Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro.

Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinson's disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4--94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0--283 pmols/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), the cells expanded over 1000-fold in 3--4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).

Reactivation of human neurotropic JC virus expressing oncogenic protein in a recurrent glioblastoma multiforme.

Examination of the primary tumor of glioblastoma multiforme and its recurrence for their association with JC virus revealed that, while the viral genome is present in both initial and recurrent tumors, expression of the viral oncoprotein T-antigen occurs only in the recurrent tumor cells. Accordingly, the level of inducible cellular transcription factors, including the p65 subunit of NF-kappaB and YB-1, which have the ability to stimulate JCV gene expression, was found to be higher in the recurrent tumor cells. These observations suggest that induction of the regulatory factors after resection of the primary tumor may have reactivated JC virus gene expression and led to redevelopment of the tumor in brain.

Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system.

A subset of stem-like cells from bone marrow that are referred to as marrow stromal cells (MSCs) have been shown to be capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes, astrocytes and perhaps neurons. Recently, conditions have been developed where human MSCs can be expanded almost without limit in culture without apparently losing their multipotentiality for differentiation. The cells appear to be potentially useful for the repair of extracellular matrix and the central nervous system.

Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease.

Bone marrow stromal cells can be used as an alternative source of cells for neural transplantation and repair. Here, the efficacy of genetically modified marrow stromal cells was examined in a rat model of Parkinson disease. Rat marrow stromal cells (rMSCs) and human marrow stromal cells (hMSCs) were genetically engineered by transduction with retroviruses encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase I, the enzyme necessary for production of the tetrahydrobiopterin cofactor for TH (BH4). Transduced cells synthesized 3,4-dihydroxyphenylalanine (L-DOPA) in vitro and maintained their multipotentiality after retroviral transduction. To examine the cells in vivo, transduced rMSCs were injected into the striatum of 6-hydroxydopamine-lesioned rats. L-DOPA and metabolites were detected by microdialysis in the denervated striatum of rats that received doubly transduced rMSCs. Also, there was a significant reduction in apomorphine-induced rotation when compared with controls. The cells engrafted and survived for at least 87 days. However, expression of the transgenes ceased at about 9 days, an observation consistent with reports from other laboratories in which similar retroviruses were used to express transgenes in the brain.

Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts.

Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson's disease. Here, we examine the effects of direct injection into rat brain of human marrow stromal cells (MSCs), a subset of cells from bone marrow that include stem-like precursors for nonhematopoietic tissues. Human MSCs isolated by their adherence to plastic were infused into the corpus striatum. Five to 72 days later, brain sections were examined for the presence of the donor cells. About 20% of the infused cells had engrafted. There was no evidence of an inflammatory response or rejection. The cells had migrated from the injection site along known pathways for migration of neural stem cells to successive layers of the brain. After infusion into the brain, the human MSCs lost their immunoreactivity to antibodies for collagen I. Initially, the human cells continued to stain with antibodies to fibronectin but the region of staining with fibronectin was significantly decreased at 30 and 72 days. The results suggest that MSCs may be useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.

Principles of treatment of malignant gliomas in adults: an overview.

The cornerstone of conventional treatments of malignant gliomas in adults has been surgical debulking, radiation therapy and chemotherapy. Almost always a combination of these treatments is used. With these conventional treatments the outcome, as measured by survival and quality of life, has remained universally dismal. Novel treatments, which are at different stages of laboratory and clinical trials, may offer a ray of hope for treatment of malignant gliomas. Development of these methods are directly related to the discoveries, over the past two decades, of cellular and molecular mechanisms involved in the genesis of brain tumors. Understanding of the mechanisms of tumor genesis may open new avenues of effective treatments for this devastating cancer.

Clivus and cervical spinal osteomyelitis with epidural abscess presenting with multiple cranial neuropathies.

A 65-year-old diabetic man with a history of otitis was admitted with headache, neck and shoulder pain and cranial nerve abnormalities including sixth, seventh and twelfth nerve palsies, hearing loss and ptosis. Lumbar puncture revealed an elevated CSF protein and pleocytosis. Imaging procedures demonstrated osteomyelitis of the clivus that involved the epidural space and extended within the prevertebral space to the cervical spine. The patient improved after treatment with antibiotics and immobilization of the neck. This case illustrates the importance of recognizing infections of the clivus in patients with cranial nerve abnormalities.

Human immunodeficiency virus-associated motor axonal polyradiculoneuropathy.

