Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils.

PrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher Mr forms (Mr 34,000 and 29,000) of PrP can be reduced in apparent Mr to a lower Mr form (Mr 25,000) with Peptide-N-glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher Mr forms whereas others bind more strongly to the lower Mr form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in Mr. The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.

Regional distribution of progesterone and 5 alpha-pregnane-3,20-dione in rat brain during progesterone-induced "anesthesia".

Progesterone and 5 alpha-pregnane-3,20-dione (5 alpha-DHP) concentrations were measured in seven brain areas and in plasma during "anesthesia" induced by progesterone (1-2 mg IV) in female rats. The highest levels of progesterone were detected in the striatum and hypothalamus (23.3 +/- 5.27 and 22.7 +/- 4.30 micrograms/g +/- SEM, respectively); these concentrations were approximately 1000 times higher than those during the post-ovulatory phase. Highest levels of 5 alpha-DHP were detected in the striatum and hippocampus (11.5 +/- 1.74 and 10.4 +/- 3.15 micrograms/g +/- SEM, respectively). The ratio of 5 alpha-DHP to progesterone was approximately 100 times higher in brain tissue than in plasma. We conclude that a conversion of progesterone to 5 alpha-DHP occurs in the brain during the course of progesterone-induced "anesthesia". This metabolic step may be an important contributory factor to the anesthetic potency of progesterone.

Are adrenergic neurons subject to a serotoninergic influence in the nucleus tractus solitarii? A morphological and biochemical study in the rat.

The possible relationships between adrenaline-synthesizing neurons and serotoninergic afferent fibers in the nucleus tractus solitarii of the rat were investigated both morphologically and biochemically. Adrenergic elements (cell bodies, dendrites and nerve endings) were detected simultaneously with serotoninergic axonal varicosities in the same electron-microscopic sections by means of combined phenylethanolamine-N-methyltransferase immunocytochemistry and [3H]serotonin-uptake radioautography. Among some 500 serotoninergic varicosities scanned in the areas of significant overlap between the 2 types of labeling, only 3 were directly apposed to an adrenergic process, identified as a dendrite in each case. No synaptic membrane differentiations were seen at these occasional sites of contact. Destruction of the serotonin input by 5,7-dihydroxytryptamine had no significant effect on the tyrosine hydroxylase dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase enzymatic activities in the C2 adrenergic region, but induced 22% and 38% increases of tyrosine hydroxylase and dopamine-beta-hydroxylase activities, respectively, in the neighboring A2 noradrenergic area. Taken together, these results suggest that serotoninergic and adrenergic neurons do not significantly interact in the nucleus tractus solitarii; this implies that the possible catecholaminergic relays for the action of serotonin in autonomic regulation at this level could consist of noradrenergic neurons rather than of their adrenergic counterparts.

Chronic benzodiazepine administration. IV. Rapid development of tolerance and receptor downregulation associated with alprazolam administration.

The triazolobenzodiazepine compound alprazolam may have unique clinical effects compared to other benzodiazepines, and both behavioral and neurochemical studies have indicated unusual results after acute doses of alprazolam. To determine the effects of chronic dosage in mice, alprazolam (2 mg/kg/day) was administered via osmotic pumps for 1-14 days, and open-field activity, plasma and brain concentrations, benzodiazepine receptor binding in vivo and in vitro, [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding, and muscimol-stimulated chloride uptake were determined. Alprazolam decreased motor activity after 1 and 2 days, but tolerance developed by day 4 and persisted to day 14. Plasma and brain concentrations remained constant during the 2-week period. Benzodiazepine receptor binding in vivo was decreased at day 4 compared to day 1 in cortex (CX) and hypothalamus (HYPO), and remained depressed to day 14 in CX but not HYPO. Benzodiazepine binding in vitro and [35S]TBPS binding were decreased in CX at day 7. Muscimol-stimulated [36Cl-] uptake was decreased at days 4 and 7 compared to day 1, but at day 14 uptake was similar to day 1. These results indicate that behavioral tolerance and receptor downregulation develop rapidly during chronic alprazolam administration. Behavioral and neurochemical changes were similar to those associated with lorazepam administration, but occurred more rapidly and with different regional specificity.

Neuronal RNA in nucleus ambiguus and nucleus hypoglossus of patients with amyotrophic lateral sclerosis.

To establish objectively the involvement of neurons in the medulla oblongata in patients with amyotrophic lateral sclerosis (ALS), ribonucleic acid (RNA) content was determined in neurons of the hypoglossal nucleus and the nucleus ambiguus. Neurons from those two nuclei showed a significant loss of RNA content in patients with ALS; only 57% and 38% of the normal RNA content was found in hypoglossal and ambiguus neurons, respectively. This marked loss of neuronal RNA suggests changes in functional states of neurons, which may contribute to fasciculations in the tongue and difficulties in swallowing often associated with ALS.

