Frequency stabilization of an optically pumped far-infrared laser to the harmonic of a microwave synthesizer.

Measurements of the frequency stability of a far-infrared molecular laser have been made by mixing the harmonic of an ultrastable microwave source with a portion of the laser output signal in a terahertz (THz) Schottky diode balanced mixer. A 3 GHz difference-frequency signal was used in a frequency discriminator circuit to lock the laser to the microwave source. Comparisons of the short- and long-term laser frequency stability under free-running and locked conditions show a significant improvement with locking. Short-term frequency jitter was reduced by an order of magnitude, from approximately 40 to 4 kHz, and long-term drift was reduced by more than three orders of magnitude, from approximately 250 kHz to 80 Hz. The results, enabled by the efficient Schottky diode balanced mixer downconverter, demonstrate that ultrastable microwave-based frequency stabilization of THz optically pumped lasers (OPLs) will now be possible at frequencies extending well above 4.0 THz.

Leukocyte Gene Expression in Patients with Medication Refractory Depression before and after Treatment with ECT or Isoflurane Anesthesia: A Pilot Study.

Objective. To evaluate leukocyte gene expression for 9 selected genes (mRNAs) as biological markers in patients with medication refractory depression before and after treatment with ECT or isoflurane anesthesia (ISO). Methods. In a substudy of a nonrandomized open-label trial comparing effects of ECT to ISO therapy, blood samples were obtained before and after treatment from 22 patients with refractory depression, and leukocyte mRNA was assessed by quantitative PCR. Patients' mRNAs were also compared to 17 healthy controls. Results. Relative to controls, patients before treatment showed significantly higher IL10 and DBI and lower ADRA2A and ASIC3 mRNA (P < 0.025). Both ECT and ISO induced significant decreases after treatment in 4 genes: IL10, NR3C1, DRD4, and Sult1A1. After treatment, patients' DBI, ASIC3, and ADRA2A mRNA remained dysregulated. Conclusion. Significant differences from controls and/or significant changes after ECT or ISO treatment were observed for 7 of the 9 mRNAs studied. Decreased expression of 4 genes after effective treatment with either ECT or ISO suggests possible overlap of underlying mechanisms. Three genes showing dysregulation before and after treatment may be trait-like biomarkers of medication refractory depression. Gene expression for these patients has the potential to facilitate diagnosis, clarify pathophysiology, and identify potential biomarkers for treatment effects.

Functional properties of tooth pulp neurons responding to thermal stimulation.

The response properties of tooth pulp neurons that respond to noxious thermal stimulation of the dental pulp have been not well-studied. The present study was designed to characterize the response properties of tooth pulp neurons to noxious thermal stimulation of the dental pulp. Experiments were conducted on 25 male ferrets, and heat stimulation was applied by a computer-controlled thermode. Only 15% of tooth pulp neurons (n = 39) responded to noxious thermal stimulation of the teeth. Tooth pulp neurons were found in both the superficial and deep nuclear regions of the subnucleus caudalis (Vc) and in the interface between the nucleus caudalis and interpolaris (Vc/Vi). Thirty-seven neurons had cutaneous receptive fields and were classified as either NS (16) or WDR (21) neurons. Repeated heat stimulation of the dental pulp sensitized and increased the number of electrically evoked potentials of tooth pulp neurons. These results provide evidence that both the Vc and Vc/Vi regions contain neurons that respond to noxious thermal stimulation of the dental pulp, and that these cells may contribute to the sensitization process associated with symptomatic pulpitis.

Gene expression alterations at baseline and following moderate exercise in patients with Chronic Fatigue Syndrome and Fibromyalgia Syndrome.

