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PubMed1997

An enzyme linked immunosorption assay for tissue factor pathway inhibitor.

An assay for the quantification of full-length and carboxy-terminus truncated tissue factor pathway inhibitor (TFPI) has been developed. The assay is a classical two-antibody sandwich assay with a monoclonal capture antibody directed against the third Kunitz-type domain of human TFPI and a polyclonal rabbit peroxidase-labelled anti-human TFPI detecting antibody. The assay is sensitive to full-length and carboxy-terminus truncated TFPI with intact third Kunitz-type domain, but not to two-domain TFPI. TFPI associated with lipoproteins is not or only sparsely detected and TFPI in complex with factor Xa only partially measured. The assays gives linear reference curves in the dose range of 5 to 100 ng/ml in a double logarithmic plot. The normal range assessed from analyses on citrated plasma from 81 normal human donors is 7.8 to 26.0 ng/ml (average +/- 2 SD, log-normal distribution). There is no statistically significant difference between TFPI levels measured in 10 fasting and 10 non-fasting individuals. The reproducibility of the assay is about 5.6-5.9% (relative standard error) and the within-days and between-day reproducibilities are 4.7-5.1% and 5.9-8.5%, respectively. The assay is in very good agreement with a commercial ELISA assay recently marketed. A robust, reproducible and convenient ELISA assay for the determination of full-length and three-domain TFPI has been developed.

Animals↗
PubMed2004

Molecular wheel B8(2-) as a new inorganic ligand. photoelectron spectroscopy and ab initio characterization of LiB8(-).

The bare B(8) cluster was previously reported to be a D(7h) molecular wheel with a triplet group state. The B(8)(2-) dianion was predicted to be a closed-shell singlet and double aromatic D(7h) molecular wheel. Here we report the experimental observation of B(8)(2-) stabilized by a Li(+) cation in LiB(8)(-) and its experimental characterization using photoelectron spectroscopy. Theoretical searches lead to a C(7v) LiB(8)(-) global minimum structure, and its calculated photodetachment transitions are in good agreement with the experimental values. Except for a small out-of-plane distortion due to the asymmetric Li(+) capping, the B(8)(2-) unit in LiB(8)(-) is nearly identical to the bare B(8)(2-), suggesting it is a robust and stable structural unit and may be used as a new ligand and building block in chemistry.

Journal Article↗
PubMed2004

Characterization of RNA interference in rat PC12 cells: requirement of GERp95.

Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system to achieve RNAi in a cultured rat neuronal cell line, PC12. Targeting of neuropeptide Y mRNA by synthetic siRNA results in knock down of the mRNA levels with an IC50 of approximately 0.1 nM. The mRNA knockdown lasts for at least 96 h and is not dependent on protein synthesis. Further, PC12 cells were ablated of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells.

Animals↗
PubMed1990

Theoretical basis for an anomalous temperature coefficient in swelling pressure of rabbit corneal stroma.

In the rabbit corneal stroma, the swelling pressure, P, has been reported to have an anomalous (negative) temperature coefficient, alpha P, contradicting traditional Donnan swelling theory. A parallel-plate, diffuse double layer Gouy-Chapman model was used to resolve this discrepancy. The present model incorporates the possibility that surface charge, sigma, is temperature dependent. It is shown that negative, zero, or positive coefficients of swelling pressure change with temperature are not mutually exclusive conditions, but can be attributed to the same underlying mechanism. For likely values of alpha P(range -7 x 10(-3) K-1 to +3.2 x 10(-3)K-1), the effective stromal charge has a negative temperature dependency, or dln sigma/dT less than 0. The present formalism is robust against variation in assumed alpha P, and is able to simultaneously satisfy the known values of swelling pressure, its thermal dependency, and stromal charge. These results implicate significant coulombic forces behind P. Predicted stromal surface charge is approximately 0.01 Cm-2. The predictions were confirmed with macrocontinuum Donnan swelling theory, suggesting that Donnan osmotic swelling is the principal macroscopic component of P.

Animals↗
PubMed1997

Determination of (pseudo)plastic flow properties of pharmaceutical semisolids using rheological AUC parameters.

