyte activation; and (iii) whether this modulation of astrocyte activity is associated with MA development in CCI mice. KEY RESULTS: The expression of σ1 receptors was significantly increased in astrocytes on day 3 following CCI surgery. Sustained intrathecal treatment with the σ1 antagonist, BD-1047, attenuated CCI-induced increase in GFAP-immunoreactive astrocytes, and the treatment combined with fluorocitrate, an astrocyte metabolic inhibitor, synergistically reduced the development of MA, but not thermal hyperalgesia. The number of p-p38-ir astrocytes and neurons, but not microglia was significantly increased. Interestingly, intrathecal BD-1047 attenuated the expression of p-p38 selectively in astrocytes but not in neurons. Moreover, intrathecal treatment with a p38 inhibitor attenuated the GFAP expression, and this treatment combined with fluorocitrate synergistically blocked the induction of MA. CONCLUSIONS AND IMPLICATIONS: Spinal σ1 receptors are localized in astrocytes and blockade of σ1 receptors inhibits the pathological activation of astrocytes via modulation of p-p38, which ultimately prevents the development of MA in neuropathic mice.

Suppression of adrenal gland-derived epinephrine enhances the corticosterone-induced antinociceptive effect in the mouse formalin test.

BACKGROUND: There is both clinical and experimental evidence to support the application of corticosterone in the management of inflammation and pain. Corticosterone has been used to treat painful inflammatory diseases and can produce antinociceptive effects. Epinephrine is synthesized from norepinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT) and works as an endogenous adrenoceptor ligand secreted peripherally by the adrenal medulla. It is currently unclear whether corticosterone's antinociceptive effect is associated with the modulation of peripheral epinephrine. METHODS: We first determined whether exogenous corticosterone treatment actually produced an antinociceptive effect in a formalin-induced pain model, and then examined whether this corticosterone-induced antinociceptive effect was altered by suppression of adrenal-derived epinephrine, using the following three suppression methods: (1) inhibition of the PNMT enzyme; (2) blocking peripheral epinephrine receptors; and (3) adrenalectomy. RESULTS: Exogenous treatment with corticosterone at a high dose (50 mg/kg), but not at lower doses (5, 25 mg/kg), significantly reduced pain responses in the late phase. Moreover, injection of 2,3-dichloro-a-methylbenzylamine, a PNMT enzyme inhibitor, (10 mg/kg) before corticosterone treatment caused a leftward shift in the dose-response curve for corticosterone and injection of propranolol (5 mg/kg), but not phentolamine, also shifted the dose-response curve to the left during the late phase. Chemical sympathectomy with 6-hydroxydopamine had no effect on corticosterone-induced antinociceptive effect, but injection of a low dose of corticosterone produced an antinociceptive effect in adrenalectomized animals. CONCLUSIONS: These results demonstrate that suppression of epinephrine, derived from adrenal gland, enhances the antinociceptive effect of exogenous corticosterone treatment in an inflammatory pain model.

Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice.

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

Nociceptive characteristics of tumor necrosis factor-alpha in naive and tumor-bearing mice.

A nociceptive role for tumor necrosis factor-alpha (TNF-alpha) in naive mice and in mice with fibrosarcoma tumor-induced primary hyperalgesia was investigated. The presence of TNF-alpha mRNA was confirmed in tumor site homogenates by reverse transcription-polymerase chain reaction (RT-PCR), and examination of TNF-alpha protein levels in tumor-bearing mice indicated a significantly higher concentration of this cytokine in tumor microperfusates and tumor site homogenates compared with that obtained from a similar site on the contralateral limb or in naive mice. Intraplantar injection of TNF-alpha into naive or fibrosarcoma tumor-bearing mice induced mechanical hypersensitivity, as measured by withdrawal responses evoked by von Frey monofilaments. This hypersensitivity suggests that TNF-alpha can excite or sensitize primary afferent fibers to mechanical stimulation in both naive and tumor-bearing mice. In addition, the hyperalgesia produced by TNF-alpha was completely eliminated when the injected TNF-alpha was pre-incubated with the soluble receptor antagonist TNFR:Fc. Importantly, pre-implantation systemic as well as post-implantation intra-tumor injection of TNFR:Fc partially blocked the mechanical hyperalgesia, indicating that local production of TNF-alpha may contribute to tumor-induced nociception.

The anti-inflammatory effect of bee venom stimulation in a mouse air pouch model is mediated by adrenal medullary activity.

Cutaneous electrical or chemical stimulation can produce an anti-inflammatory effect, which is dependent on adrenal medullary-sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti-inflammatory effect that is neurally mediated. In the present study, we examined whether this anti-inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)-alpha concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF-alpha elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan-induced air-pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti-inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV-induced inhibition of leucocyte migration. By contrast, pretreatment with the beta-adrenergic antagonist propranolol reversed the BV-induced inhibitory effect on leucocyte migration. These results suggest that the anti-inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.

Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain.

This paper describes a model of tumor-induced bone destruction and hyperalgesia produced by implantation of fibrosarcoma cells into the mouse calcaneus bone. Histological examination indicates that tumor cells adhere to the bone edge as early as post-implantation day (PID) 3, but osteolysis does not begin until PID 6, correlating with the development of hyperalgesia. C3H/He mice exhibit a reproducible hyperalgesia to mechanical and cold stimuli between PID 6 and 16. These behaviors are present but significantly reduced with subcutaneous implantation that does not involve bone. Systemic administration of morphine (ED(50) 9.0 mg/kg) dose-dependently attenuated the mechanical hyperalgesia. In contrast, bone destruction and hypersensitivity were not evident in mice implanted with melanoma tumors or a paraffin mass of similar size. A novel microperfusion technique was used to identify elevated levels of the putative algogen endothelin (ET) in perfusates collected from the tumor sites of hyperalgesic mice between PID 7 and 12. Increased ET was evident in microperfusates from fibrosarcoma tumor-implanted mice but not from melanoma tumor-implanted mice, which are not hyperalgesic. Intraplantar injection of ET-1 in naive and, to a greater extent, fibrosarcoma tumor-bearing mice produced spontaneous pain behaviors, suggesting that ET-1 activates primary afferent fibers. Intraplantar but not systemic injection of the ET-A receptor antagonist BQ-123 partially blocked tumor-associated mechanical hyperalgesia, indicating that ET-1 contributes to tumor-induced nociception. This model provides a unique approach for quantifying the behavioral, biochemical, and electrophysiological consequences of tumor-nerve interactions.

Transtympanic tetrodotoxin alters the VOR and Fos labeling in the vestibular complex.

The sodium channel blocker tetrodotoxin (TTX) is an effective tool for blockade of action potentials. Unilateral transtympanic administration of 3 mM TTX produced behavioral symptoms similar to those following unilateral peripheral vestibular ablation. Complete resolution of visible symptoms occurred between 48 and 72 h post-TTX. Eye-coil recordings indicated a spontaneous nystagmus and a decrease in the VOR in TTX-treated animals. Neuronal activity in the central vestibular complex (VC), as monitored with Fos immunocytochemistry, revealed an asymmetric pattern of Fos labeling in the medial, inferior and superior vestibular nuclei and the prepositus hypoglossal nucleus. Although the spatio-temporal pattern of Fos labeling was consistent and reproducible at each time-point, changes were noted among time-points. Transient blockade with TTX may be useful for studying the central vestibular response to recurrent or episodic vestibular disruption in the intact system.

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

The analgesic efficacy of bee venom acupuncture for knee osteoarthritis: a comparative study with needle acupuncture.

The aim of this investigation was to determine whether bee venom (BV) administered directly into an acupoint was a clinically effective and safe method for relieving the pain of patients with knee osteoarthritis (OA) as compared to traditional needle acupuncture. We evaluated the efficacy of BV acupuncture using both pain relief scores and computerized infrared thermography (IRT) following 4 weeks of BV acupuncture treatment. We observed that a significantly higher proportion of subjects receiving BV acupuncture reported substantial pain relief as compared with those receiving traditional needle acupuncture therapy. Furthermore, the IRT score was significantly improved and paralleled the level of pain relief.

Visceral antinociception produced by bee venom stimulation of the Zhongwan acupuncture point in mice: role of alpha(2) adrenoceptors.

The goal of the present study was to determine whether bee venom (BV) injection into the Zhongwan acupoint (CV12), compared to injection into a non-acupoint, produced antinociception in an acetic acid-induced visceral pain model. This was accomplished by injecting BV subcutaneously into the Zhongwan acupoint or into a non-acupoint 30 min before intraperitoneal injection of acetic acid in ICR mice. BV injection into the acupoint produced a dose dependent suppression of acetic acid-induced abdominal stretches and of acetic acid-induced Fos expression in the spinal cord and the nucleus tractus solitarii. In contrast BV injection into the non-acupoint only produced antinociception at the highest dose of BV tested. Naloxone pretreatment did not alter the antinociceptive effect of BV acupoint injection on the abdominal stretch reflex. On the other hand, pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine completely blocked the antinociceptive effect of BV acupoint injection. These results imply that BV acupoint stimulation can produce visceral antinociception that is associated with activation of alpha 2-adrenoceptors, but not with naloxone-sensitive opioid receptors.