OBJECTIVES: To determine mRNA expression differences in genes involved in signalling and modulating sensory fatigue, and muscle pain in patients with chronic fatigue syndrome (CFS) and fibromyalgia syndrome (FM) at baseline, and following moderate exercise. DESIGN: Forty-eight patients with CFS only, or CFS with comorbid FM, 18 patients with FM that did not meet criteria for CFS, and 49 healthy controls underwent moderate exercise (25 min at 70% maximum age-predicted heart rate). Visual-analogue measures of fatigue and pain were taken before, during and after exercise. Blood samples were taken before and 0.5, 8, 24 and 48 h after exercise. Leucocytes were immediately isolated from blood, number coded for blind processing and analyses and flash frozen. Using real-time, quantitative PCR, the amount of mRNA for 13 genes (relative to control genes) involved in sensory, adrenergic and immune functions was compared between groups at baseline and following exercise. Changes in amounts of mRNA were correlated with behavioural measures and functional clinical assessments. RESULTS: No gene expression changes occurred following exercise in controls. In 71% of patients with CFS, moderate exercise increased most sensory and adrenergic receptor's and one cytokine gene's transcription for 48 h. These postexercise increases correlated with behavioural measures of fatigue and pain. In contrast, for the other 29% of patients with CFS, adrenergic α-2A receptor's transcription was decreased at all time-points after exercise; other genes were not altered. History of orthostatic intolerance was significantly more common in the α-2A decrease subgroup. FM-only patients showed no postexercise alterations in gene expression, but their pre-exercise baseline mRNA for two sensory ion channels and one cytokine were significantly higher than controls. CONCLUSIONS: At least two subgroups of patients with CFS can be identified by gene expression changes following exercise. The larger subgroup showed increases in mRNA for sensory and adrenergic receptors and a cytokine. The smaller subgroup contained most of the patients with CFS with orthostatic intolerance, showed no postexercise increases in any gene and was defined by decreases in mRNA for α-2A. FM-only patients can be identified by baseline increases in three genes. Postexercise increases for four genes meet published criteria as an objective biomarker for CFS and could be useful in guiding treatment selection for different subgroups.

Myalgic encephalomyelitis: International Consensus Criteria.

The label 'chronic fatigue syndrome' (CFS) has persisted for many years because of the lack of knowledge of the aetiological agents and the disease process. In view of more recent research and clinical experience that strongly point to widespread inflammation and multisystemic neuropathology, it is more appropriate and correct to use the term 'myalgic encephalomyelitis' (ME) because it indicates an underlying pathophysiology. It is also consistent with the neurological classification of ME in the World Health Organization's International Classification of Diseases (ICD G93.3). Consequently, an International Consensus Panel consisting of clinicians, researchers, teaching faculty and an independent patient advocate was formed with the purpose of developing criteria based on current knowledge. Thirteen countries and a wide range of specialties were represented. Collectively, members have approximately 400 years of both clinical and teaching experience, authored hundreds of peer-reviewed publications, diagnosed or treated approximately 50 000 patients with ME, and several members coauthored previous criteria. The expertise and experience of the panel members as well as PubMed and other medical sources were utilized in a progression of suggestions/drafts/reviews/revisions. The authors, free of any sponsoring organization, achieved 100% consensus through a Delphi-type process. The scope of this paper is limited to criteria of ME and their application. Accordingly, the criteria reflect the complex symptomatology. Operational notes enhance clarity and specificity by providing guidance in the expression and interpretation of symptoms. Clinical and research application guidelines promote optimal recognition of ME by primary physicians and other healthcare providers, improve the consistency of diagnoses in adult and paediatric patients internationally and facilitate clearer identification of patients for research studies.

Brain activation in multiple sclerosis: a BOLD fMRI study of the effects of fatiguing hand exercise.

BACKGROUND: Multiple sclerosis (MS) patients experience fatigue as a chronic symptom that decreases quality of life. Commonly, fatigue in MS patients is manifested as decreased motor function during or after physical activity and is associated with changes in brain metabolism. OBJECTIVE: To determine brain activation patterns in MS patients and healthy controls during a simple motor task before and after fatiguing hand-grip exercise. METHODS: Functional magnetic resonance imaging (fMRI) scans were conducted on 10 MS patients and 13 healthy controls during 4-finger flexion and extension in rested and fatigued states. RESULTS: Before the fatigue protocol, MS patients had greater activation in the contralateral primary motor cortex, insula, and cingulate gyrus than controls. Following fatiguing exercise, controls showed increased activation of precentral gyrus and insula while patients did not show any activation increases and actually decreased activity to the insula. CONCLUSION: Results indicate that before fatiguing exercise, MS patients marshaled more brain activation compared to controls, which may represent functionally adaptive changes in response to demyelination. This increased activation may suggest that patients require more effort to perform even simple motor tasks, possibly because peripheral or central signals for fatigue are chronically enhanced. When fatigued further by muscle contraction, brain activation cannot be further increased.

