Injections Of Fluoro-Gold Research Articles

Anatomical tracer injections into the lower urinary tract may compromise cystometry and external urethral sphincter electromyography in female rats

Physiological and anatomical investigations are commonly combined in experimental models. When studying the lower urinary tract (LUT), it is often of interest to perform both urodynamic studies and retrogradely labeled neurons innervating the peripheral target organs. However, it is not known whether the use of anatomical tracers for the labeling of, e.g. spinal cord neurons may interfere with the interpretation of the physiological studies on micturition reflexes. We performed cystometry and external urethral sphincter (EUS) electromyography (EMG) under urethane anesthesia in adult female rats at 5–7 days after injection of a 5% fluorogold (FG) solution or vehicle into the major pelvic ganglia (MPG) or the EUS. FG and vehicle injections into the MPG and EUS resulted in decreased voiding efficiency. MPG injections increased the duration of both bladder contractions and the inter-contractile intervals. EUS injections decreased EUS EMG bursting activity during voiding as well as increased both the duration of bladder contractions and the maximum intravesical pressure. In addition, the bladder weight and size were increased after either MPG or EUS injections in both the FG and vehicle groups. We conclude that the injection of anatomical tracers into the MPG and EUS may compromise the interpretation of subsequent urodynamic studies and suggest investigators to consider experimental designs, which allow for physiological assessments to precede the administration of anatomical tracers into the LUT.

γ-Aminobutyric Acid Cells with Cocaine-Induced ΔFosB in the Ventral Tegmental Area Innervate Mesolimbic Neurons

The transcription factor DeltaFosB is implicated in the plasticity induced by drugs of abuse. We showed that psychostimulants induce DeltaFosB in gamma-aminobutyric acid (GABA) cells of a caudal subregion of the ventral tegmental area (VTA) that was named tail of the VTA (tVTA). Although tVTA mostly shares VTA inputs, its outputs remain to be characterized. The tVTA efferents were studied by iontophoretic injections of the anterograde tracer biotinylated dextran amine (BDA). To further study VTA inputs arising from tVTA, injections of the retrograde tracer Fluoro-Gold were combined with multiple labeling by immunohistochemistry in rats treated with cocaine. Indirect projections from the tVTA to the nucleus accumbens were assessed with a double-tracing approach, cholera toxin B subunit (CTB) being delivered in the nucleus accumbens and BDA in the tVTA. Tract-tracing studies showed that tVTA heavily projects to the midbrain dopaminergic system and revealed terminal appositions with dopamine cells in the VTA. Double-labeling studies demonstrated that this tVTA output is mostly GABAergic, includes cells in which cocaine exposure induces DeltaFosB, and displays appositions to dopamine cells projecting to the nucleus accumbens. The GABA neurons expressing DeltaFosB in the tVTA after cocaine exposure project to the dopamine mesolimbic neurons.

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1–2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with, e.g., neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.

Morphological and electrophysiological features of motor neurons and putative interneurons in the dorsal vagal complex of rats and mice

The dorsal motor nucleus of the vagus (DMV) contains preganglionic motor neurons that control viscera along the subdiaphragmatic digestive tract, but may also contain neurons that do not project to the viscera. Neurons that expressed EGFP 60–72 h subsequent to PRV-152 inoculation of vagal terminals in the stomach wall were targeted for whole-cell patch-clamp recording and biocytin filling in transverse brainstem slices from rats and their quantitative morphological and electrophysiological characteristics were compared with uninfected cells. Over 90% of PRV-152 labeled neurons were also labeled subsequent to intraperitoneal injection of FluoroGold, indicating that most were preganglionic motor neurons. In reconstructed neurons with an identifiable axon trajectory, two cellular subtypes were distinguished. The axon projected ventrolaterally from the DMV in 44 of 49 cells and these were likely to be vagal motor neurons. Axons of other neurons ramified within the nucleus tractus solitarius (NTS) or DMV. These cells were smaller and otherwise morphologically distinct from putative motor neurons. Transgenic mice with GFP-expressing inhibitory neurons (i.e., GIN mice) were used to identify a GABAergic subset of DMV neurons. These neurons had locally ramifying axons and formed a morphologically distinct subset of DMV cells, which were similar in size and axon trajectory to GABAergic neurons in the NTS. Most neurons in the DMV therefore possess morphological features of motor neurons, but locally projecting cells and inhibitory neurons with distinct morphological features are also found within the DMV. These cells likely contribute to regulation of vagal function.

