Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata.

Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-BötC, or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.

The tree shrew cerebellum atlas: Systematic nomenclature, neurochemical characterization, and afferent projections.

The cerebellum is involved in the control of movement, emotional responses, and reward processing. The tree shrew is the closest living relative of primates. However, little is known not only about the systematic nomenclature for the tree shrew cerebellum but also about the detailed neurochemical characterization and afferent projections. In this study, Nissl staining and acetylcholinesterase histochemistry were used to reveal anatomical features of the cerebellum of tree shrews (Tupaia belangeri chinensis). The cerebellar cortex presented a laminar structure. The morphological characteristics of the cerebellum were comprehensively described in the coronal, sagittal, and horizontal sections. Moreover, distributive maps of calbindin-immunoreactive (-ir) cells in the Purkinje cell layer of the cerebellum of tree shrews were depicted using coronal, sagittal, and horizontal schematics. In addition, 5th cerebellar lobule (5Cb)-projecting neurons were present in the pontine nuclei, reticular nucleus, spinal vestibular nucleus, ventral spinocerebellar tract, and inferior olive of the tree shrew brain. The anterior part of the paramedian lobule of the cerebellum (PMa) received mainly strong innervation from the lateral reticular nucleus, inferior olive, pontine reticular nucleus, spinal trigeminal nucleus, pontine nuclei, and reticulotegmental nucleus of the pons. The present results provide the first systematic nomenclature, detailed atlas of the whole cerebellum, and whole-brain mapping of afferent projections to the 5Cb and PMa in tree shrews. Our findings provide morphological support for tree shrews as an alternative model for studies of human cerebellar pathologies.

[Value of 1H-MRS on SCA3/MJD diagnosis and clinical course].

OBJECTIVE: To investigate the value of proton magnetic resonance spectroscopy (1H-MRS) on the diagnosis of SCA3/MJD, and to calculate the correlation between 1H-MRS ratio and the clinical score.
 Methods: Sixteen patients with SCA3/MJD and 19 healthy volunteers were scanned with 1H-MRS. The data of N-acetyl aspartate, creatine, choline-containing compounds, myoinositol, NAA/Cr, Cho/Cr, and mI/Cr ratio were collected, which were grouped for comparative study. The onset patients with SCA3/MJD were evaluated with the International Cooperative Ataxia Rating Scale and Scale for the Assessment and Rating of Ataxia, the correlation between NAA/Cr, Cho/Cr or mI/Cr ratio and the clinical score was calculated.
 Results: The NAA/Cr in the pons and cerebellar dentate nucleus from the onset patients with SCA3/MJD was significantly reduced compared to that in the normal control group. The NAA/Cr in the cerebellar dentate nucleus of onset patients with SCA3/MJD was obviously correlated with ICARS.
 Conclusion: SCA3/MJD lesions are mainly located in the cerebellum and brainstem, where gray and white mater are also involved. The cerebellar dentate nucleus may be the earliest involved area. There is a correlation between the ICARS and the cerebellar lesion degree. The ICARS reflects the severity of clinical manifestations. 1H-MRS is useful in the diagnosis of SCA3/MJD.

Barrington's nucleus: Neuroanatomic landscape of the mouse "pontine micturition center".

Barrington's nucleus (Bar) is thought to contain neurons that trigger voiding and thereby function as the "pontine micturition center." Lacking detailed information on this region in mice, we examined gene and protein markers to characterize Bar and the neurons surrounding it. Like rats and cats, mice have an ovoid core of medium-sized Bar neurons located medial to the locus coeruleus (LC). Bar neurons express a GFP reporter for Vglut2, develop from a Math1/Atoh1 lineage, and exhibit immunoreactivity for NeuN. Many neurons in and around this core cluster express a reporter for corticotrophin-releasing hormone (BarCRH ). Axons from BarCRH neurons project to the lumbosacral spinal cord and ramify extensively in two regions: the dorsal gray commissural and intermediolateral nuclei. BarCRH neurons have unexpectedly long dendrites, which may receive synaptic input from the cerebral cortex and other brain regions beyond the core afferents identified previously. Finally, at least five populations of neurons surround Bar: rostral-dorsomedial cholinergic neurons in the laterodorsal tegmental nucleus; lateral noradrenergic neurons in the LC; medial GABAergic neurons in the pontine central gray; ventromedial, small GABAergic neurons that express FoxP2; and dorsolateral glutamatergic neurons that express FoxP2 in the pLC and form a wedge dividing Bar from the dorsal LC. We discuss the implications of this new information for interpreting existing data and future experiments targeting BarCRH neurons and their synaptic afferents to study micturition and other pelvic functions.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Pontine reticulospinal projections in the neonatal mouse: Internal organization and axon trajectories.