The purpose of this study was to develop a simple and unique method of data reduction for pharmaceutical semisolids. Application and accuracy of different regression models for rate-controlled rotating cylinder and T-shaped spindle data was compared in a conventional analysis of variance. Methods employed to determine area under the flow curve (AUC) parameters included integration of fitting function, planimetration, and calculation of trapezoids. Complex rheological behavior such as structure breakdown at low rates of shear (D) and deflocculation of particle aggregates at high values for D is not sufficiently described by conventional fitting functions. Iterating the nonlinear power law function tau = tau 0 + k2. Dn0, however, gives the best fit of data for pharmaceutical samples, estimating a yield value, tau 0.1 calculated for D = 0.1 s-1 following double logarithmic transformation. Alternatively, rheological AUC parameters are determined for D: 0 < or = D < or = 98 s-1. Validating models, maximum deviation for different methods of determination is smaller than Srel of 5 consecutive planimetration experiments. In case of sufficient fit of experimental data, integration of a nonlinear power law function is acceptable. Convenient fitting parameters tau and n0 can be substituted by area parameters such as AT, Arel, or R for all liquids, and hydrophilic and lipophilic gels, as well as o/w and w/o emulsions being investigated. The new AUC method offers robust data reduction and universal pharmaceutical application for different Newtonian and (pseudo)plastic materials. The approach is, however, limited to instrumental conditions and the range of D.

Analysis of Variance↗
PubMed2000

Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints.

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowle's Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.

Blood Chemical Analysis↗
PubMed2004

Evidence for semantic learning in profound amnesia: an investigation with patient H.M.

Until recently, it seemed unlikely that any semantic knowledge could be acquired following extensive bilateral damage to the medial temporal lobes (MTL). Although recent studies have demonstrated some semantic learning in amnesic patients, questions remain regarding the limits of this capacity and the extent to which it relies on those patients' residual MTL function. The present study examined whether detailed, semantic memory could be acquired by a patient with no functioning hippocampus. We used cued recall and forced-choice recognition tasks to investigate whether the patient H.M. had acquired knowledge of people who became famous after the onset of his amnesia. Results revealed that, with first names provided as cues, he was able to recall the corresponding famous last name for 12 of 35 postoperatively famous personalities. This number nearly doubled when semantic cues were added, suggesting that his knowledge of the names was not limited to perceptual information, but was incorporated in a semantic network capable of supporting explicit recall. In forced-choice recognition, H.M. discriminated 87% of postmorbid famous names from foils. Critically, he was able to provide uniquely identifying semantic facts for one-third of these recognized names, describing John Glenn, for example, as "the first rocketeer" and Lee Harvey Oswald as a man who "assassinated the president." Although H.M.'s semantic learning was clearly impaired, the results provide robust, unambiguous evidence that some new semantic learning can be supported by structures beyond the hippocampus proper.

Aged↗
PubMed2005

Determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in foods by high performance liquid chromatography-atmospheric pressure chemical ionisation-medium-resolution mass spectrometry.

A robust and sensitive method for the determination of ethylenethiourea (ETU) and iso-propylenethiourea (i-PTU) in foods is reported. ETU and i-PTU were extracted by blending with dichloromethane (DCM) in the presence of sodium sulphate, sodium carbonate, thiourea and ascorbic acid. 2H4-ETU and n-PTU were used as internal standards. After filtration the DCM was removed by rotary evaporation and the extract re-dissolved in water before analysis by reversed-phase liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry using a double focusing mass spectrometer at a resolution of 5000. Mean recoveries of ETU and i-PTU from fruit-based, cereal-based and meat-based infant foods, potato chips and tinned potatos at 0.01 mg kg(-1) and from pizza and yoghurt at 0.02-0.1 mg kg(-1) were 95% and 97% respectively. Precision, including both repeatability and internal reproducibility, was in the range of 3.1-13.1%.

Chromatography, High Pressure Liquid↗
PubMed2003

The third intracellular loop and the carboxyl terminus of beta2-adrenergic receptor confer spontaneous activity of the receptor.

