The role of 5-hydroxytryptophan (5-HTP) in the regulation of the sleep/wake cycle in parachlorophenylalanine (p-CPA) pretreated rat: a multiple approach study

Summary In the rat, the insomnia which follows the administration of parachlorophenylalanine (p-CPA), a serotonin synthesis inhibitor, is transiently reversed either by intra-cisternal injection of L-5-HTP or by an associated injection of 5-HTP and an L-aromatic-aciddecarboxylase inhibitor (benserazide). Histochemical, immunohistochemical and chemical investigations showed that 5-HTP administration does not lead to a detectable increase in cerebral 5-HT. These findings suggest that the restoration of sleep after p-CPA treatment could be mediated by the central action of 5-HTP.     

5-Hydroxytryptophan (5-HTP)-immunoreactive neurons in the rat brain tissue

We demonstrated the presence of 5-hydroxytryptophan (5-HTP), the immediate precursor of serotonin (5-HT), in the rat brain tissue using a glutaraldehyde-coupled immunohistochemical technique. The immunoreactivity of 5-HTP was intensified in the colchicine-pretreated rat. The distribution of labelled cells was the same as for 5-HT-immunoreactive cells, but they were fewer in number.     

5-Hydroxytryptophan uptake and decarboxylating neurons in the cat hypothalamus

Parachlorophenylalanine, an inhibitor of tryptophan hydroxylase, induced a virtually total disappearance of serotonin-immunoreactivity in the hypothalamus of the cat. After intrahypothalamic injection of 5-hydroxytryptophan, an immediate precursor of serotonin in cats pretreated with parachlorophenylalanine, serotonin-immunoreactivity was detected in many fibers surrounding the injection site. Furthermore, when 5-hydroxytryptophan was injected with inhibitor of monoamine oxidase, a large number of small neurons immunoreactive to serotonin was identified in many discrete regions: the anterior and lateral hypothalamic areas, preoptic area, suprachiasmatic nucleus, dorsal hypothalamic area, dorsomedial nucleus, posterior hypothalamic area and nucleus of the fields of Forel. Serotonin-immunoreactivity was also evident in the thick axon bundles in the lateral hypothalamus. The distribution pattern of these cells was quite similar to that of aromatic L-amino acid decarboxylase, which catalyses the conversion of 5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. However, we failed to demonstrate serotonin-immunoreactivity in these parvocellular neurons without monoamine oxidase inhibitor. It is possible that 5-hydroxytryptophan is decarboxylated to serotonin by aromatic L-amino acid decarboxylase but rapidly degraded by monoamine oxidase-A, the enzyme which preferentially deaminates serotonin. In contrast, serotonin-immunostaining was always demonstrable after intrahypothalamic injection of 5-hydroxytryptophan without monoamine oxidase inhibitor in magnocellular neurons located in the ventrolateral posterior hypothalamus and which contain exclusively monoamine oxidase-B and histidine decarboxylase. It appears that in these cells and axons, serotonin, possibly formed by histidine decarboxylase, is not rapidly oxidized by monoamine oxidase-B. Possible roles of serotonin as a neurohormone in sleep-waking regulation and of trace amines in the brain are discussed.     

Paradoxical (REM) Sleep Deprivation Causes a Large and Rapidly Reversible Decrease in Long-Term Potentiation,Synaptic Transmission,Glutamate Receptor Protein Levels,and ERK/MAPK Activation in the Dorsal Hippocampus

Study Objectives:

It has been shown that wake (W) and slow wave sleep (SWS) modulate synaptic transmission in neocortical projections. However the impact of paradoxical sleep (PS) quantities on synaptic transmission remains unknown. We examined whether PS modulated the excitatory transmission and expression of glutamate receptor subtypes and phosphorylated extracellular signal-regulated kinases (p-ERK1/2).

Design:

PS deprivation (PSD) was carried out with the multiple platforms method on adult male Sprague-Dawley rats. LTP, late-LTP, and synaptic transmission were studied in the dorsal and ventral hippocampus of controls, 75-h PSD and 150-min PS rebound (PSR). GluR1 and NR1 protein and mRNA expression were evaluated by western blot and real-time PCR. P-ERK1/2 level was quantified by western blot and immunohistochemistry.