We recently characterized physiologically a pontine reticulospinal (pRS) projection in the neonatal mouse that mediates synaptic effects on spinal motoneurons via parallel uncrossed and crossed pathways (Sivertsen et al. [2014] J Neurophysiol 112:1628-1643). Here we characterize the origins, anatomical organization, and supraspinal axon trajectories of these pathways via retrograde tracing from the high cervical spinal cord. The two pathways derive from segregated populations of ipsilaterally and contralaterally projecting pRS neurons with characteristic locations within the pontine reticular formation (PRF). We obtained estimates of relative neuron numbers by counting from sections, digitally generated neuron position maps, and 3D reconstructions. Ipsilateral pRS neurons outnumber contralateral pRS neurons by threefold and are distributed about equally in rostral and caudal regions of the PRF, whereas contralateral pRS neurons are concentrated in the rostral PRF. Ipsilateral pRS neuron somata are on average larger than contralateral. No pRS neurons are positive in transgenic mice that report the expression of GAD, suggesting that they are predominantly excitatory. Putative GABAergic interneurons are interspersed among the pRS neurons, however. Ipsilateral and contralateral pRS axons have distinctly different trajectories within the brainstem. Their initial spinal funicular trajectories also differ, with ipsilateral and contralateral pRS axons more highly concentrated medially and laterally, respectively. The larger size and greater number of ipsilateral vs. contralateral pRS neurons is compatible with our previous finding that the uncrossed projection transmits more reliably to spinal motoneurons. The information about supraspinal and initial spinal pRS axon trajectories should facilitate future physiological assessment of synaptic connections between pRS neurons and spinal neurons.

Profiling differences in chemical composition of brain structures using Raman spectroscopy.

Raman spectroscopy enables non-invasive investigation of chemical composition of biological tissues. Due to similar chemical composition, the analysis of Raman spectra of brain structures and assignment of their spectral features to chemical constituents presents a particular challenge. In this study we demonstrate that standard and independent component analysis of Raman spectra is capable of assessment of differences in chemical composition between functionally related gray and white matter structures. Our results show the ability of Raman spectroscopy to successfully depict variation in chemical composition between structurally similar and/or functionally connected brain structures. The observed differences were attributed to variations in content of proteins and lipids in these structures. Independent component analysis enabled separation of contributions of major constituents in spectra and revealed spectral signatures of low-concentration metabolites. This provided finding of discrepancies between structures of striatum as well as between white matter structures. Raman spectroscopy can provide information about variations in contents of major chemical constituents in brain structures, while the application of independent component analysis performed on obtained spectra can help in revealing minute differences between closely related brain structures.

Cerebrospinal fluid BACE1 activity and brain amyloid load in Alzheimer's disease.

The secretase BACE1 is fundamentally involved in the development of cerebral amyloid pathology in Alzheimer's disease (AD). It has not been studied so far to what extent BACE1 activity in cerebrospinal fluid (CSF) mirrors in vivo amyloid load in AD. We explored associations between CSF BACE1 activity and fibrillar amyloid pathology as measured by carbon-11-labelled Pittsburgh Compound B positron emission tomography ([¹¹C]PIB PET). [¹¹C]PIB and CSF studies were performed in 31 patients with AD. Voxel-based linear regression analysis revealed significant associations between CSF BACE1 activity and [¹¹C]PIB tracer uptake in the bilateral parahippocampal region, the thalamus, and the pons. Our study provides evidence for a brain region-specific correlation between CSF BACE1 activity and in-vivo fibrillar amyloid pathology in AD. Associations were found in areas close to the brain ventricles, which may have important implications for the use of BACE1 in CSF as a marker for AD pathology and for antiamyloid treatment monitoring.

