Phospholipidosis in neurons caused by posaconazole, without evidence for functional neurologic effects.

The azole antifungal drug posaconazole caused phospholipidosis in neurons of the central nervous system, dorsal root ganglia of the spinal cord, and myenteric plexus in chronic toxicity studies in dogs. The time of onset, light and electron microscopic features, neurologic and electrophysiologic effects on the central and peripheral nervous systems, and potential for regression were investigated in a series of studies with a duration of up to one year. Nuclei of the medulla oblongata were the prominently affected areas of the brain. Neurons contained cytoplasmic vacuoles with concentrically whorled plasma membrane-like material (i.e., multilamellar bodies) morphologically identical to that commonly caused in other tissues by cationic amphiphilic drugs. Some axons in the brain and spinal cord were swollen and contained granular eosinophilic, electron-dense lysosomes. There were no features suggesting degeneration or necrosis of neurons or any associated elements of nervous tissue. The earliest and most consistent onset was in neurons of dorsal root ganglia. The observed neural phospholipidosis did not result in any alteration in the amplitude or latency of the auditory, visual, or somatosensory evoked potentials. The histopathologic changes did not progress or regress within the three-month postdose period. The results indicate that phospholipidosis can be induced in central and peripheral neurons of dogs by administration of posaconazole, but this change is not associated with functional effects in the systems evaluated.

Effect of neonatal capsaicin treatment on neural activity in the medullary dorsal horn of neonatal rats evoked by electrical stimulation to the trigeminal afferents: an optical, electrophysiological, and quantitative study.

To elucidate which glutamate receptors, NMDA or non-NMDA, have the main role in synaptic transmission via unmyelinated afferents in the trigeminal subnucleus caudalis (the medullary dorsal horn), and to examine the early functional effects of neonatal capsaicin treatment to the subnucleus caudalis, optical recording, field potential recording, and quantitative study using electron micrographs were employed. A medulla oblongata isolated from a rat 5--7 days old was sectioned horizontally 400-microm thick or parasagittally and stained with a voltage-sensitive dye, RH482 or RH795. Single-pulse stimulation with high intensity to the trigeminal afferents evoked optical responses mainly in the subnucleus caudalis. The optical signals were composed of two phases, a fast component followed by a long-lasting component. The spatiotemporal properties of the optical signals were well correlated to those of the field potentials recorded simultaneously. The fast component was eliminated by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10 microM), while the long-lasting component was not. The latter increased in amplitude under a condition of low Mg(2+) but was significantly reduced by DL-2-amino-5-phosphonovaleric acid (AP5; 30 microM). Neonatal capsaicin treatment also reduced the long-lasting component markedly. In addition, the decreases in the ratio of unmyelinated axons to myelinated axons and in the ratio of unmyelinated axons to Schwann cell subunits of trigeminal nerve roots both showed significant differences (P<0.05, Student's t-test) between the control group and the neonatal capsaicin treatment group. This line of evidence indirectly suggests that synaptic transmission via unmyelinated afferents in the subnucleus caudalis is mediated substantially by NMDA glutamate receptors and documented that neonatal capsaicin treatment induced a functional alteration of the neural transmission in the subnucleus caudalis as well as a morphological alteration of primary afferents within several days after the treatment.

Cuneothalamic relay neurons are postsynaptic to glycine-immunoreactive terminals in the rat cuneate nucleus.