Long-lasting hyperalgesia and sympathetic dysregulation after formalin injection into the rat hind paw.

Subcutaneous formalin injection has been used extensively to evaluate acute effects (over several hours) of chemical nociceptive stimulation on nociceptive reflexes. Also, a persistent hyperreflexia for mechanical and thermal stimulation, lasting 3 weeks after formalin injection, has been revealed and related to microglial activation in the spinal dorsal horn. The present study demonstrates more prolonged effects of formalin injection, lasting 6 weeks, on operant escape from nociceptive thermal stimulation. Operant escape requires cerebral processing of nociceptive input and can detect effects that are not limited to spinal or spinal-brain stem-spinal reflex circuits. Compared with rats injected with saline, escape responding to 44.5 degrees C and 47 degrees C stimulation was increased after bilateral s.c. injection of 5% formalin into the dorsal hind paws. The hyperalgesia outlasted visible signs of trauma (e.g. paw edema). Responses to 36 degrees C were not altered after formalin injection, providing a control for effects of the peripheral injury on activity levels or exploratory tendencies. Skin temperature recordings from the forepaws and contralateral hind paw during 44.5 degrees C stimulation of the left hind paw provided an indirect measure of cutaneous blood flow in formalin- and saline-injected animals. Normal reductions in skin temperature during thermal stimulation were attenuated (nearly eliminated) at 1 and 2 weeks after formalin injection and partially recovered by 10 weeks. Thus, formalin-induced tissue injury produced a long-term secondary hyperalgesia, accompanied by a reduced sympathetic responsivity. The similar time-course for these phenomena suggests that there are mechanistic linkages between focal injury, autonomic dysregulation and enhanced pain sensitivity.

Estrous cycle modulation of nociceptive behaviors elicited by electrical stimulation and formalin.

The impact of circulating ovarian hormones on nociceptive behaviors elicited by phasic and tonic stimuli was evaluated in rats using two behavioral tests: an operant escape task and the formalin test. The operant escape task was structured to separately evaluate hindlimb flexion reflexes, the latency of escape, and the amplitude of peak vocalization to a series of phasic electrocutaneous stimuli (0.05-0.8 mA), whereas the formalin test evaluated nociceptive behaviors elicited by tonic stimulation following a subcutaneous injection of dilute formalin (1%). Hindlimb reflex amplitude, escape latency, and peak vocalization varied across the estrous cycle, such that rats were most sensitive to electrical stimuli during proestrus (reflex and escape latency) and diestrus (vocalization). Furthermore, morphine-induced (3 mg/kg sc) attenuation of hindlimb reflex amplitude was sensitive to estrous cycling. During proestrus, morphine produced less attenuation of hindlimb reflex amplitude than during nonproestrus phases. However, estrous cycling did not alter nociceptive behaviors elicited by 1% formalin. These data support the notion that circulating ovarian hormones may differentially modulate behaviors associated with phasic and tonic pain.

Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons.

In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a significantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by mu-, delta(1)-, and kappa-selective opioids was significantly reduced when 1% but not < or =0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a significantly higher proportion of neurons fired spontaneous action potentials with either 0.05-0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward rectification were each significantly altered for neurons recorded with 1% but not 0.05-0.2% biocytin. These effects may be due to a relatively specific blockade of diverse potassium channel types. Because efficient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated.

Long-lasting inflammation and long-term hyperalgesia after subcutaneous formalin injection into the rat hindpaw.

Subcutaneous formalin injection is widely used as a nociceptive stimulus in the rat. This procedure evokes overt behaviors that last about 90 minutes. However, little is known about the duration of paw inflammation and alterations in pain sensitivity to noxious stimuli after 2 hours. We studied the nociceptive responses to thermal and mechanical stimuli 2 hours to 4 weeks after formalin injection into the dorsal or plantar side of the hindpaw. Thirty-two adult male Sprague-Dawley rats were divided into 3 groups: In group I, 50 microL of 5% formalin was injected into the plantar side (n = 12); in group II, 50 microL of 5% formalin was injected into the dorsal side (n = 12); in group III, 50 microL saline was injected into the dorsal or plantar side of the hindpaw (n = 8). Nociceptive responses to thermal and mechanical stimuli applied to the dorsal or plantar surfaces of the injected and the contralateral hindpaws were recorded. The injection of formalin into the rat's hindpaw produced a hypoalgesic region around the injection site. In contrast, hyperalgesic responses to thermal and mechanical stimulation were induced on the opposite surface of the injected hindpaw as well as in the contralateral noninjected hindpaw. The hyperalgesic responses, which were observed 2 hours after formalin administration, were enhanced 1 to 3 days after injection and lasted 3 to 4 weeks. These results suggest that peripheral inflammation after subcutaneous formalin injection produces a long-lasting sensitization. Possible mechanisms for these changes in nociception are discussed.