Antinociceptive effects of bee venom acupuncture (apipuncture) in rodent animal models: a comparative study of acupoint versus non-acupoint stimulation.

From a clinical perspective, the alternative forms of acupoint stimulation including electroacupuncture, moxibustion and acupressure appear to have more potent analgesic effects than manual needle acupuncture. Bee venom (BV) injection has also been reported to produce persistent nociceptive stimulation and to cause neuronal activation in the spinal cord. In previous study, we observed that BV stimulation into acupoint, namely BV acupuncture or Apipuncture, produced more potent anti-inflammatory and antinociceptive potency in rodent arthritis model as comparing with that of non-acupoint injection. Based on previous report, we decided to further investigate that BV injection into an acupoint produces antinociception as a result of its potent chemical stimulatory effect in both abdominal stretch assay and formalin test. Different doses of BV were injected into an acupoint or a non-acupoint 30 min prior to intraplantar formalin injection or intraperitoneal acetic acid injection. Using the abdominal stretch assay, we found that the high dose of BV (1:100 diluted in 20microl saline) produced a potent antinociceptive effect irrespective of the site of BV injection. In contrast the antinociceptive effect observed in both the writhing and formalin tests following administration of a low dose of BV (1:1000 diluted in 20microl saline) was significantly different between acupoint and non-acupoint sites. BV injection into an acupoint (Zhongwan, Cv. 12) was found to produce significantly greater antinociception than non-acupoint injection (10 mm from Zhongwan, Cv. 12) in the abdominal stretch assay. Similarly, in the formalin test, acupoint (Zusanli, St. 36) injection of BV produced more potent antinociception than non-acupoint injection (gluteal muscle). In contrast, BV injection into an arbitrary non-acupoint site on the back did not produce antinociception in either the writhing or formalin test. These results indicate that BV injection directly into an acupoint can produce a potent antinociceptive effect and suggest that this alternative form of acupoint stimulation (Apipuncture) may be a promising method for the relief of pain.

Bee venom pretreatment has both an antinociceptive and anti-inflammatory effect on carrageenan-induced inflammation.

Although the injection of bee venom (BV) has been reported to evoke tonic pain and hyperalgesia, there is conflicting evidence in the literature indicating that BV can also exert an anti-inflammatory and antinociceptive effects on inflammation. In this regard, BV has been traditionally used in Oriental medicine to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis. The present study was designed to test the hypothesis that BV induces acute nociception under normal conditions, but that it can serve as a potent anti-inflammatory and antinociceptive agent in a localized inflammatory state. The experiments were designed to evaluate the effect of BV pretreatment on carrageenan (CR)-induced acute paw edema and thermal hyperalgesia. In addition, spinal cord Fos expression induced by peripheral inflammation was quantitatively analyzed. In normal animals subcutaneous BV injection into the hindlimb was found to slightly increase Fos expression in the spinal cord without producing detectable nociceptive behaviors or hyperalgesia. In contrast pretreatment with BV (0.8 mg/kg) 30 min prior to CR injection suppressed both the paw edema and thermal hyperalgesia evoked by CR. In addition, there was a positive correlation between the percent change in paw volume and the expression of Fos positive neurons in the spinal cord. These results indicate that BV pretreatment has both antinociceptive and anti-inflammatory effects in CR-induced inflammatory pain. These data also suggest that BV administration may be useful in the treatment of the pain and edema associated with chronic inflammatory diseases.

Functional interactions between tumor and peripheral nerve in a model of cancer pain in the mouse.

Cancer is usually accompanied by pain, which tends to increase in relation to metastatic infiltration and destruction. In the United States, 30% to 40% of newly diagnosed cancer patients and 67% to 90% of patients with advanced cancer report moderate to severe pain. Relief for approximately 90% of patients with cancer-related pain may be provided by the World Health Organization's "analgesic ladder," which involves progressing from non-opioid (e.g., acetaminophen, ibuprofen) to weak opioid (e.g., codeine), to strong opioid (e.g., morphine, fentanyl) intervention for pain relief. The severity of cancer pain is affected by diverse factors. In addition to the obvious factors of tumor size and degree of metastatic destruction, the type of tumor and its location are also important factors that contribute to pain severity. Severe cancer pain is especially associated with tumors involving bone destruction and nerve infiltration. Cancer pain seems to involve diverse mechanisms, including characteristics of both nociceptive and neuropathic pain. Unfortunately, even opioid analgesics often produce poor pain relief against neuropathic pain derived from peripheral nerve or root damage common to cancers involving bone metastases and nerve infiltration. In addition, these drugs may induce adverse side effects since they affect various physiological functions, including hormone secretion, neurotransmitter release, feeding, gastrointestinal motility, and respiratory activity. Currently, drug therapies utilizing antidepressants and anticonvulsants are being used to relieve neuropathic pain whereas cancer pain is treated largely with opiods in cancer patients.