Comparison of synenkephalin and methionine enkephalin immunocytochemistry in rat brain.

Immunocytochemistry using an antiserum to the C-terminal octapeptide of synenkephalin, proenkephalin(63-70), was performed throughout the rat brain and revealed numerous immunopositive fibers and some cell bodies. The morphology and distribution of synenkephalin immunoreactivity was extremely similar to that of a commercial methionine enkephalin (Met-ENK) antiserum. Colchicine pretreatment allowed the immunostaining of cell bodies not otherwise possible without pretreatment, but did not affect the distribution of immunoreactive fibers. Using 6 microns serial sections, we were able to colocalize synenkephalin and Met-ENK immunoreactivities in gigantocellular neurons of the medullary reticular formation. Preabsorption of the antiserum with [Tyr63]proenkephalin(63-70) octapeptide (YEESHLLA) completely eliminated immunoreactivity in the rat brain, while preabsorption with all other peptides used had no detectable effect. We conclude that our antiserum to synenkephalin is specific for enkephalinergic cell bodies, fibers and terminals. The synenkephalin antiserum used in these studies may have advantages over other antisera utilized for immunocytochemical detection of proenkephalin gene expression.

Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat.

Thyrotropin-releasing hormone-like immunoreactivity (TRH-LI) was localized at the ultrastructural level in the dorsal vagal complex (DVC: dorsal motor nucleus of the vagus (DMV) and the nucleus of the solitary tract (NST] in rat. TRH-LI was concentrated in large granular vesicles in axons, presynaptic terminals, and non-synaptic axon varicosities. TRH-LI presynaptic terminals established both asymmetric and symmetric synaptic contacts with dendrites. These observations are consistent with recently described direct inhibitory and facilitatory effects of TRH on the electrical activity of neurons in the DVC.

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

ECF pH dynamics within the ventrolateral medulla: a microelectrode study.

Using pH-sensitive microelectrodes, we evaluated pH dynamics of extracellular fluid (ECF) within the ventrolateral medulla (VLM) beneath the central chemoceptive areas in anesthetized, spontaneously breathing cats. Static ECF pH was acid in the superficial layers (less than 1 mm), compared with the overlying cerebrospinal fluid pH that became alkaline gradually during the experiments. In the deeper VLM areas (1-3 mm), no systematic gradients of ECF pH were observed. We found various, isolated regions where intravertebral artery injections of CO2-saturated saline evoked acidic shift of ECF pH in the time course analogous to ventilatory augmentation. Those responsive regions were found to be scattered not only in the superficial layers but also in the deeper VLM areas, although many nonresponsive regions were also intermingled among them. Occlusions of the principal vessels supplying the tested VLM regions diminished but failed to abolish the ECF pH responses to the CO2 loadings, suggesting a collateral blood flow by fine pial vessels. The present study suggests a possibility that the pH-dependent central chemoreceptors, if any, would be scattered in the deeper VLM areas as well as the superficial layers.

Serotonin and substance P colocalization in medullary projections to the nucleus tractus solitarius: dual-colour immunohistochemistry combined with retrograde tracing.

Serotonin (5HT) and substance P (SP) are colocalized in terminals within the nucleus tractus solitarius (NTS). The purpose of the present study was to determine the origin of these terminals. 5HT- and SP-immunoreactivities (IR) were visualized using dual-colour immunofluorescence histochemistry with amino-4-methylcoumarin-3-acetic acid- and fluorescein isothiocyanate-conjugated secondary antisera, while NTS-afferent neurons were visualized by retrograde labelling with rhodamine beads. Extensive colocalization of 5HT- and SP-IR was seen in NTS-afferent neurons located in the nucleus raphe pallidus, nucleus raphe obscurus, nucleus raphe magnus, and in the parapyramidal region. Over 80 per cent of the SP-IR NTS-afferent neurons contained 5HT-IR, while 68 per cent of the 5HT-IR neurons contained SP-IR. Thus, 5HT- and SP-IR are extensively colocalized in NTS-afferent neurons in the medullary raphe nuclei and associated areas of the ventral medulla.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the medulla oblongata of the cat, in relation to choline acetyltransferase-immunoreactive motoneurones and substance P-immunoreactive fibres.