It is well established that the beta2-adrenergic receptor (beta2-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related beta1-AR subtype. To identify the potential domain(s) of beta2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (beta1/beta2-Li3) or the carboxyl terminus (beta1/beta2-CT) or both domains (beta1/beta2-Li3CT) of beta1-AR are replaced by the corresponding parts of the beta2-AR. Using adenoviral gene transfer, we individually expressed these beta1/beta2-AR chimeras in mouse cardiomyocytes lacking both native beta1-AR and beta2-AR (beta1/beta2 double knockout), and examined their possible spontaneous activities. Overexpression of these beta1/beta2-AR chimeras markedly elevated basal cAMP accumulation and myocyte contractility in the absence of agonist stimulation compared with those infected by a control adenovirus expressing beta-galactosidase or an adenovirus expressing wild type beta1-AR. These effects were fully reversed by a beta2-AR inverse agonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 x 10-7 M), regardless of inhibition of Gi with pertussis toxin, but not by a panel of beta1-AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate (CGP20712A), betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of beta1-AR is not responsible for the lack of beta1-AR spontaneous activation, although it has been known that the beta1-AR PDZ motif prevents the receptor from undergoing agonist-induced trafficking and Gi coupling in cardiomyocytes. Taken together, the present results indicate that both the third intracellular loop and the C terminus are involved in beta2-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.

Adrenergic beta-Antagonists↗
PubMed2003

Facilitation from ventral premotor cortex of primary motor cortex outputs to macaque hand muscles.

We demonstrate that in the macaque monkey there is robust, short-latency facilitation by ventral premotor cortex (area F5) of motor outputs from primary motor cortex (M1) to contralateral intrinsic hand muscles. Experiments were carried out on two adult macaques under light sedation (ketamine plus medetomidine HCl). Facilitation of hand muscle electromyograms (EMG) was tested using arrays of fine intracortical microwires implanted, respectively, in the wrist/digit motor representations of F5 and M1, which were identified by previous mapping with intracortical microstimulation. Single pulses (70-200 microA) delivered to F5 microwires never evoked any EMG responses, but small responses were occasionally seen with double pulses (interval: 3 ms) at high intensity. However, both single- and double-pulse stimulation of F5 could facilitate the EMG responses evoked from M1 by single shocks. The facilitation was large (up to 4-fold with single and 12-fold with double F5 shocks) and occurred with an early onset, with significant effects at intervals of only 1-2 ms between conditioning F5 and test M1 stimuli. A number of possible pathways could be responsible for these effects, although it is argued that the most likely mechanism would be the facilitation, by cortico-cortical inputs from F5, of corticospinal I wave activity evoked from M1. This facilitatory action could be of considerable importance for the coupling of grasp-related neurons in F5 and M1 during visuomotor tasks.

Animals↗
PubMed2004

Chandelier cells control excessive cortical excitation: characteristics of whisker-evoked synaptic responses of layer 2/3 nonpyramidal and pyramidal neurons.

Chandelier cells form inhibitory axo-axonic synapses on pyramidal neurons with their characteristic candlestick-like axonal terminals. The functional role of chandelier cells is still unclear, although the preferential loss of this cell type at epileptic loci suggests a role in epilepsy. Here we report an examination of whisker- and spontaneous activity-evoked responses in chandelier cells and other fast-spiking nonpyramidal neurons and regular-spiking pyramidal neurons in layer 2/3 of the barrel cortex. Fast-spiking nonpyramidal neurons, including chandelier cells, basket cells, neurogliaform cells, double bouquet cells, net basket cells, bitufted cells, and regular-spiking pyramidal neurons all respond to stimulation of multiple whiskers on the contralateral face. Whisker stimulation, however, evokes small, delayed EPSPs preceded by an earlier IPSP and no action potentials in chandelier cells, different from other nonpyramidal and pyramidal neurons. In addition, chandelier cells display a larger receptive field with lower acuity than other fast-spiking nonpyramidal neurons and pyramidal neurons. Notably, simultaneous dual whole-cell in vivo recordings show that chandelier cells, which rarely fire action potentials spontaneously, fire more robustly than other types of cortical neurons when the overall cortical excitation increases. Thus, chandelier cells may not process fast ascending sensory information but instead may be reserved to prevent excessive excitatory activity in neuronal networks.