The effects of noxious dental heating on the jaw-opening reflex and trigeminal Fos expression in the ferret.

Previous studies have established that the activation of peripheral nociceptors alters the central processing of nociceptive stimuli. In this study, we examined whether noxious heating of the dental pulp enhances the nociceptive jaw-opening reflex (JOR) and the expression of the immediate early gene c-fos in chloral hydrate/pentobarbital-anesthetized ferrets. We hypothesized that the application of noxious heat to the dental pulp, a procedure that evokes a preferential activation of pulpal C-fibers, will enhance JOR responses to electrical stimulation of the tooth pulp and that this enhanced response will be associated with the expression of Fos protein in discrete regions of the trigeminal nucleus. Consistent with our predictions, we observed that noxious heat conditioning enhanced the JOR as indicated by an increase in the magnitude of the signal averaged digastric electromyogram response evoked by electrical stimuli applied to either a heat-conditioned maxillary canine or the contralateral nonconditioned canine. The enhancement in JOR responses was independent of temporal summation of the electrical stimulus for test stimuli delivered at either 1.0 or 0.1 Hz. Sensitization of the JOR was associated with an increase in the number of immunohistochemically identified Fos-positive nuclei in trigeminal caudalis (Vc) and the transition zone between trigeminal interpolaris and caudalis (Vi/Vc) ipsilateral to the site of stimulation compared with sham stimulated animals. These findings suggest that neuronal populations in Vc and Vi/Vc play a role in the enhanced reflex responses to tooth pulp stimulation and may contribute to the pain and hyperalgesia associated with a symptomatic pulpitis.

Effects of systemic morphine on escape latency and a hindlimb reflex response in the rat.

The present study uses focal electrical stimulation of myelinated nociceptors to simultaneously assess behavioral responses that are organized at spinal and supraspinal sites in the rat. Hindlimb reflex amplitude and the latency to operant escape responses by a forelimb were recorded for each stimulus presentation to a hindlimb across a wide range of intensities. This paradigm provided a tool whereby effects of morphine on conscious escape responses could be delineated from effects on a segmental flexion reflex over a range of doses. Administration of morphine (3 mg/kg and 10 mg/kg, subcutaneously) increased the latency of escape responses and decreased the amplitude of reflex responses in a dose-dependent manner. However, morphine produced a greater suppression of reflex responses compared with the increase in effects on escape latencies. The effects of morphine on escape latency were not expressed at the highest stimulus intensities (0.6 to 0.8 mA), whereas reflex responses were attenuated at all suprathreshold stimulus intensities. Thus, electrically evoked, spinal-mediated responses of rats are not affected by morphine in the same manner as electrically evoked supraspinal-mediated nociceptive behaviors. However, both measures confirm evidence that responses elicited by activation of myelinated afferents are less powerfully affected by morphine than responses to input from unmyelinated nociceptors.

Relationship between nociceptor activity, peripheral edema, spinal microglial activation and long-term hyperalgesia induced by formalin.