The immunohistochemical distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (ir) in the cat medulla oblongata was examined using an antiserum to rat alpha-CGRP. Comparative distributions of substance P (SP)-like and choline acetyltransferase (ChAT)-like ir were also studied on sections adjacent to those stained for CGRP, and on sections simultaneously stained for CGRP by double staining techniques. The vast majority of ChAT-ir motoneurones in somatomotor or branchiomotor cranial nuclei (of VI, VII and XII nerves) and their accessory nuclei also displayed a coarsely granular CGRP-ir, shown by electron microscopic examination to represent immunoreactive Golgi bodies. The nucleus ambiguus (IX and X nerves), a mixed branchiomotor and visceromotor nucleus, showed CGRP-ir in a lower proportion of its motoneurones, whereas the purely viseromotor dorsal motor vagal nucleus (X nerve) contained no CGRP-ir cells. A few CGRP-ir but ChAT-negative cells were seen in the ventromedial reticular formation, lateral cuneate nucleus, infratrigeminal nucleus and nucleus of the solitary tract. Coarse, often varicose CGRP-ir fibres were most prominent in the X and IX cranial nerve rootlets, the spinal tract of the V nerve and the solitary tract, and also in the V spinal nucleus and nucleus of the solitary tract. Many of these also appeared to contain SP-ir. The central patterns of CGRP and SP-ir fibres thus reflect the previously reported coexistence of these peptides in sensory afferent cells of the trigeminal and nodose ganglia. These results are consistent with a role for CGRP as a transmitter or modulator in efferents to striated muscle, sensory afferents and intrinsic neurones in the cat brain stem.

The connections from botzinger expiratory neurons to upper cervical inspiratory neurons in the cat.

These experiments examined possible inhibitory inputs to upper cervical inspiratory neurons from the expiratory neurons of the Botzinger complex. Eighty-one Botzinger neurons were tested with antidromic mapping for a projection to the C1 segment of the spinal cord; 44/81 (54%) were found to project, 27/79 (34%) contralaterally, 17/68 (25%) ipsilaterally, and 1/66 (2%) both contralaterally and ipsilaterally. Antidromic mapping in contralateral C1 demonstrated the presence of a collateral in 3/15 (20%) of the Botzinger neurons tested, while 3/9 (33%) had collateral arborizations in ipsilateral C1. The collaterals mapped were not localized to the region of the upper cervical inspiratory neurons. Microstimulation in C3 (12-17 microA, 0.2-ms duration) at locations which produced short-latency (2.7-3.5 ms) inhibition of phrenic nerve discharge resulted in the short latency (3.0 ms) inhibition of 1/27 (3.7%) upper cervical inspiratory neurons as demonstrated by cross-correlation. It was concluded that while some upper cervical inspiratory neurons may be inhibited during expiration by the Botzinger expiratory neurons, this connection is not a strong one.

The concentration of neuropeptide Y immunoreactivity falls with age selectively in microdissected regions of the ventrolateral medulla of spontaneously hypertensive and normotensive Wistar-Kyoto rats.

The concentrations of neuropeptide Y immunoreactivity were measured in microdissected regions enriched in noradrenergic (A1, A2, A6) and adrenergic (C1, C2, C3) nuclei of the brainstem, and in the nucleus of the spinal tract of the trigeminal nerve (Sp5C) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at 8, 18 and 31 weeks of age. The aim of this study was to compare the manner in which changes in neuropeptide Y immunoreactivity levels related to increases in blood pressure with ageing in each rat strain. The concentration of neuropeptide Y immunoreactivity in the A1 nucleus progressively fell with increasing age in both SHR and WKY. In contrast, the levels of neuropeptide Y immunoreactivity in the C1 region fell at 18 weeks of age but did not fall further by 31 weeks. No significant age-related changes occurred in the concentrations of neuropeptide Y immunoreactivity in the A2, C2, A6 and Sp5C nucleus. The levels of neuropeptide Y immunoreactivity in the C3 region were below assay sensitivity. The neurochemical changes that occur in the A1 nucleus are consistent with the increase in blood pressure observed with ageing in both rat strains. However, this observation alone does not account for the elevated blood pressure measured in the SHR strain.

Distribution of serotonin 5-HT1C receptor mRNA in adult rat brain.

Based on in situ hybridization histochemistry (ISHH), we describe the anatomical distribution of the serotonin 5-HT1C receptor mRNA. In addition to the very high levels in epithelial cells of the choroid plexus, 5-HT1C receptor mRNA is found throughout the limbic system, in catecholaminergic cells and in serotonergic neurons. Receptor transcripts are also present in the hypothalamus, numerous motor nuclei and the subthalamus. Our results correlate well with serotonin (5-HT) innervation and receptor binding. Receptor mRNA is present in many brain structures in addition to regions previously shown to have 5-HT1C receptor binding. The distribution of this receptor mRNA suggests that the 5-HT1C receptor may mediate a number of the central effects of 5-HT.

Decreased density of alpha 2-adrenoceptors in medulla oblongata of spontaneously hypertensive rats.