Characterization of the Projections to the Hypothalamic Paraventricular and Periventricular Nuclei in the Female Sheep Brain, Using Retrograde Tracing and Immunohistochemistry

The paraventricular nucleus (PVN) and the periventricular nucleus (Pe) are important neuroendocrine centers, but the neuronal input to these regions is poorly defined in nonrodent species. We utilized the retrograde transport of injected tracers to determine the neural input to these two nuclei in the ovine brain. Adult Corriedale ewes were studied following FluoroGold injection into either the PVN (n = 5) or the Pe (n = 3). Both the PVN and the Pe were found to receive neuronal input from a number of hypothalamic nuclei. Projections to the PVN from the lateral hypothalamic area were from neurons that produce melanin-concentrating hormone or orexins and a subset of those from the arcuate nucleus were immunopositive for neuropeptide Y and γ-melanocyte stimulating hormone. This pathway was verified by staining of terminals in the PVN. Input to the PVN from the brain stem was seen to originate from the catecholaminergic and serotoninergic neurons. The projections to the PVN and Pe from hypothalamic and brain stem regions in the sheep brain are generally similar to those in the rat, with some minor differences. These studies highlight the differences in the afferent input to these two closely related nuclei in the ovine brain.

A morphologic study of Fluorogold labeled tensor tympani motoneurons in mice

The tensor tympani is one of two middle ear muscles that regulates the transmission of sound through the middle ear. Contraction of the tensor tympani in response to both auditory and non-auditory stimulation is mediated by the tensor tympani motoneurons (TTMNs). There are interesting differences among species in the acoustic thresholds for contraction of the middle ear muscles, which may be a reflection of underlying anatomical differences such as the number of TTMNs. However anatomical data for mice are lacking, even though the mouse is becoming the most common animal model for auditory and neuroscience research. We investigated the number and morphology of TTMNs in mice using Fluorogold, a retrograde neuronal tracer. After injections of Fluorogold into the tensor tympani muscle, a column of labeled TTMNs was identified ventro-lateral to the ipsilateral trigeminal nucleus. The labeled TTMNs were classified according to their morphological characteristics into three subtypes: “octopus-like”, “fusiform” and “stellate”, suggesting underlying differences in function. All three subtypes formed sparsely branched and radiating dendrites, some longer than 600 μm. Dendrites were longest and most numerous in the dorso-medial direction. In 18 cases, the mean number of mouse TTMNs was 51; the largest numbers were 70, 74 and 90 ( n = 3 injections). The mean size of mouse TTMNs was 13.0 μm (minor axis) and 23.5 μm (major axis). Compared with studies of TTMNs in larger species (cats and rats), mouse TTMNs are both fewer in number and smaller in size.

Distribution of premotoneurons for jaw-closing and jaw-opening motor nucleus receiving contacts from axon terminals of primary somatosensory cortical neurons in rats

To clarify features of direct projections from the primary somatosensory cortex (S1) to premotoneurons for the jaw-closing (JC) and jaw-opening (JO) components of the trigeminal motor nucleus, biotinylated dextranamine (BDA) and Fluorogold (FG) were used as the anterograde and retrograde tracers. The BDA and FG injections were made in the S1 and the JC or JO component, respectively, in rats. The distribution of FG-labeled JC and JO premotoneurons receiving contact(s) from BDA-labeled axon terminals of S1 neurons was quantitatively examined; the contacts were identified microscopically by using a X100 oil immersion objective. The largest and second largest numbers of JC and JO premotoneurons with contact(s) were found in the lateral reticular formation at the levels of the caudal pons and the medulla oblongata (cpmLRt) and trigeminal oral nucleus (Vo) bilaterally, and they comprised about 80% of the total premotoneurons with contact(s). The percentage of premotoneurons with contact(s) was higher in the Vo than in the cpmLRt for both JC and JO premotoneurons. Most of the JC or JO premotoneurons found in the nucleus of the solitary tract, inter- and supratrigeminal regions, mesencephalic trigeminal nucleus, parabrachial nucleus and reticular formation medial to the JO component of the trigeminal motor nucleus hardly received contact(s) from S1 neurons. This suggests that the contribution of S1 to the control of jaw movements is mediated via JC and JO premotoneurons located primarily in the cpmLRt and Vo areas of the brainstem.