Action Potentials↗
PubMed1995

High speed 1H spectroscopic imaging in human brain by echo planar spatial-spectral encoding.

We introduce a fast and robust spatial-spectral encoding method, which enables acquisition of high resolution short echo time (13 ms) proton spectroscopic images from human brain with acquisition times as short as 64 s when using surface coils. The encoding scheme, which was implemented on a clinical 1.5 Tesla whole body scanner, is a modification of an echo-planar spectroscopic imaging method originally proposed by Mansfield Magn. Reson. Med. 1, 370-386 (1984), and utilizes a series of read-out gradients to simultaneously encode spatial and spectral information. Superficial lipid signals are suppressed by a novel double outer volume suppression along the contours of the brain. The spectral resolution and the signal-to-noise per unit time and unit volume from resonances such as N-acetyl aspartate, choline, creatine, and inositol are comparable with those obtained with conventional methods. The short encoding time of this technique enhances the flexibility of in vivo spectroscopic imaging by reducing motion artifacts and allowing acquisition of multiple data sets with different parameter settings.

Artifacts↗
PubMed2004

Perfusion MRI in CNS disease: current concepts.

Today there are several indications for cerebral perfusion MRI. The major indications routinely used in increasing numbers of imaging centers include cerebrovascular disease, tumor imaging and recently psychiatric disorders. Perfusion MRI is based on the injection of a gadolinium chelate and the rapid acquisition of images as the bolus of contrast agent passes through the blood vessels in the brain. The contrast agent causes a signal change; this signal change over time can be analysed to measure cerebral hemodynamics. The quality of brain perfusion studies is very dependent on the contrast agent used: a robust and strong signal decrease with a compact bolus is needed. MultiHance (gadobenate dimeglumine, Gd-BOPTA) is the first of a new class of paramagnetic MR contrast agents with a weak affinity for serum proteins. Due to the interaction of Gd-BOPTA with serum albumin, MultiHance presents with significantly higher T1- and T2-relaxivities enabling a sharper bolus profile. This article reviews the indications of perfusion MRI and the performance of MultiHance in MR perfusion of different diseases. Previous studies using perfusion MRI for a variety of purposes required the use of double dose of contrast agent to achieve a sufficiently large signal drop to enable the acquisition of a clear input function and the calculation of perfusion rCBV and rCBF maps of adequate quality. Recent studies with Multi-Hance suggest that only a single dose of this agent is needed to cause a signal drop of about 30% which is sufficient to allow the calculation of high quality rCBV and rCBF maps.

Brain Diseases↗
PubMed2005

Is the prophylactic antidepressant efficacy of lithium in bipolar I disorder dependent on study design and lithium level?

In the 1970s, several randomized controlled trials demonstrated significant antimanic and antidepressant properties of lithium in the prophylactic treatment of bipolar disorder. However, a recent meta-analysis of randomized, placebo-controlled trials of lithium in bipolar disorder found that its protective effect against depressive relapse/recurrence was equivocal. By examining potentially relevant parameters of recent randomized controlled trials with regard to lithium's prophylactic antidepressant efficacy, we try to identify factors which might help to explain these discrepant results across the different trials. Lithium's efficacy against manic relapse/recurrence appears rather robust at plasma levels between 0.8 and 1.2 mmol/L, whereas lithium's efficacy against depressive relapse/recurrence may be more modest and dependent on whether a response during the preceding acute episode was achieved by lithium treatment. Furthermore, it might be advisable to continue lithium without interruption at the same dose/plasma level, which yielded the initial response. A lithium level between 0.5 and 0.8 mmol/L may be equally efficacious against overall relapse and associated with equal or even superior efficacy regarding depressive relapse/recurrence. To provide evidence-based guidelines on this issue, large prospective, randomized, double-blind, placebo-controlled trials are needed comparing the efficacy of lithium at different plasma levels against manic and depressive relapse/recurrence. In these trials, factors previously associated with predicting response to lithium should also be assessed.

Antidepressive Agents↗
PubMedDate not supplied

Mechanoelectrical and voltage-gated ion channels in mammalian vestibular hair cells.