OBJECTIVE: To report two cases of an axonal motor polyradiculoneuropathy associated with human immunodeficiency virus infection in otherwise asymptomatic subjects. DESIGN: Case series. SETTING: Tertiary care hospital neurology inpatient service. RESULTS: Electrophysiologic testing showed acute denervation with almost normal sensory potentials and no evidence of demyelination. CONCLUSIONS: These cases present an example of an acute axonal polyradiculoneuropathy in persons infected with human immunodeficiency virus without acquired immunodeficiency syndrome.

Interactions of visual and auditory mossy fiber inputs in the paraflocculus of the rat: a gating action of multimodal inputs.

We have demonstrated previously that visual and auditory inputs reach the rat paraflocculus via dorsolateral pontine gray from the secondary regions of the visual and auditory cortices. It has also been demonstrated that mossy fiber responses can be evoked to physiological acoustic stimuli in unanesthetized preparations. In this study, we investigated the interaction of auditory and visual inputs in the paraflocculus. Activity of parafloccular neurons was recorded in immobilized, locally anesthetized hooded rats. Selected images and tones were presented to animals. Orientation, position, and velocity of visual stimuli and different parameters of acoustic stimuli were controlled by a computer. Visual and auditory stimuli were also presented in combination or in different temporal sequences. We discovered that visual and auditory stimuli have coextensive termination zones in paraflocculus, and influence the same neurons in 60% of the cases. Combined auditory and visual stimuli produced synergistic responses in parafloccular neurons in comparison with single modality stimuli. Augmentation of responses could be observed even when one of the stimuli was subthreshold and did not alter per se the neuronal activity. Our findings suggest that within the cerebro-cerebellar system, subthreshold inputs are capable of powerful control over the neuronal activity and may alter responses to subsequent stimuli when properly biased by inputs from paired modalities.

Modulation of rat cortical area 17 neuronal responses to moving visual stimuli during norepinephrine and serotonin microiontophoresis.

The present study was conducted to examine the actions of norepinephrine (NE) and serotonin (5-HT) on the multiphasic, visually evoked discharges of cells recorded from the visual cortex (area 17) of anesthetized Long-Evans pigmented rats. Visual responses of 51 cells, evoked by computer controlled presentation of moving visual stimuli, were examined before, during and after low level microiontophoretic application of NE (1-55 nA) or 5-HT (1-50 nA). Drug-induced changes in stimulus-evoked and spontaneous discharges were quantitatively assessed by computer analysis of peri-event histograms. In the majority of cases tested, NE produced a net enhancement of visually evoked responses by facilitating excitatory and inhibitory components of stimulus-bound discharges. By contrast, 5-HT tended to suppress stimulus-evoked excitation and inhibition in many cases to the extent that neurons were no longer responsive to appropriate visual stimuli. In selected cases we were able to demonstrate additional effects of NE and 5-HT on response threshold, direction selectivity and discrimination of receptive field borders. For example, in some cells NE was capable of revealing evoked responses to visual stimuli which were previously ineffective in eliciting stimulus-bound discharges. In other instances, changes in cell activity evoked by stimulus movement across the visual field were accentuated during NE application in such a way that unit discharges at receptive field borders were more sharply defined in comparison to control conditions. 5-HT, on the other hand, was capable of decreasing the contrast between spontaneous and visually evoked discharge at receptive field boundaries. In summary, these results suggest that endogenously released NE and 5-HT may modulate, by complimentary actions, the magnitude of responses of visual cortical neurons to afferent synaptic inputs. Moreover, these monoaminergic projection systems may also have the capacity to modify the threshold of detection of afferent signals within a neuronal network as well as alter feature extraction properties of the circuit.

Survey of noncortical afferent projections to the basilar pontine nuclei: a retrograde tracing study in the rat.

The retrograde transport of the conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was used in the rat to identify the cell bodies of origin for all subcortical projections to the basilar pontine nuclei (BPN). A parapharyngeal surgical approach was used to allow the injection micropipette to enter the BPN from the ventral aspect of the brainstem and thus avoid any potential for false-positive labeling due to transection and injury-filling of axonal systems located dorsal to the basilar pontine gray. A surprisingly large number of BPN afferent cell groups were identified in the present study. Included were labeled somata in the lumbar spinal cord and a large variety of nuclei in the medulla, pons, and midbrain, as well as labeled cells in diencephalic and telencephalic nuclei such as the zona incerta, ventral lateral geniculate, hypothalamus, amygdala, nucleus basalis of Meynert, and the horizontal nucleus of the diagonal band of Broca. Quite a number of cell groups known to project directly to the cerebellum also exhibited labeled somata in the present study. To explore the possibility that such neurons were labeled because their axons were transected and injury-filled as they coursed through the BPN injection site to enter the cerebellum via the brachium pontis, a series of rats received complete, bilateral lesions of the brachium pontis followed 30-60 minutes later with multiple, diffuse injections of WGA-HRP (12-16 placements per animal) throughout the cerebellar cortex. In another series of animals, the massive cerebellar WGA-HRP injections were not preceded by brachium pontis lesions. In the latter cases, each of the cell groups in question that were known to project directly to the cerebellum exhibited labeled somata. However, when the cerebellar HRP injections were preceded by brachium pontis lesions, each of the cell groups in question continued to exhibit labeled somata in numbers comparable to that observed in the nonlesion cases. This implies that such neurons project to the BPN and the cerebellar cortex and that the axons of these particular neurons do not project to the cerebellum via the brachium pontis.