Changes in GABA A receptor subunit gamma 2 in extensor and flexor motoneurons and astrocytes after spinal cord transection and motor training

GABA signaling plays an important role in the spinal cord response to injury and subsequent motor training. Since benzodiazepines are commonly used to treat muscle spasticity in spinal cord injured subjects and the γ2 subunit of the GABA A receptor is necessary for benzodiazepine binding, this subunit may be an important factor modulating sensorimotor function after an injury. Changes in γ2 levels in muscle-specific motoneurons and surrounding astrocytes were determined ∼ 3 months after a complete mid-thoracic spinal cord transection at P5 in non-trained and in step-trained spinal rats. Soleus (ankle extensor) and tibialis anterior (TA, ankle flexor) motor pools were identified using retrograde labeling via intramuscular injections of Fast Blue or Fluoro Gold, respectively. Lumbar spinal cord sections showed γ2 immunostaining in both soleus and TA motoneurons and astrocytes. γ2 immunoreactivity on the soma of soleus and TA motoneurons in spinal rats was differentially modulated. Compared to intact rats, spinal rats had higher levels of γ2 in TA, and lower levels in soleus motoneurons. Step training restored GABA A γ2 levels towards control values in motoneuronal pools of both muscles. In contrast, the γ2 levels were elevated in surrounding astrocytes of both motor pools in spinal rats, and step training had no further effect. Thus, motor training had a specific effect on those neurons that were directly involved with the motor task. Since the γ2 subunit is involved with GABA A receptor trafficking and synaptic clustering, it appears that this subunit could be an important component of the activity-dependent response of the spinal cord after a spinal injury.

Neurochemistry of Olivocochlear Neurons in the Hamster

The present study was conducted to characterize the superior olivary complex (SOC) of the lower brain stem in the pigmented Djungarian hamster Phodopus sungorus. Using Nissl-stained serial cryostat sections from fresh-frozen brains, we determined the borders of the SOC nuclei. We also identified olivocochlear (OC) neurons by retrograde neuronal tracing upon injection of Fluoro-Gold into the scala tympani. To evaluate the SOC as a putative source of neuronal nitric oxide synthase (nNOS), arginine-vasopressin (AVP), oxytocin (OT), vasoactive intestinal polypeptide (VIP), or pituitary adenylate cyclase-activating polypeptide (PACAP) that were all found in the cochlea, we conducted immunohistochemistry on sections exhibiting retrogradely labeled neurons. We did not observe AVP-, OT-, or VIP-immunoreactivity, neither in OC neurons nor in the SOC at all, revealing that cochlear AVP, OT, and VIP are of nonolivary origin. However, we found nNOS, the enzyme responsible for nitric oxide synthesis in neurons, and PACAP in neuronal perikarya of the SOC. Retrogradely labeled neurons of the lateral olivocochlear (LOC) system in the lateral superior olive did not contain PACAP and were only infrequently nNOS-immunoreactive. In contrast, some shell neurons and some of the medial OC (MOC) system exhibited immunofluorescence for either substance. Our data obtained from the dwarf hamster Phodopus sungorus confirm previous observations that a part of the LOC system is nitrergic. They further demonstrate that the medial olivocochlear system is partly nitrergic and use PACAP as neurotransmitter or modulator.

Parvalbumin-immunoreactive neurons and GABAergic neurons of the basal forebrain project to the rat basolateral amygdala