Mammalian vestibular afferents respond robustly to head movements at low frequencies and provide input to reflexes that control eye, head and body position. Vestibular organs have distinctive regions and hair cells: Type II cells receive bouton afferent endings and type I cells receive large calyx afferent endings. In the rodent utricle, type II cells are broadly tuned to frequencies between 10 and 30 Hz. Other recent data suggest that otolith organs function in this frequency range, which is higher than previously imagined. Some of the tuning derives from adaptation of the transducer current, which is best fitted with a double exponential decay with time constants of approximately 4 and 40 ms. Further tuning is provided by basolateral conductances, principally outwardly rectifying, voltage-gated K+ conductances. The kinetics of the K+ currents tend to vary with location in the sensory epithelium and therefore may contribute to regional variation in afferent physiology. Type I hair cells have a large, negatively activating K+ conductance, g(K,L), that confers a very low input resistance and therefore attenuates the receptor potential. This may reduce nonlinearity in the receptor potential, a possibly useful feature for the motor reflexes served by the vestibular system. On the other hand, the small receptor potentials together with unusually negative resting potentials are hard to reconcile with calcium-mediated quantal transmission. This problem may be overcome by factors that inhibit g(K,L)'s activation at resting potential. Also, the calyx may support nonquantal transmission.

Animals↗
PubMed2006

Validation plots in antenatal screening for Down's syndrome.

OBJECTIVE: To validate empirically the accuracy of antenatal Down's syndrome screening using the Integrated test, to compare this with other screening tests (including the Integrated test with the addition of cross trimester [CT] marker ratios) and to suggest how such validation analyses should be presented and interpreted. METHODS: Using data from 7809 unaffected and 27 Down's syndrome pregnancies that had had an Integrated test, risk estimates for various screening tests (maternal age, Double, Triple, Quadruple, Combined, Integrated and serum Integrated tests) that use Integrated test markers were categorized according to quintile categories of risk estimates of the 27 affected pregnancies. For each screening test, the median risk estimate for each category was plotted against the observed prevalence within each category. Such validation plots were also produced for the Integrated test with CT marker ratios by measuring the level of the serum markers in the trimester of pregnancy not already measured in stored samples of all affected and a one-in-five sample of unaffected pregnancies. The robustness of the method was assessed by repeating the analysis for the Integrated test after re-classifying affected pregnancies with low risk estimates as unaffected, simulating the underascertainment of cases. RESULTS: The validation plots (i) confirmed the accuracy of risk estimation for the different tests (by how close the points lay to the line of identity between predicted risk and observed prevalence), (ii) demonstrated the differences in screening performance of the different tests (by the range of risk spanned by the points and, in particular, the separation between the points representing the lowest risk and the next point), and (iii) are robust to underascertainment of affected pregnancies (by having little influence on the closeness of the points to the line of identity). CONCLUSION: The validation plot is a useful, simple and robust way to assess the validity of new screening methods, to assess the accuracy of risk estimation and to audit the performance of screening programmes.

Biomarkers↗
PubMed1994

Evaluating diffusion of light in the eye by objective means.

PURPOSE: The authors have developed an index of diffusion that describes the relative spread of light inside and outside the region of image focus of the living human eye. It provides in numerical terms a measure of light scatter and can be used to characterize the optical deficit in eyes with age- and disease-related abnormalities of the anterior segment. METHOD: An improved version of the double-pass method of examining the aerial image formed by reflection of the retinal image of a point source is employed, together with a new way of analyzing the image. Experimental estimation shows the contaminating effect of back scatter from the media and corneal reflection to be negligible. Measurements are objective and do not require any responses on the part of the patient. Data become available practically on-line. RESULTS: Index of diffusion values were obtained on 13 patients and varied from 0.22 to 1.04, strongly tending to increase with age. They are rather robust to pupil size, exposure duration, and small amounts of defocus. CONCLUSION: The index appears to provide a promising measure of optical performance of the media of the anterior segment of the eye, which might be useful in studying the effect of aging, injury, and disease.

Adult↗
PubMed1999

Hypothalamic hypocretin (orexin): robust innervation of the spinal cord.

Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.

Animals↗