New evidence for a gating action of norepinephrine in central neuronal circuits of mammalian brain.

Many previous studies have examined the effects of norepinephrine (NE) on neuronal responsiveness to synaptic inputs and putative transmitter substances and have described differential depressant actions of NE on stimulus evoked versus spontaneous discharge such that the "signal to noise" ratio of threshold responses was increased. In the present studies, similar experimental strategies employing a combination of microiontophoresis, single unit recording and afferent pathway stimulation in intact anesthetized and brain tissue slice preparations have revealed noradrenergic "gating" actions whereby weak or subthreshold synaptic stimuli can evoke threshold neuronal responses in the presence of iontophoretically applied NE or following electrical stimulation of the locus coeruleus. Overall, these results suggest that potentially threshold excitatory and inhibitory synaptic inputs may normally arrive at central neurons but appear weak or absent except during behavioral conditions favoring the synaptic release of NE. As such, these findings provide evidence that signal to noise ratio may not be the only potential modulatory action expressed by NE in noradrenergic target circuits of the mammalian brain.

Electrophysiological actions of norepinephrine in rat lateral hypothalamus. II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to gamma-aminobutyric acid (GABA).

The preceding studies demonstrated that norepinephrine (NE) can consistently augment synaptically mediated (70%) and gamma-aminobutyric acid (GABA)-induced (69%) inhibitory responses of lateral hypothalamic (LH) neurons in vivo. The present experiments further characterized the interactions of NE with LH neuronal responses to GABA in terms of alpha- and beta-receptor mechanisms and demonstrated the utility of the in vitro LH tissue slice preparation as a model for future extra- and intracellular studies of NE modulatory phenomena. Extracellular activity of LH cells was recorded from diencephalic slices (450 microns) incubated in artificial cerebrospinal fluid at 33 degrees C. Interactions between iontophoretically applied NE, isoproterenol (ISO) or phenylephrine (PE) and responses of LH neurons (n = 64) to GABA microiontophoresis were quantitated and characterized using computer-generated ratemeter and histogram records. This analysis revealed two distinct actions of NE on GABA-induced responses of LH neurons. In 8 of 32 cells tested (25%), locally applied NE markedly enhanced inhibitory responses to GABA iontophoresis in a manner identical to that observed in vivo. However, in 20 cells (62.5%), iontophoretic application of NE produced a clear antagonism of GABA responses. NE also exerted dual effects on the background firing rate of LH neurons, causing both inhibition and excitation. Overall, in those cells where NE administration increased spontaneous discharge, it either antagonized or had no effect on GABA-mediated inhibition. In contrast, spontaneous firing rate was never elevated above control levels in those cases where NE potentiated GABA responses. Additional experiments demonstrated that the GABA potentiating actions of the benzodiazepine, flurazepam, were preserved in LH tissue slice preparations. In addition, iontophoretic application of the beta-agonist, ISO, routinely suppressed the spontaneous activity of LH neurons and mimicked the facilitating action of NE on GABA. Likewise, microiontophoretic application of 8-bromo cyclic adenosine monophosphate (AMP) enhanced GABA-induced inhibition of LH firing rate in each of 11 cells tested. On the other hand, local administration of the alpha agonist, PE, routinely produced NE-like antagonism of GABA inhibition along with increases in spontaneous firing rate. Taken together these findings indicate that the commonly observed in vivo phenomena of NE augmentation of GABA and suppression of LH neuron spontaneous firing can be demonstrated in vitro, and most likely result from activation of beta adrenoceptors and subsequent elevation of cyclic AMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Convergence of cortico- and cuneopontine projections onto components of the pontocerebellar system in the rat: an anatomical and electrophysiological study.