This study was aimed to clarify whether the cuneothalamic relay neurons (CTNs) in the rat cuneate nucleus contained glycine or whether the neurons were modulated directly by presynaptic glycine-IR terminals. For this purpose, retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and immunoperoxidase labelling for glycine have been used to ascertain if the CTNs in the rat are glycine-immunoreactive (glycine-IR). Our results have shown that the WGA-HRP-labelled CTNs (mean area = 318 +/- 6.5 microm(2)) were not reactive for glycine. Glycine immunoreactivity, however, was localized in some small-sized neurons (mean area = 210 +/- 6.2 microm(2)) and axon terminals associated with the CTNs. The synaptic organization between the glycine-IR terminals and CTNs was further analyzed using anti-glycine postembedding immunogold labelling. By electron microscopy, the immunogold-labelled glycine-IR terminals containing pleomorphic synaptic vesicles formed symmetrical synaptic contacts with the dendrites, dendritic spines, and somata of CTNs. Quantitative estimation showed that the mean ratios of glycine-IR terminals to total terminals associated with the soma, proximal dendrites and distal dendrites of the CTN were 49.5, 45.2, and 45.8%, respectively. The higher incidence of glycine-IR terminals on the soma, however, was not significantly different from that of the proximal and distal dendrites. Notwithstanding the above, this study has shown a large number of glycine-IR terminals making direct synaptic contacts with CTNs, suggesting that glycine is one of the important neurotransmitters involved in postsynaptic inhibition on the cuneothalamic relay neurons to modulate incoming somatosensory information from forelimb areas in the rat.

Neurons in the caudal ventrolateral medulla projecting to the paraventricular hypothalamic nucleus receive synaptic inputs from the nucleus of the solitary tract: a light and electron microscopic double-labeling study in the rat.

We used light and electron microscopic techniques to investigate the possibility that neurons in the nucleus of the solitary tract (NST) might send projection fibers to neurons in the caudal ventrolateral medulla (CVLM) projecting to the paraventricular hypothalamic nucleus (PVN) by the anterograde and retrograde double labeling method in the rat. The retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, was injected into the PVN, and the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the NST of each rat. Many retrogradely labeled neurons were encountered in the CVLM, including the A1 region. On the other hand, a dense network of anterogradely labeled terminals was found in the CVLM. Electron microscopic examination revealed synaptic contacts between PHA-L-immunoreactive nerve terminals and dendrites of retrogradely labeled neurons in the CVLM. The results indicate that CVLM neurons projecting to the PVN receive axon terminals of NST neurons.

Anatomic patterns of Fos immunostaining in rat brain following systemic endotoxin administration.

To identify brain neurons that participate in the acute phase response, rat brains were examined immunocytochemically for Fos protein following the intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS). Two to three hours after the injection of LPS, 150 micrograms/kg body weight, to adult male Long-Evans rats, a consistent anatomic pattern of Fos immunostained cell nuclei is seen. In the brain stem, prominant Fos immunostaining is induced in tyrosine hydroxylase immunoreactive neurons of the caudal ventral-lateral medulla (the A1 cell group), in both tyrosine hydroxylase positive and negative neurons of nu. tractus solitarius, in the parabrachial nu., and in a few neurons of the locus ceruleus. In the hypothalamus, endotoxin induces Fos expression in magnocellular neurons of the paraventricular and supraoptic nuclei and internuclear cell groups. A higher percentage of oxytocin-immunoreactive cells is double labeled for Fos nuclear immunostaining than vasopressin-immunoreactive cells. A minority of somatostatin immunoreactive periventricular hypothalamic neurons are Fos positive. Other hypothalamic nuclei that contain endotoxin-induced Fos nuclear immunostaining include the parvocellular neurons of the paraventricular nu., the dorsomedial and arcuate nuclei, the lateral hypothalamus, the dorsal hypothalamic area (zona incerta), and the median nucleus of the preoptic area. LPS induces numerous Fos-positive neurons in regions known to respond to a variety of stressful stimuli; these regions include the preoptic area, bed nucleus of the stria terminalis, lateral septum, and the central and medial nuclei of the amygdala. Moreover, Fos nuclear immunostaining is seen in neurons of circumventricular organs: the organum vasculosum of the lamina terminalis, the subfornical organ, and the area postrema. The maximum intensity of Fos nuclear immunostaining occurs 2-3 h after endotoxin administration and declines thereafter. It is attenuated by pretreatment with indomethacin, 25 mg/kg body weight Sc, or dexamethasone, 1 mg/kg IP. These observations are consistent with the participation of a variety of brain neuronal systems in the acute phase response and elucidate the functional neuroanatomy of that response at the cellular level.