Metabolic assessment of the human pons by in vivo proton magnetic resonance spectroscopy.

OBJECTIVE: To determine the normal mean reference normal value for metabolic ratios in the pons of healthy adult Chinese subjects by using proton magnetic resonance spectroscopy (1HMRS). MATERIALS AND METHODS: Eighty healthy Chinese subjects, ranging in age from 21 to 60 years, were divided into four groups, each containing 20 subjects per decade. The pons of every subject was scanned on single-voxel 1HMRS by using the point-resolved proton spectroscopy sequence (PRESS) with echo time (TE)=144 ms and repetition time (TR)=1500 ms. RESULTS: The total mean ratios of N-acetylasparate/creatine-phosphocreatine (NAA/Cr), NAA/choline-containing compounds (Cho) and Cho/Cr in subjects ranging from 21 to 60 years were 2.13+/-0.07, 1.22+/-0.11 and 1.81+/-0.09 respectively. The highest metabolite ratios were seen in the 41-50 year group. There was no significant difference with respect to age or gender. CONCLUSIONS: The ratios of NAA/Cr, NAA/Cho or Cho/Cr in the pons did not correlate with the age or gender of healthy subjects.

Precerebellar and vestibular nuclei of the short-beaked echidna (Tachyglossus aculeatus).

The monotremes are a unique group of living mammals, which diverged from the line leading to placental mammals at least 125 million years ago. We have examined the organization of pontine, inferior olivary, lateral reticular and vestibular nuclei in the brainstem of the short-beaked echidna (Tachyglossus aculeatus) to determine if the cyto- and chemoarchitecture of these nuclei are similar to that in placental mammals and marsupials. We have used Nissl staining in conjunction with enzyme-histochemistry for acetylcholinesterase, cytochrome oxidase and NADPH diaphorase as well as immunohistochemistry for non-phosphorylated neurofilament protein (SMI-32 antibody) and calcium binding proteins (parvalbumin, calbindin, calretinin). Homologies could be established between the arch shaped inferior olivary complex of the echidna and the principal, dorsal and medial accessory subdivisions of the therian inferior olivary complex. The pontine nuclei of the echidna included basilar and reticulotegmental components with similar cyto- and chemarchitectural features to therians and there were magnocellular and subtrigeminal components of the lateral reticular nucleus, also as seen in therians. Subdivisions and chemoarchitecture of the vestibular complex of the echidna were both similar to that region in rodents. In all three precerebellar nuclear groups studied and in the vestibular nucleus organization, the cyto- and chemoarchitecture of the echidna was very similar to that seen in therian mammals and no "primitive" or "reptilian" features were evident.

Effects of feeding grains naturally contaminated with Fusarium mycotoxins on brain regional neurochemistry of laying hens, turkey poults, and broiler breeder hens.

Three experiments were conducted to compare the effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on brain regional neurochemistry of laying hens, turkey poults, and broiler breeder hens. In Experiment 1, thirty-six 45-wk-old laying hens were fed diets including the following for 4 wk: 1) control, 2) contaminated grains, and 3) contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent (GMA). Concentrations of brain neurotransmitters and metabolites were analyzed in pons, hypothalamus, and cortex by HPLC with electrochemical detection. Neurotransmitters and the metabolites measured included dopamine, 3,4-dihydroxylphenyacetic acid, homovanillic acid, serotonin [5-hydroxytryptamine (5-HT)], 5-hydroxyindolacetic acid, epinephrine, and norepinephrine. The feeding of contaminated grains significantly increased concentrations of 5-HT and decreased the 5-hydroxyindolacetic acid:5-HT in the pons region in the brain stem. Dietary supplementation with GMA prevented these effects. There was no effect of diet on concentrations of other neurotransmitters or metabolites in the pons, hypothalamus, or cortex. In Experiment 2, thirty-six 1-d-old turkey poults were fed diets including the following for 4 wk: 1) control, 2) contaminated grains, and 3) contaminated grains + 0.2% GMA. Hypothalamic, pons, and cortex neurotransmitter concentrations were not affected by diet. In Experiment 3, forty-two 26-wk-old broiler breeder hens were fed diets including the following for 15 wk: 1) control, 2) contaminated grains, and 3) contaminated grains + 0.2% GMA. There was no effect of diet on neurotransmitter concentrations in the pons, hypothalamus, or cortex. It was concluded that differences in intraspecies effects of these mycotoxins on brain neurotransmitter concentrations might explain the intraspecies differences in the severity of Fusarium mycotoxin-induced reductions in feed intake.