To determine whether initial nociceptive inputs caused by subcutaneous injection of formalin into the hindpaw are necessary and/or sufficient for allodynic behavior and microglial activation observed at one week following behavior, we examined Sprague-Dawley rats under five test conditions. Test condition 1. Formalin alone group (six rats), 5% formalin was injected subcutaneously into the dorsal side of the right hind paw. Test condition 2. Bupivacaine/Formalin group (six rats), bupivacaine was injected into the ankle area and into the site of formalin injection 10 min before formalin injection. Test condition 3. Saline/Formalin group (six rats), saline was injected 10min before formalin in the same manner as bupivacaine. Test condition 4. Formalin/Bupivacaine group 1 (six rats), bupivacaine was injected 10 min after formalin. Test condition 5. Formalin/Bupivacaine group 2 (six rats), bupivacaine was injected similarly 1h after formalin. The magnitude of paw edema and paw withdrawal thresholds to mechanical stimuli applied to the plantar surface of the injected paw and on the dorsal surface of the contralateral side were evaluated prior to and one week after formalin injection. The lumbar spinal cord was immunohistochemically processed at one week to assess the expression of a marker for activated microglia. The results showed: (i) pre-treatment with bupivacaine blocked both phases of formalin-evoked pain behaviors and the mechanical allodynia that developed one week post-formalin injection, but did not block microglial activation; (ii) treatment with bupivacaine 1h after formalin injection reduced paw edema and prevented skin ulceration, but one week allodynia and microglial activation were still present; and (iii) prolonged spinal microglial activation was not dependent on acute formalin-induced nociceptor activity, but was strongly associated with the amount of tissue destruction. Our studies suggest that: (i) the central sensitization associated with the phase II of formalin-evoked behaviors and spinal microglial activation are both necessary to permit the development of the long-term hyperalgesia produced by the subcutaneous administration of formalin into the rat's hindpaw; and (ii) acute nociceptive inputs following formalin injection are not necessary for central microglial activation that may be triggered by nerve damage or prolonged signals from peripherally inflamed tissue

Spinal laminae I-II neurons in rat recorded in vivo in whole cell, tight seal configuration: properties and opioid responses.

Using the in vivo whole cell recording procedure described previously, we recorded 73 neurons in laminae I and II in the lumbar spinal cord of the rat. Input impedances averaged 332 MOmega, which indicated that prior sharp electrode recordings contained a significant current shunt. Characterization of the adequate stimuli from the excitatory hindlimb receptive field indicated that 39 of 73 neurons were nociceptive, 6 were innocuous cooling cells, 20 responded maximally to brush, and 8 cells were not excited by stimulation of the skin of the hindlimb. The locations of 15 neurons were marked with biocytin. Nociceptive neurons were mostly found in lamina I and outer II, cooling cells in lamina I, and innocuous mechanoreceptive cells were mostly found in inner II or in the overlying white matter. The mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-Enkephalin (DAMGO) hyperpolarized 7 of 19 tested neurons with a conductance increase. This hyperpolarization was reversed by naloxone in the neurons in which it was applied. DAMGO also decreased the frequency of spontaneous PSPs in 13 neurons, 7 of which were also hyperpolarized by DAMGO. Five of the seven hyperpolarized neurons were nociceptive, responding to both heat and mechanically noxious stimuli, whereas two responded to slow, innocuous brush. These results indicate that whole cell, tight seal recordings sample a similar population of lamina I and II neurons in the rat as those found with sharp electrode recordings in cat and monkey. They further indicate that DAMGO hyperpolarizes a subset of the nociceptive neurons that have input from both heat and mechanical nociceptors and that presynaptic DAMGO effects can be observed in nociceptive neurons that are not hyperpolarized by DAMGO.

Microglial reactions after subcutaneous formalin injection into the rat hind paw.

Microglia in primary afferent projection territories are activated and proliferate after peripheral nerve injury. However, it is not known whether stimulation of peripheral nerves by noxious stimuli applied to their receptive fields activates microglial cells in the spinal cord. This study was designed to investigate the response of microglia in the lumbar spinal cord and in the brainstem to a tonic noxious stimulus. Thirty-two male Sprague-Dawley rats received subcutaneous injections of 5% formalin (50 microliter) into the plantar surface of the right hind paw, and 24 rats were injected with 50 microliter saline as a control. The lumbar spinal cord and brainstem were evaluated for immunoreactivity (IR) to complement receptor C3bi (monoclonal antibody OX-42) and major histocompatibility complex class II (monoclonal antibody OX-6) on postinjection hours 0, 2, 4 and 8 and days 1, 3, 7, 14 and 28. A qualitative and quantitative increase of OX-42-IR microglial cells were observed in the medial portion of the dorsal horn and in the gracile nucleus of the brainstem on the side ipsilateral to the formalin injection, starting on days 1-3 and peaking on day 7 postinjection. OX-6-positive cells were scattered both in gray and white matter, but no difference was detected between the two sides of the spinal cord or between formalin-injected and control animals. This is the first study that reports that subcutaneous injection of formalin into the rat's hind paw induces microglial activation in the spinal cord as well as in the brainstem. Although we have not determined whether these responses result from nociceptor activity, peripheral inflammation, or degeneration of primary afferents and/or central neurons, this method provides a simple, effective and stable animal model that will permit the future study of the mechanisms that contribute to microglial activation and its pathophysiological consequences.