Medullary synaptic inputs to thyrotropin-releasing hormone (TRH)-containing neurons in the hypothalamus: an ultrastructural study combining WGA-HRP anterograde tracing with TRH immunocytochemistry.

Ascending projections from the A1/C1 cell group and from the A2 cell group in the medulla oblongata was studied in the light microscope by anterograde tracing of Phaseolus vulgaris leucoagglutinin and in the electron microscope by anterograde tracing of wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) combined with thyrotropin-releasing hormone (TRH) immunocytochemistry in the hypothalamic paraventricular nucleus (PVN). WGA-HRP-labeled axon terminals originating from neurons in the A1/C1 or the A2 cell group were found to make synaptic contacts with TRH-containing cell bodies and dendrites in the medial parvocellular part of the PVN, usually forming axo-dendritic synapses. Of all the afferent synapses on TRH neurons in the PVN, 9.8-20.9% of the presynaptic axon terminals were WGA-HRP-positive. This indicates that each brain stem catecholaminergic cell group that contribute to innervation of the PVN is in a position to modulate the activity of TRH neurons.

Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Vasopressinergic mechanisms in the nucleus reticularis lateralis in blood pressure control.

We sought to determine whether arginine vasopressin (AVP) modulates arterial pressure (AP) by a receptor-mediated action in the nucleus reticularis rostroventrolateralis (nRVL). Immunocytochemical labeling with an antiserum against a synthetic AVP conjugate revealed a discrete although modest presumptive neuropeptidergic innervation of the nRVL. Electron microscopic analysis of vasopressinergic processes in the nRVL revealed that AVP-like immunoreactivity (AVP-LI) was primarily in axons and axon terminals. Immunoreactive terminals contained numerous small clear vesicles and large dense core vesicles and formed synapses with unlabeled dendrites. In the nRVL, retrograde transport-immunofluorescence data demonstrated close appositions between vasopressinergic beaded processes and a compact subambigual column of reticulospinal neurons labeled by deposits of cholera toxin beta-subunit into the thoracic spinal cord. Similar methods were used to define the origins of the AVP-afferent projection to nRVL. These retrograde transport-immunofluorescence studies demonstrated numerous retrogradely labeled neurons in the hypothalamus, including the paraventricular nucleus (PVN), after injections of a retrograde tracer, Fluoro-Gold into the ventrolateral medulla. However, double-labeled neurons were rare and confirmed a diffuse AVP afferent innervation of the sympathoexcitatory area. Microinjection of AVP into the nRVL in anesthetized rats produced a large dose-related increase in AP different from control at a dose of 1 pmol or higher. AVP injected intravenously elevated AP only at significantly higher doses. Microinjections of AVP into the nucleus tractus solitarii (NTS) had a smaller effect whereas into the caudal ventrolateral medulla exerted no effect on AP. Bilateral microinjections of an AVP antagonist, d(CH2)5[Tyr(Me)2]AVP into the nRVL produced no change in AP but blocked the increase produced by subsequent injections of AVP. An acute hemorrhage produced by withdrawal of 2 ml of blood from the femoral vein did not alter AP. However, bilateral microinjections of the AVP antagonist into the nRVL 5 min after hemorrhage decreased AP. In contrast, the AVP-antagonist injected intravenously after hemorrhage had no effect on AP. Our data suggest that under conditions demanding increased sympathetic drive to maintain AP, such as hemorrhage, a functional AVP receptor mechanism via terminals in the nRVL may be activated to restore normal levels of AP.

[The effect of stimulating the auricular liver-gall acupoint with electrode on synaptic of nucleus originis dorsalis nervi vagi in rabbits].

In this experiment, several ultrastructural parameters of Gray's type I synapses in nucleus originis dorsalis nervi vagi were observed and quantitatively analysed by use of electron microscopy in condition gallbladder contraction by stimulating the auricular liver-gall acupoint of rabbits with electrode. It was found that the synaptic contact length was increased extremely significantly, and the postsynaptic thickening length lengthened significantly. There were no changes in the area and the perimeter of presynaptic bouton as well as synaptic vesicle density. The percentages of synaptic perforation and negative synaptic interface tended towards increase. But there were no differences in synapses of positive and straight interface. It is suggested that the nucleus is involved in the regulation of gallbladder activity induced by auricular acupoint stimulation.