Age-related metabolic changes in the upper brainstem tegmentum by MR spectroscopy.

Several neurodegenerative disorders have a profound metabolic and structural impact on the brainstem. MR spectroscopy provides metabolic information non-invasively and has the potential to characterize the changes associated with normal aging and differentiate them from neurodegenerative alterations. The present work was aimed at studying the upper brainstem tegmentum at the midbrain and pontine levels in 57 adult normal volunteers, aged 23-79 years, with long-echo time proton MR spectroscopy to evaluate possible regional differences and the effect of age. Higher ratios of N-acetyl aspartate (NAA)/total creatine (Cr) and choline-containing compounds (Cho)/Cr were observed in the pons compared to the midbrain, resulting from higher net NAA and Cho content. In the midbrain, there was a linear decline of NAA and Cho with age in subjects over 50, most probably related to neuronal tissue loss. In the pons, such an aging effect was not observed, with subjects over 50 showing higher Cr and Cho than the under-50 subjects. Our findings provided evidence of regional differences and suggest different effects of age on the two studied brainstem segments, hitherto undescribed.

Characteristics of two distinct clinical phenotypes in pathologically proven progressive supranuclear palsy: Richardson's syndrome and PSP-parkinsonism.

The clinical diagnosis of progressive supranuclear palsy (PSP) relies on the identification of characteristic signs and symptoms. A proportion of pathologically diagnosed cases do not develop these classic features, prove difficult to diagnose during life and are considered as atypical PSP. The aim of this study was to examine the apparent clinical dichotomy between typical and atypical PSP, and to compare the biochemical and genetic characteristics of these groups. In 103 consecutive cases of pathologically confirmed PSP, we have identified two clinical phenotypes by factor analysis which we have named Richardson's syndrome (RS) and PSP-parkinsonism (PSP-P). Cases of RS syndrome made up 54% of all cases, and were characterized by the early onset of postural instability and falls, supranuclear vertical gaze palsy and cognitive dysfunction. A second group of 33 (32%) were characterized by asymmetric onset, tremor, a moderate initial therapeutic response to levodopa and were frequently confused with Parkinson's disease (PSP-P). Fourteen cases (14%) could not be separated according to these criteria. In RS, two-thirds of cases were men, whereas the sex distribution in PSP-P was even. Disease duration in RS was significantly shorter (5.9 versus 9.1 years, P < 0.001) and age at death earlier (72.1 versus 75.5 years, P = 0.01) than in PSP-P. The isoform composition of insoluble tangle-tau isolated from the basal pons also differed significantly. In RS, the mean four-repeat:three-repeat tau ratio was 2.84 and in PSP-P it was 1.63 (P < 0.003). The effect of the H1,H1 PSP susceptibility genotype appeared stronger in RS than in PSP-P (odds ratio 13.2 versus 4.5). The difference in genotype frequencies between the clinical subgroups was not significant. There were no differences in apolipoprotein E genotypes. The classic clinical description of PSP, which includes supranuclear gaze palsy, early falls and dementia, does not adequately describe one-third of cases in this series of pathologically confirmed cases. We propose that PSP-P represents a second discrete clinical phenotype that needs to be clinically distinguished from classical PSP (RS). The different tau isoform deposition in the basal pons suggests that this may ultimately prove to be a discrete nosological entity.

Behavioral effects of 8-OH-DPAT injections into pontine and mesencephalic areas containing 5-HT-immunoreactive perikarya in the pigeon.