Ultrastructural analysis of ectopic synaptic boutons arising from peripherally regenerated primary afferent fibers.

The central axons of peripherally regenerated Abeta primary sensory neurons were impaled in the dorsal columns of alpha-chloralose-anesthetized cats 9-12 mo after axotomy. The adequate peripheral stimulus was determined, and the afferent fibers intracellularly stimulated while simultaneously recording the resulting cord dorsum potentials (CDPs). Fibers that successfully had reinnervated the skin responded to light tactile stimulation, and evoked CDPs that suggested dorsally located boutons were stained intracellularly with horseradish peroxidase (HRP). Two HRP-stained regenerated Abeta afferent fibers were recovered that supported large numbers of axon collaterals and swellings in laminae I, IIo, and IIi. Sections containing the ectopic collateral fibers and terminals in the superficial dorsal horn were embedded in plastic. Analyses of serial ultrathin sections revealed that ectopic projections from both regenerated fibers supported numerous synaptic boutons filled with clear round vesicles, a few large dense core vesicles (LDCVs) and several mitochondria (>3). All profiles examined in serial sections (19) formed one to three asymmetric axo-dendritic contacts. Unmyelinated portions of ectopic fibers giving rise to en passant and terminal boutons often contained numerous clear round vesicles. Several boutons (47%) received asymmetric contacts from axon terminals containing pleomorphic vesicles. These results strongly suggest that regenerated Abeta fibers activated by light tactile stimuli support functional connections in the superficial dorsal horn that have distinct ultrastructural features. In addition, the appearance of LDCVs suggests that primary sensory neurons are capable of changing their neurochemical phenotype.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954-2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the mu-opioid agonist (-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1-5 microM DAMGO caused a robust and repeatable hyperpolarization in membrane potential (Vm) and decrease in neuronal input resistance (RN) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5'-triphosphate (GTP; 100 microM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 microM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S; 500 microM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate mu-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord.

The localization of GABAA receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the beta 2 and beta 3 subunits of the receptor. beta 2/beta 3-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I-III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that beta 2/beta 3-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of beta 2/beta 3-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of beta 2/beta 3-subunit immunoreactivity were also detected using immunofluorescence. Focal "hot spots" correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of beta 2/beta 3-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane beta 2/beta 3 immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of "classical" (synaptic) and "nonclassical" (paracrine) actions mediated via spinal cord GABAA receptors. The study also revealed the relative paucity of beta 2/beta 3-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals.

Guanylin, an endogenous ligand for C-type guanylate cyclase, is produced by goblet cells in the rat intestine.

BACKGROUND & AIMS: Guanylin activates an intestinal guanylate cyclase (GCC) and stimulates electrolyte movement across the gut epithelium. Cells expressing guanylin messenger RNA have been localized to the epithelial cell layer of the intestine; however, the identity of the guanylin-producing cells has not been determined. The aim of this study was to identify cells that express guanylin in the rat intestine. METHODS: Antibodies were raised against defined proguanylin epitopes, evaluated by Western blotting, and used for immunoperoxidase histochemistry. RESULTS: Guanylin-like immunoreactivity was localized to a subset of goblet cells. In the small intestine, most, perhaps all, goblet cells in the villi were immunopositive, as were some goblet cells in upper crypts; however, goblet cells deep within crypts were unlabeled. In the colon, goblet cells clustered in the necks and around the openings of crypts were immunopositive, whereas (as in the small intestine) goblet cells in deeper crypt regions were unlabeled. In some animals, immunoreactive columnar epithelial cells were also observed in the colon (although such cells were not apparent in the small intestine). Relative labeling of columnar cells varied from animal to animal. CONCLUSIONS: Guanylin is expressed in mature goblet cells. If secreted in conjunction with mucin, it could play a role in the hydration of mucus.