Stellate neurons mediate functional hyperemia in the cerebellar molecular layer.

Mice lacking cyclin D2 have a profound reduction in the number of stellate neurons in the cerebellar molecular layer. We used cyclin D2-null mice to study the contribution of stellate neurons in the increase of cerebellar blood flow (BFcrb) produced by neural activation. Crus II, a region of the cerebellar cortex that receives trigeminal sensory afferents, was activated by stimulation of the upper lip (5-30 V; 10 Hz), and BFcrb was recorded at the activated site by the use of a laser-Doppler flow probe. In wild-type mice, upper lip stimulation increased BFcrb in crus II by 32 +/- 2%. The rise in BFcrb was attenuated by 19% in heterozygous mice and by 69% in homozygous mice. In contrast to the cerebellum, the increases in somatosensory cortex blood flow produced by upper lip stimulation was not attenuated in D2-null mice. The field potentials evoked in crus II by upper lip stimulation did not differ between wild-type and D2-null mice. Stellate neurons are a major source of nitric oxide (NO) in the cerebellar molecular layer. The neuronal NO synthase inhibitor 7-nitroindazole attenuated the vascular response to crus II activation in wild-type mice but not in D2-null mice, suggesting that stellate neurons are the major source of NO mediating the vascular response. The data provide evidence that stellate neurons are a critical link between neural activity and blood flow in the activated cerebellum and that NO is the principal effector of their vascular actions.

The normal distribution and projections of constitutive NADPH-d/NOS neurons in the brainstem vestibular complex of the rat.

The vestibular system is a highly conserved sensory system in vertebrates that is largely responsible for maintenance of one's orientation in space, posture, and balance and for visual fixation of objects during motion. In light of the considerable literature indicating an involvement of nitric oxide (NO) in sensory systems, it is important to determine whether NO is associated with vestibular pathways. To study the relationship of NO to vestibular pathways, we first examined the normal distribution of constitutive NADPH-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), in the vestibular complex (VC) and then examined its association with selected vestibular projection neurons. Survey of the four major vestibular nuclei revealed that only the medial vestibular nucleus contained significant numbers of perikarya stained for NADPH-d/NOS. By contrast, all the vestibular nuclei contained a network of fine processes that stained positive for NADPH-d, although the density of this network varied among the individual nuclei. To determine whether NADPH-d/NOS neurons project to vestibular efferent targets, injections of the retrograde tracer Fluoro-Gold were made into known targets of second-order vestibular neurons. Vestibular neurons containing constitutive NADPH-d/NOS were found to project predominantly to the oculomotor nucleus. A small number of neurons also participate in vestibulothalamic and intrinsic vestibular connections. These results indicate that NADPH-d/NOS neurons are prevalent in the MVN and that a subpopulation of these neurons project to the oculomotor complex. Nitric oxide is probably released locally from axons located throughout the vestibular complex but may play a particularly important role in vestibulo-ocular pathways.

Catecholaminergic, cholinergic and peptidergic innervation of gut-associated lymphoid tissue in porcine jejunum and ileum.

With its abundance of neurons and immunocytes, the gut is a potentially important site for the study of the interaction between the nervous and immune systems. Using immunohistochemical techniques, we tested the hypothesis that gut-associated lymphoid tissue in the porcine small intestine might receive catecholaminergic, cholinergic and peptidergic innervation. Antibodies against protein gene product (PGP) 9.5 were employed to detect neuronal membranes; antibodies against tyrosine hydroxylase (TH), type 2 vesicular monoamine transporter (VMAT-2) and choline acetyltransferase (ChAT) were used to detect catecholaminergic and cholinergic neurons; and antibodies to neuromedin U-8 (NMU-8), substance P (SP) and vasoactive intestinal peptide (VIP) were also used. PGP9.5-immunoreactive nerve fibers were observed between jejunal Peyer's patch (PP) follicles and in submucosal ganglia localized at the base of continuous ileal PP. Many ChAT-positive and a few TH-/VMAT-2-immunoreactive neurons or axons adjacent to jejunal and ileal PP were observed. Neurons and fibers from ganglia situated between or at the base of PP follicles manifested robust immunoreactivities to VIP and NMU-8; relatively less SP immunoreactivity was observed at these locations. All neuromedin-U 8-positive neurons observed exhibited immunoreactivity to ChAT as did some VIP-positive neurons. The specific chemical coding of enteric neurons in close apposition to jejunal and ileal PP and the differential localization of neuropeptides within the jejunal and ileal PP are indicative of neuroimmunomodulation at these sites.