Measurement and Results:

PSD decreased synaptic transmission and LTP selectively in dorsal CA1 and PSR rescued these deficits. PSD-induced synaptic modifications in CA1 were associated with a decrease in GluR1, NR1, and p-ERK1/2 levels in dorsal CA1 without change in GluR1 and NR1 mRNA expression. Regression analysis shows that LTP is positively correlated with both PS quantities and SWS episodes duration, whereas synaptic transmission and late-LTP are positively correlated with PS quantities and negatively correlated with SWS quantities.

Conclusions:

These findings unveil previously unrecognized roles of PSD on synaptic transmission and LTP in the dorsal, but not in the ventral, hippocampus. The fact that the decrease in protein expression of GluR1 and NR1 was not associated with a change in mRNA expression of these receptors suggests that a sleep-induced modulation of translational mechanisms occurs in dorsal CA1.

Citation:

Ravassard P; Pachoud B; Comte JC; Mejia-Perez C; Scoté-Blachon C; Gay N; Claustrat B; Touret M; Luppi PH; Salin PA. Paradoxical (REM) sleep deprivation causes a large and rapidly reversible decrease in long-term potentiation, synaptic transmission, glutamate receptor protein levels, and ERK/MAPK activation in the dorsal hippocampus. SLEEP 2009;32(2):227–240.     

Ultrasonographic control of the puncture level for lumbar neuraxial block in obstetric anaesthesia

BACKGROUND: Errors in the judgement of puncture level during neuraxial anaesthesia can lead to significant complications. The aim of this study was to assess, in obstetric anaesthesia, the accuracy of clinical determination of the lumbar spinal interspace level, using surface ultrasound imaging as control. METHODS: At the anaesthesia follow-up visit, women who had received lumbar neuraxial anaesthesia during labour were prospectively included. The intervertebral level of needle insertion, located by the needle scar position, was identified by ultrasonography and compared with the clinical level reported on the chart by the anaesthetist who performed the block. RESULTS: Ninety-nine women were studied. The clinical puncture level was accurate in 36.4% of patients. Ultrasound examination showed the puncture level to be more cephalad than the level noted in the anaesthetic record in almost 50% of patients. In 15% of patients, the puncture level was more caudad than the anaesthetist had assessed. Factors including type of anaesthesia, indication, time period, level of anaesthetic experience, BMI, and spinal pathology did not seem to influence the frequency of errors. CONCLUSIONS: The observed differences between clinical and ultrasonic identification of spinal puncture level highlight the potential for serious complications associated with the performance of neuraxial blocks above the spinous process of L3 in the parturient. With the increase in popularity of techniques involving puncture of the dura mater for labour anaesthesia, we feel that awareness of this risk is important.     

In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force

Phagosomes employ lytic enzymes, cationic peptides, and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these microbicidal mechanisms is potentiated by the acidic pH created by H(+)-pumping vacuolar-type ATPases (V-ATPases) on the phagosomal membrane. The degree of phagosomal acidification varies greatly among neutrophils, macrophages, and dendritic cells and can be affected by diseases like cystic fibrosis. The determinants of phagosomal pH are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. When counterion conductance is limiting, generation of a large membrane potential will dominate the proton-motive force (pmf), with a proportionally diminished pH gradient. Validation of this notion requires direct measurement of the electrical potential that develops across the phagosomal membrane (Psi(Phi)). We describe a noninvasive procedure to estimate Psi(Phi) in intact cells, based on fluorescence resonance energy transfer. This approach, in combination with measurements of phagosomal pH, enabled us to calculate the pmf across phagosomes of murine macrophages and to analyze the factors that limit acidification. At steady state, Psi(Phi) averaged 27 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A. The comparatively small contribution of the potential to the pmf suggests that proton pumping is not limited by the counterion permeability, a notion that was validated independently by using ionophores. Instead, phagosomal pH stabilizes when the rate of proton pumping, which decreases gradually as the lumen acidifies, is matched by the passive leak of proton equivalents.     