Previous studies in the rat have demonstrated that corresponding peripheral tactile and somatosensory cortical inputs converge within the granule cell layer of various cerebellar lobules and further that descending corticopontine projections from the forelimb sensory cortex (FLSCx) partially overlap with the projection zones of ascending basilar pontine afferents from nucleus cuneatus (NC). The present study employed anatomical and electrophysiological procedures to determine whether cortical and dorsal column nuclear afferent projections converge on pontine neurons that, in turn, provide mossy fiber input to the granule cell layer of the paramedian lobule (PML), i.e., that portion of the rodent cerebellum shown to receive forelimb peripheral inputs. The combination of the orthograde and retrograde axonal transport of horseradish peroxidase (HRP) conjugated to wheat germ agglutinin (WGA) was used light microscopically to demonstrate that orthogradely labeled projections from injections of the FLSCx and NC converged with ponto-paramedian projection neurons that were retrogradely labeled from injections of the PML. These studies were also repeated in conjunction with ablations of either the FLSCx or NC which resulted in the ultrastructural identification of degenerating, as well as WGA-HRP labeled axonal boutons of these pontine afferent projections thus confirming that such projections actually formed synaptic contacts with the retrogradely labeled pontoparamedian projection neurons. Single unit recording analyses of neurons in the ventromedial region of the basilar pons following combined electrical stimulation of various regions of the sensorimotor cortex and the contralateral body surface indicated that approximately 40% of all cells recorded responded to electrical stimulation of corresponding regions of the cortex and periphery, particularly the FLSCx and the forepaw. Natural cutaneous stimuli applied to the forepaw that also elicited responses in these same groups of basilar pontine neurons and were associated with relatively small receptive fields. Taken together, these observations indicate that the previously observed convergence of peripheral and somatosensory cortical inputs within the granule cell layer of the cerebellar cortex may be at least partially organized at the level of the basilar pons.

Inferior olivary nuclear complex of the rat: morphology and comments on the principles of organization within the olivocerebellar system.

This study was undertaken (1) to provide a description of the normal morphology and anatomical interrelationships within the inferior olivary cell groups and (2) to determine the topographical organization of projections from this nuclear complex to the cerebellum. Conventional histological methods and the technique of retrograde transport of horseradish peroxidase (HRP) and WGA-HRP in conjunction with the sensitive chromogen tetramethyl benzidine were used in this series of experiments. In common with that of other animals, the inferior olivary nucleus of the rat consists of three subdivisions: the medial accessory olive (MAO), dorsal accessory olive (DAO), and principal olive (PO). The MAO is made of several subnuclei including a, b, c, and nucleus beta. On the basis of their common connections, the smaller subnuclei, dorsal cap, ventrolateral outgrowth, and dorsomedial cell column can be considered as parts of the MAO. The DAO is made of two subdivisions or lamellae--the dorsal and ventral folds--joined together laterally to form a twisted V-shaped structure. The principal olive consists of dorsal and ventral lamellae. A point-to-point projection was determined for four areas of the cerebellum: the anterior lobe, the posterior vermis, the intermediate and the lateral cerebellum. Analysis of the details of the projection system revealed that distinct groups of cells, here referred to as lamellae, project to sagittal zones of the cerebellum. (1) The medial accessory olive appears to be composed of three lamellae: horizontal, vertical, and rostral. The horizontal lamella (elsewhere denoted as groups a and b) projects to a sagittal zone in the vermal anterior lobe. The vertical lamella (groups c, beta, dorsal cap, ventrolateral outgrowth, and dorsomedial cell column) projects to a sagittal zone in the posterior vermis and the flocculus, and the rostral lamella projects to the lateral cerebellum. (2) The dorsal accessory olive is composed of two distinct lamellae which we have previously denoted as the dorsal and ventral folds. The dorsal fold projects to the vermal anterior lobe and receives afferents from the spinal cord, whereas the ventral fold projects to a sagittal zone in the intermediate cerebellum and receives afferents primarily from dorsal column nuclei. (3) The principal olive contains the two familiar lamellae: dorsal and ventral lamellae, each of which projects to a specific sagittal strip in the lateral cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

Origin and ultrastructural identification of dorsal column nuclear synaptic terminals in the basilar pontine gray of rats.

The ultrastructural characteristics of HRP-WGA-labeled or degenerating axon terminals arising from neurons in the dorsal column nuclei (DCN) were identified within the contralateral basilar pontine nuclei (BPN) following unilateral HRP-WGA injections or ablations of the DCN. The cells of origin of these projections were also identified through the application of the retrograde tracer HRP-WGA. Two groups of degenerating DCN-pontine terminals were identified. Both formed asymmetrical synaptic contacts with dendritic shafts and/or dendritic appendages of pontine neurons. One group of degenerating terminals contained small, round synaptic vesicles, while the other exhibited a mixture of dense core and pleomorphic vesicles. The former group, which clearly represented the majority of degenerating terminals observed, was interpreted to progress from an early filamentous form of degeneration to a later electron-dense variety and to originate from dorsally located DCN cells distributed primarily at the level of and just caudal to the area postrema. Other DCN-labeled neurons were more ventrally located and were postulated to give rise to those degenerative boutons that contained a mixture of dense core and pleomorphic-shaped vesicles. The present study also identified the cells of origin of two additional projections to the basilar pons: one from cells in the external cuneate nucleus and another from neurons of the medullary reticular formation.