This study examined the distribution of 5-HT-immunoreactive perikarya (5-HT-IRp) and the effects of local injections of 8-OH-DPAT into 5-HT-IRp-containing pontine and mesencephalic regions on feeding and drinking behaviors in free-feeding pigeons. When infused into the midline 5-HT-IRp-containing areas, 8-OH-DPAT (6.1 nmol) reliably elicited drinking and, to a lesser extent, feeding responses during the first hour after injection. These responses were significantly higher than the ingestive indexes observed (1) after vehicle (ascorbic acid 0.1%, 200 nl) injections at the same sites and (2) after 8-OH-DPAT injections into adjacent sites devoid of 5-HT-IRp. Increases in drinking were proportionally higher than those observed in feeding and a significant negative correlation was observed between water and food after midline 8-OH-DPAT injections. Similar dipsogenic responses were observed after injections of different 8-OH-DPAT doses (0.6, 2.0, and 6.1 nmol). Pretreatment with local injections of p-MPPI (an antagonist of 5-HT1A receptors) attenuated the ingestive responses evoked by 8-OH-DPAT injections. Injections of 8-OH-DPAT into lateral 5-HT-IRp-containing sites evoked only inconsistent and weak ingestive responses. These results indicate that 5-HT1A receptor-mediated circuits located in the midline superior raphe system of the pigeon may play an important role in mechanisms controlling water intake, similar to that observed in mammals.

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat.

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase-wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus alpha and peri-alpha, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.

Pathways controlling trigeminal and auditory nerve-evoked abducens eyeblink reflexes in pond turtles.

An in vitro brain stem preparation from turtles exhibits a neural correlate of eyeblink classical conditioning during pairing of auditory (CS) and trigeminal (US) nerve stimulation while recording from the abducens nerve. The premotor neuronal circuits controlling abducens nerve-mediated eyeblinks in turtles have not been previously described, which is a necessary step for understanding cellular mechanisms of conditioning in this preparation. The purpose of the present study was to neuroanatomically define the premotor pathways that underlie the trigeminal and auditory nerve-evoked abducens eyeblink responses. The results show that the principal sensory trigeminal nucleus forms a disynaptic pathway from both the trigeminal and auditory nerves to the principal and accessory abducens motor nuclei. Additionally, the principal abducens nucleus receives vestibular inputs, whereas the accessory nucleus receives input from the cochlear nucleus. The late R2-like component of abducens nerve responses is mediated by the spinal trigeminal nucleus in the medulla. Both the principal sensory trigeminal nucleus and the abducens motor nuclei receive CS-US convergence and therefore both, or either, might be considered potential sites of synapse modification during in vitro abducens conditioning. Further data are required to determine the role of the principal sensory trigeminal nucleus in in vitro conditioning.

Rubratoxin B elicits antioxidative and DNA repair responses in mouse brain.

Rubratoxin B (RB) is a mycotoxin with potential neurotoxic effects that have not yet been characterized. Based on existing evidence that RB interferes with mitochondrial electron transport to produce oxidative stress in peripheral tissues, we hypothesized that RB would produce oxidative damage to macromolecules in specific brain regions. Parameters of oxidative DNA damage and repair, lipid peroxidation, and superoxide dismutase (SOD) activity were measured across six mouse brain regions 24 h after administration of a single dose of RB. Lipid peroxidation and oxidative DNA damage were either unchanged or decreased in all brain regions in RB-treated mice compared with vehicle-treated mice. Concomitant with these decreased indices of oxidative macromolecular damage, SOD activity was significantly increased in all brain regions. Oxyguanosine glycosylase activity (OGG1), a key enzyme in the repair of oxidized DNA, was significantly increased in three brain regions--cerebellum (CB), caudate/putamen (CP), and cortex (CX)--but not in the hippocampus (HP), midbrain (MB), and pons/medulla (PM). The RB-enhanced OGG1 catalytic activity in these brain regions was not due to increased OGG1 protein expression, but was a result of enhanced catalytic activity of the enzyme. In conclusion, specific brain regions responded to an acute dose of RB by significantly altering SOD and OGG1 activities to maintain the degree of oxidative DNA damage equal to, or less than, that of normal steady-state levels.