Molecular and cellular mechanisms underlying iron transport deficiency in microcytic anemia

A mutation of the iron transporter Nramp2 (DMT1, Slc11a2) causes microcytic anemia in mk mice and in Belgrade rats by impairing iron absorption in the duodenum and in erythroid cells, causing severe iron deficiency. Both mk and Belgrade animals display a glycine-to-arginine substitution at position 185 (G185R) in the fourth predicted transmembrane domain of Nramp2. To study the molecular basis for the loss of function of Nramp2(G185R), we established cell lines stably expressing extracellularly tagged versions of wild-type (WT) or mutated transporters. Like WT Nramp2, the G185R mutant was able to reach the plasmalemma and endosomal compartments, but with reduced efficiency. Instead, a large fraction of Nramp2(G185R) was detected in the endoplasmic reticulum, where it was unstable and was rapidly degraded by a proteasome-dependent mechanism. Moreover, the stability of the mutant protein that reached the plasma membrane was greatly reduced, further diminishing its surface density at steady state. Last, the specific metal transport activity of plasmalemmal Nramp2(G185R) was found to be significantly depressed, compared with its WT counterpart. Thus, a singlepoint mutation results in multiple biosynthetic and functional defects that combine to produce the impaired iron deficiency that results in microcytic anemia.     

The locus coeruleus: a quantitative and genetic study in mice

A fluorescence histochemical study of the pontine catecholaminergic (CA) neurons of two inbred strains of mice, BALB/c (C) and C57BL/6 (B6) and their F1 hybrids revealed that the total number of thefluorescent cells in the C strain was 38% lower than that of the B6 strain. In F1 hybrids, the total number of neurons was not statistically different from that of the B6 parent. During the development from birth to adulthood, the same quantitative differences between strains were observed.     

Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. I. Origins of the reticulospinal tracts and their funicular trajectories

Using a retrograde tracer technique with horseradish peroxidase (HRP) attempts were made to determine the origins of reticulospinal tracts and their funicular trajectories. Reticulospinal tracts originating from the mesencephalic reticular formation (RF) were composed of: (1) descending projections arising from the cluster of cells located just lateral to the periaqueductal gray that course in the anterior funiculus (AF) and ventral part of the lateral funiculus (LF) with ipsilateral predominance; and (2) projections from the cluster of cells located dorsal to the brachium conjunctivum that course in the ipsilateral LF. Origins of the pontine reticulospinal tracts arising from the n. reticularis pontis oralis (Poo) have been divided qnto three parts: (1) medial one-third; (2) middle; and (3) ventrolateral. The axons from the medial part descend ipsilaterally via the medial part of the AF, while the axons from the ventrolateral part of the Poo give rise to diffuse descending projections in the AF and LF. The middle part of the Poo has been further subdivided into: (1) dorsal part that gives rise to spinal projections ipsilaterally in the ventrolateral funiculus (VLF); and (2) ventral, particularly its upper part, whose axons descend bilaterally via the DLF. Origins of reticulospinal tracts from the n. reticularis pontis caudalis (Poc) could be divided into three parts: (1) medial; (2) dorsolateral; and (3) ventrolateral. The medial part of the Poc is a source of axons via the medial part of the ipsilateral AF, while the ventrolateral part of the nucleus is a source of axons via the contralateral LF. The spinal projections from the dorsolateral part of the Poc appears to course diffusely in the AF and LF, but with DLF predominance. The n. reticularis gigantocellularis (Gc) was found to be a main medullary source of the spinal projections in the ipsilateral AF, while n. reticularis magnocellularis (Mc) is the major source of the fibers coursing ipsilaterally in the VLF. The most medial part of the Mc descends ipsilaterally via the medial part of the AF, while the ventrolateral part of the nucleus together with the n. reticularis lateralis of Meesen and Olszewski descends ipsilaterally via the DLF. It has also been found that the axons from the n. reticularis paramedianus pass via both the AF and LF with ipsilateral predominance, while the n. reticularis dorsalis and ventralis course via the LF with ipsilateral predominance.