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI).

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.

Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem.

The efferent connections of two types neurons in the dorsal motor nucleus of the vagus nerve (DMV) were studied in the cat by light and electron microscopy following horseradish peroxidase (HRP) injections into the cervical vagus nerve or brainstem. After injections of HRP into the vagus nerve, up to 80% of medium-sized neurons averaging 26 x 20 micrometers in 1-micrometer-thick sections were retrogradely labeled while no small neurons were labeled in the DMV. Incubation with either diaminobenzidene (DAB) or p-phenylenediamine-pyrocatechol (PPD-PC) chromogens yielded electron-dense reaction products localized mainly in lysosomes. Identification of label at the ultrastructural level was facilitated by omitting lead citrate staining and by counting numbers of lysosomes, which were higher in labeled neurons. Quantitative comparisons of the dimensions of labeled and unlabeled somata demonstrated that retrograde transport and incorporation of HRP had no effect on cell size within the 2-3-day survival times used in this study. In order to determine whether neurons in the DMV project to higher levels of the brain stem, large injections of HRP (1-3 microliters) were made into the pons, mesencephalon, hypothalamus, and amygdala. After injections of HRP into the brainstem, only small neurons, measuring 17 x 10 micrometers, were retrogradely labeled. Approximately 90% of the small neurons remained unlabeled following the HRP injections. The ultrastructural features of the labeled small neurons included an invaginated nucleus, low cytoplasmic/nuclear ratio, and relatively fewer organelles than the medium-sized neurons. A quantitative analysis of labeled and unlabeled small neurons demonstrated that the labeled neurons were significantly larger than the unlabeled small neurons. Thus, two populations of small neurons may exist in the DMV. One population appears to have ascending projections to higher levels of the brainstem while the other more numerous population may be interneurons or project for only short distances.

Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat.

Fine structural localization of an ACTH-like substance was obtained in neurons of the rat arcuate nucleus using immuno-electron microscopy, whereas it could not be confirmed that ACTH-containing cell bodies are present in the supraoptic nucleus. The immunoreactive cells of the arcuate nucleus appeared to be more numerous than the unreactive neurons. Immunostaining was carried out before embedding in resin. Empty vesicles of irregular shape were found in dendrites of immunoreactive arcuate neurons, but their significance and nature remain enigmatic. The reaction product was distributed uniformly throughout the cytoplasm of the ACTH-positive cells, except that the mitochondria, rough endoplasmic reticulum and Golgi vesicles and cisternae were devoid of PAP molecules. This distribution differed from the localization reported in ACTH-secreting cells of the rat anterior pituitary, where the reaction product was found in the rough endoplasmic reticulum and Golgi complex as well as in secretory granules.

Magnocellular hypothalamic projections to the lower brain stem and spinal cord of the rat. Immunocytochemical evidence for predominance of the oxytocin-neurophysin system compared to the vasopressin-neurophysin system.

The paraventricular nucleus of the rat hypothalamus has been shown to project to the medulla and spinal cord. The proportion of oxytocin-neurophysin (OTNP) axons to vasopressin-neurophysin (VPNP) axons in these structures is unknown. A major difficulty in resolving this problem in previous immunocytochemical studies was the lack of a specific antiserum to each rat neurophysin. In this study two approaches have been used: (1) comparison of immunostaining for neurophysin in normal versus homozygous Brattleboro rats with diabetes insipidus (HODI) which lack VPNP, and (2) application of an antiserum to both rat neurophysins absorbed with HODI rat hypothalamic-pituitary extracts which contain only OTNP. The latter would result in an antiserum specific for VPNP. Our results indicate that the axons which constitute the caudal projections from the paraventricular nucleus are predominately oxytocinergic, the vasopressinergic innervation being limited to the nucleus tractus solitarius, the dorsal motor nucleus of vagus, and the substantia gelatinosa. A similar number of reactive fibers were seen in the medulla and spinal cord of normal and HODI rats. No positive perikarya were observed caudal to the hypothalamus. Fibers in the medulla appeared to terminate in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus nerve. Positive fibers throughout the cord were present in the substantia gelatinosa and in the intermediolateral grey. The possible role(s) of these projections in integrating autonomic functions and afferent information with neuroendocrine regulation is discussed.