Activation of the dorsal periaqueductal gray in the rat induces Fos-like immunoreactivity in select non-cholinergic mesopontine neurons.

Autonomic responses evoked from the dorsal periaqueductal gray (dPAG) have been reported to be mediated in part by acetylcholine release in the medulla. To identify the possible origin of cholinergic neurons activated by dPAG stimulation, the pattern of Fos-like immunoreactivity (FLI) in the mesopontine cholinergic cell groups was examined in three groups of urethane anesthetized rats. Relative to surgery (n=6) and blood pressure control groups (n=6), chemical disinhibition of the dPAG (n=10) induced a significant increase in FLI in the lateral dorsal tegmental nucleus (LDTg) but not the pedunculopontine tegmental nucleus. LDTg neurons stained for choline acetyltransferase immunoreactivity however did not co-label for FLI. Other pontomesencephalic regions outside of the dPAG demonstrating a significant increase in FLI relative to controls included the lateral and ventrolateral columns of the PAG, the cuneiform nucleus, dorsal raphe, and the microcellular tegmental nucleus. These findings suggest that acetylcholine release in during dPAG stimulation does not originate from mesopontine neurons.

Diabetes-induced biochemical changes in central and peripheral catecholaminergic systems.

A great variety of alterations have been described in the nervous system of diabetic animals. They are named as diabetic neuropathy and affect the brain, spinal cord and peripheral nerves. In diabetic animals, plasma and tissue catecholamine levels have been reported to be increased, decreased or unchanged, and these disparities have been explained by differences in the tissues selected, severity or duration of diabetes. Dopamine, norepinephrine and epinephrine from different tissues were extracted by absorption onto alumina, and measured by high performance liquid chromatography with electrochemical detection. We found that diabetes alters catecholaminergic systems in a highly specific manner. The dopamine content is reduced in the dopaminergic nigrostriatal system only. Norepinephrine is differently altered in several areas of the sympathetic nervous system. It is increased in cardiac ventricles, and decreased in stellate ganglia and the blood serum. However, it is not altered in the central nervous system. Finally, epinephrine is only altered in the adrenal gland where it is increased, and in the serum where it is reduced. Our results suggest that diabetes reduces the activity of the nigrostriatal dopaminergic system. Changes found at the sympathoadrenal level could be explained by reduced norepinephrine and epinephrine synthesis, with increased storage due to a reduced release from synaptic vesicles.

Analysis of protein induction in the CNS of SJL mice with experimental allergic encephalomyelitis by proteomic screening and immunohistochemistry.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by demyelination and inflammatory infiltrates in the CNS, and it is an animal model of multiple sclerosis. Piperonyl butoxide (PBO) suppresses disease in EAE mice, and it exhibits a dual effect on cytochrome P450s that manifests in a transient inhibitory phase followed by induction. In order to identify the expression of proteins associated with EAE, a proteomic screening was performed on hindbrain microsomes from control + vehicle, control + PBO, EAE + vehicle, and EAE + PBO female mice. Glucose regulated protein 94 (Grp94) and coagulation factor VIII were among the proteins identified in EAE + vehicle and EAE + PBO mice. Immunohistochemical staining of Grp94 was present in some neurons and oligodendrocytes in hindbrain sections from control animals, and in some cells within inflammatory infiltrates in EAE animals. Since Grp94 (also known as Gp96) can partake in antigen presentation and induction of proinflammatory cytokine expression, its presence in these cells suggests that it may play a role in the pathogenesis of EAE. Coagulation factor VIII is carried and protected by von Willebrand factor. Immunohistochemical staining of von Willebrand factor revealed its presence in some vessels within hindbrain sections from control animals. In EAE animals, the number of labeled vessels was significantly increased, and extracellular granular deposits were observed around labeled vessels indicating that the breakdown of the blood-brain barrier that occurs in EAE permitted its extravasation into the CNS. Additional proteins were identified in the different groups of mice by proteomic screening, but confirmation of their expression profile awaits investigations by independent measures.