Certain basilar pontine afferent systems are GABA-ergic: combined HRP and immunocytochemical studies in the rat.

Injection of the tracer substance wheat germ agglutinin-horseradish peroxidase (WGA-HRP) directly into the basilar pontine nuclei using a ventral surgical approach resulted in the labeling of somata in many areas both rostral and caudal to the basilar pons. Certain of the sections that had been reacted for HRP were also incubated in antiserum prepared against glutamic acid decarboxylase (GAD) and processed according to routine peroxidase anti-peroxidase immunocytochemical procedures. Neuronal somata exhibiting both HRP and GAD reaction products were considered to represent GABA-ergic neurons that provide axonal projections to the basilar pontine nuclei. Such double-labeled neurons were observed within the zona incerta, anterior pretectal nucleus, lateral cerebellar nucleus, perirubral area, and the pontine and medullary reticular formation.

The auditory corticopontocerebellar projection in the rat: inputs to the paraflocculus and midvermis. An anatomical and physiological study.

This study investigated afferent projections to the cerebellum, in particular those from the auditory cerebral cortex. The parafloccular lobule of the rat cerebellum is shown to be a primary target for the auditory cortical information with the midvermal region being a receiving area from the inferior colliculus. The method of anterograde transport of tritiated amino acids was employed to determine projections of the auditory cortex to the pons. Autoradiography showed that the site of termination of efferents from the auditory cortex corresponds to the location of neurons that project to the paraflocculus. Histogram analysis of neuronal activity in halothane anesthetized rats was employed to determine the response characteristics of neurons in paraflocculus and midvermis following cortical and tectal electrical stimulation. In addition, unit recordings of parafloccular neurons in immobilized, locally anesthetized animals demonstrated general characteristics of the responses of these neurons to auditory field stimulation. Electrical stimulation of the auditory cortex evoked mixed, excitatory-inhibitory and pure inhibitory mossy fiber responses in 33% of neurons in the paraflocculus, with no responses evident in the midvermis. Following inferior collicular stimulation, 12.6% of the neurons in the midvermis elicited a response. Recordings from parafloccular neurons in unanesthetized, immobilized rats showed evidence for excitatory and inhibitory mossy fiber responses, following auditory field stimulation. This spectrum of basic studies establishes the existence of a pathway in which the paraflocculus is revealed as an integrating target for cortical auditory information.

The tectopontine projection the the rat with comments on visual pathways to the basilar pons.

The projection from the superior and inferior colliculi to the basilar pons in the rat was studied with the technique of orthograde transport of labeled amino acids and autoradiography. Injections restricted to the medial or lateral regions of the superior colliculus gave rise to grain labeling representing terminal fields over the ipsilateral peduncular, dorsolateral, and ventrolateral regions of the caudal basilar pons and over the dorsomedial area of the contralateral nucleus reticularis tegmenti pontis (NRTP). The pontine projection from the superior colliculus to the lateral basilar pons is topographically organized; the medial superior colliculus projects primarily to the peduncular region, whereas the lateral superior colliculus terminates chiefly in ventrolateral pontine areas. A projection from the superior colliculus to the contralateral dorsomedial pontine and medial peduncular pontine regions, a previously undescribed finding, has also been shown. Descending fibers from the inferior colliculus do not appear to terminate extensively within the basilar pons but rather course adjacent to pontine cells of the dorsolateral region in the caudal pons. Pretectal nuclei project ipsilaterally to medial and lateral nuclei in the rostral and middle basilar pons, respectively. A rostrocaudal topography exists in the tectopontine projection; the pretectum projects to rostromiddle basilar pons, the superior colliculus to more caudal pontine regions, and the inferior colliculus (although sparsely) to further caudal areas. The pontine projection pattern from the colliculi and pretectum differs from the pontine afferents from the visual cortices. The findings of this study, when compared to our results from previous investigations on the pontocerebellar projection system, suggest that the tectal inputs to certain lateral cerebellar lobules are relayed primarily through NRTP rather than the basilar pons. The collicular projection to midvermal lobules of the cerebellum appear to be mediated in part by both NRTP and lateral pontine nuclei.