Morphological findings in granular reticulum of arcuate nucleus neurons after clomiphene citrate administration.

The action of high doses of clomiphene citrate on the rough endoplasmic reticulum of the hypothalamic arcuate neurons of male cat has been studied. Clomiphene (250 mg/kg) produces an accumulation of typical paracrystalline material in the rough endoplasmic reticulum. After the administration of a protein-synthesis inhibitor no such material was observed in clomiphene-treated cats. The possibility exists that the specific ultrastructural changes seen indicate an increased protein synthesis in hypothalamic arcuate neurons.

A comparison of epithalamic, hypothalamic and spinal neurosecretory terminals.

Nerve endings of epithalamic, hypothalamic and spinal neurosecretory areas were studied by light and electron microscopy in various vertebrates (from fishes up to mammals) including the lancelet. Areas investigated were the pineal organ, the pulvinar corporis pinealis, the neurohypophysis, the median eminence, the urophysis, the terminal filum and the medullo-spinal neurosecretory zones. We found that in all these areas the neurosecretory endings have common structures, which we call synaptic hemidesmosomes or neurohormonal terminals. These are characterized by accumulation of vesicles, and dense projections in a terminal on the basal lamina of the surface of the nervous tissue. A critical review of the literature suggests that a considerble neuroendocrine activity is associated with synaptic hemidesmosomes as special neurohormonal effector structures of the nerve cells. The cell-to-cell synapses formed by neurosecretory cells are discussed in connection with the dual capacity of these cells to function as both endocrine and "ordinary# neuronal elements. The importance of the external cerebrospinal fluid (CSF) space for the transport of materials released in the so-called neurohemal areas, is stressed.

Experimental cerebral concussion. Part 1: An electron microscopic study.

Cerebral concussion was produced in rats by an iron pendulum hitting the external occipital protuberance. This resulted in loss of consciousness lasting from 3 to 10 minutes with prompt recovery and no focal neurological signs. The energy absorbed by the head at the impact was calculated to be about 1450 gm/cm. Light microscopic survey showed only minor pathological changes. However, electron microscopic observation revealed considerable alteration which began at 30 minutes, reached a peak at 1 hour, and disappeared at 24 hours after concussion. The salient changes included severe swelling of the neuronal mitochondria at the point of impact (occipital cortex), and extracellular edema at the site of contre coup (frontal lobe). Topographically, the most severe alteration was seen in structures at the craniospinal junction (medulla oblongata and upper cervical cord), consisting of both mitochondrial and edematous changes. Although there was no visible opening of the capillary interendothelial junctions, extravasated ferritin particles were accumulated in the edematous regions, indicating a transient increase in the permeability of the blood-brain barrier.

A fine structural study of degenerative changes in the dorsal column nuclei of aging mice. Lack of protection by vitamin E.

The dorsal column nuclei of young, old, and vitamin E-supplemented old mice were examined by light and electron microscopy. Evidence of neuroaxonal dystrophy (NAD) was found in young (3-mo.-old) mice and increased with age. Vitamin E, added to the diet in the amount of 0.3%, did not protect the nuclei from age-associated degeneration. The NAD was characterized by enlarged profiles containing patches of smooth reticular networks and groups of vesicles. Various stages of mitochondrial alteration, producing multivesicular bodies as intermediate stages, were found, and other unusual forms of dense bodies were also observed. Axons, synaptic terminals, and possibly glial cells were affected, and, by 23 mo. of age, a large number of nerve fibers in nucleus gracilis were dystrophic, while nucleus cuneatus was affected to a lesser extent.