To study the role of medullary alpha-adrenoceptors in hypertension, we compared specific binding of [3H]prazosin and [3H]clonidine in different brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and normotensive Wistar-Kyoto rats (WKY). As compared with age-matched WKY, Bmax values for specific [3H]clonidine binding in the medulla oblongata were significantly lower in SHR and SHRSP at 16-24 weeks of age. In the SHRSP medulla oblongata, the decrease was more prominent in dorsomedial and ventrolateral regions than in the ventromedial region. Density of alpha 2-adrenoceptor binding sites was also decreased in the medulla oblongata of young (4-5-week-old) SHRSP. In contrast, there was no difference in Kd and Bmax values for medullary [3H]prazosin binding between WKY and SHRSP. The dorsomedial and ventrolateral regions of the SHRSP medulla oblongata showed significantly lower levels of norepinephrine (NE). Thus, the present study demonstrates that there is a specific loss of alpha 2-adrenoceptors in the medulla oblongata of SHR and SHRSP that may be partly involved in the pathogenesis of spontaneous hypertension.

Substance P-immunoreactivity in the dorsal medial region of the medulla in the cat: effects of nodosectomy.

The distribution of substance P in the vagal system of the cat was studied by immunohistochemistry. Substance P-immunoreactive cell bodies and fibres were observed in the nodose ganglion. Numerous substance P-immunoreactive terminals and fibres were localized in their bulbar projection area, i.e. throughout the caudo-rostral extent of the nucleus of the solitary tract. Four subnuclei, among the nine forming the nucleus of the solitary tract, were strongly labelled: interstitial, gelatinosus, dorsal and commissural. The dorsal motor nucleus of the vagus nerve also exhibited numerous substance P-immunoreactive terminals, sometimes closely apposed on the somata of preganglionic neurons. To determine the substance P component of the vagal afferent system a nodose ganglion was removed on one side. The ablation triggered ipsilaterally a large decrease of substance P immunoreactivity in the four subnuclei strongly labelled on normal cats. These results suggest the involvement of substance P-containing vagal fibres in integrative processes of the central regulation of cardiovascular, digestive and respiratory systems, viscerotopically organized throughout these four subnuclei. The nodose ablation also resulted in a decrease of substance P immunoreactivity in the ipsilateral dorsal motor nucleus of the vagus nerve, suggesting monosynaptic vago-vagal interactions.

Distribution of substance P, vasoactive intestinal polypeptide and serotonin immunoreactive structures in the central nervous system of the lizard, Lacerta agilis.

The distribution of substance P (SP), vasoactive intestinal polypeptide (VIP) and serotonin (5-HT) containing structures in the central nervous system of the lizard, Lacerta agilis, were investigated with the aid of the peroxidase-antiperoxidase immunohistochemical method. SP and 5-HT nerve fibers are found almost all over the brain, whereas VIP fibers are mainly observed in the limbic area, in the periventricular gray matter of the diencephalon and in the brain stem. The cells of origin are located in the telencephalon for the SP, in the mesencephalic tegmentum for the VIP, and in the midline and the basis of the brain stem for the 5-HT. In addition to the known paraventricular organ in the diencephalon VIP and 5-HT immunoreactive cells are found in the ependymal layer of the rostral part of the lateral ventricle.

Altered amino acid levels in brainstem regions of spontaneously hypertensive rats.

The contents of glutamate, gamma-aminobutyric acid (GABA), glycine, beta-alanine and taurine in brainstem regions responsible for blood pressure regulation, the rostral ventrolateral medulla (RVL), the nucleus tractus solitarii (NTS) and the caudal ventrolateral medulla (CVL) regions, were determined in spontaneously hypertensive rats (SHR) as compared with Wistar Kyoto rats. In SHR, the content of glutamate was increased in the RVL, NTS and CVL regions whereas GABA and beta-alanine contents were decreased in the RVL and all the regions studied, respectively. There was no difference between WKY and SHR in glycine and taurine contents in all the regions studied. These results provide evidence suggesting alterations of brainstem amino acid metabolism in SHR.

Cholecystokinin octapeptide immunoreactivity in the nucleus tractus solitarius of the cat.

The nucleus tractus solitarius possessed distinct patterns of cholecystokinin immunoreactive fibers and cell bodies within its various subdivisions. The commissural, medial, intermediate, parvocellular, dorsolateral and interstitial subdivisions contained relatively dense amounts of CCK immunolabelled fibers. In contrast, CCK immunoreactivity within the ventrolateral subdivision consisted of a few scattered fibers and small neurons. The commissural, intermediate, medial, dorsolateral and parvocellular subdivisions contained CCK immunoreactive neurons following colchicine treatment. The presence of CCK in the NTS suggest that it may be involved as a neuromodulator and/or neurotransmitter in circuitry that mediate cardiovascular, respiratory, gastrointestinal and taste functions.