The basal forebrain (BF) contains a diffuse array of cholinergic and non-cholinergic neurons that project to the cerebral cortex and basolateral nuclear complex of the amygdala (BLC). Previous studies have shown that the GABAergic subpopulation of non-cholinergic corticopetal BF neurons selectively innervates cortical interneurons. Although several investigations in both rodents and primates have indicated that some BF neurons projecting to the BLC are non-cholinergic, there have been no studies that have attempted to identify the neurochemical phenotype(s) of these neurons. The present study combined Fluorogold retrograde tract tracing with immunohistochemistry for two markers of BF GABAergic neurons, parvalbumin (PV) or glutamic acid decarboxylase (GAD), to determine if a subpopulation of BF GABAergic cells projects to the BLC. Injections of Fluorogold confined to the rat BLC, and centered in the basolateral nucleus, produced extensive retrograde labeling in the ventral pallidum and substantia innominata regions of the BF. Although the great majority of retrogradely labeled neurons were not double-labeled, about 10% of these neurons, located mainly along the ventral aspects of the fundus striati and globus pallidus, exhibited immunoreactivity for PV or GAD. The results of this investigation contradict the long-held belief that there is no extra-amygdalar source of GABAergic inputs to the BLC, and indicate that the cortex-like BLC, in addition to the cortex proper, receives inhibitory inputs from the basal forebrain.

Fluorogold Induces Persistent Neurological Deficits and Circling Behavior in Mice Over-Expressing Human Mutant Tau

An increasing number of applications use nanospecie-fluorescent labeling technology; however, no established guidelines are available to warrant their safety for potential clinical use. Here, rTg4510 transgenic mice and their littermate controls were injected with fluorogold, a nanospecie tracer, or phosphate buffered saline (PBS) targeted to the right amygdala. No significant abnormal behavior was detected in any mice injected with PBS. After fluorogold injection, however, rTg4510 mice displayed persistent left-sided neurological deficits and left circling behavior for up to 14 days post-injection, while control mice demonstrated a transient syndrome. Mortality occurred only in rTg4510 mice and statistically significant differences appeared independent of age. An immunofluorescent study revealed TUNEL positive cells that were heavily and extensively distributed in the periamygdalar region that overlapped with the fluorogold deposit region in rTg4510 mice, whereas control mice showed only sporadic distribution of TUNEL-positive cells. Co-localization of TUNEL and caspase-3 active peptide immunoreactivity was identified in a subset of the cells, indicating an involvement of caspase-dependent apoptotic mechanisms. In conclusion, fluorogold induces damage in the central nervous system most noticeably in mice over-expressing human mutant tau.

Optogenetics: development and application

The entorhinal cortex and other hippocampal and parahippocampal cortices are interconnected by a small number of GABAergic nonpyramidal neurons in addition to glutamatergic pyramidal cells. Since the cortical and basolateral amygdalar nuclei have cortex-like cell types and have robust projections to the entorhinal cortex, we hypothesized that a small number of amygdalar GABAergic nonpyramidal neurons might participate in amygdalo-entorhinal projections. To test this hypothesis we combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for the amygdalar nonpyramidal cell markers glutamic acid decarboxylase (GAD), parvalbumin (PV), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and the m2 muscarinic cholinergic receptor (M2R). Injections of FG into the rat entorhinal cortex labeled numerous neurons that were mainly located in the cortical and basolateral nuclei of the amygdala. Although most of these amygdalar FG+ neurons labeled by entorhinal injections were large pyramidal cells, 1–5% were smaller long-range nonpyramidal neurons (LRNP neurons) that expressed SOM, or both SOM and NPY. No amygdalar FG+ neurons in these cases were PV+ or VIP+. Cell counts revealed that LRNP neurons labeled by injections into the entorhinal cortex constituted about 10–20% of the total SOM+ population, and 20–40% of the total NPY population in portions of the lateral amygdalar nucleus that exhibited a high density of FG+ neurons. Sixty-two percent of amygdalar FG+/SOM+ neurons were GAD+, and 51% were M2R+. Since GABAergic projection neurons typically have low perikaryal levels of GABAergic markers, it is actually possible that most or all of the amygdalar LRNP neurons are GABAergic. Like GABAergic LRNP neurons in hippocampal/parahippocampal regions, amygdalar LRNP neurons that project to the entorhinal cortex are most likely involved in synchronizing oscillatory activity between the two regions. These oscillations could entrain synchronous firing of amygdalar and entorhinal pyramidal neurons, thus facilitating functional interactions between them, including synaptic plasticity.

A Glycinergic input into the intralaminar thalamic nuclei

Event Abstract Back to Event A Glycinergic input into the intralaminar thalamic nuclei Kristof Giber1*, Viktor Plattner1, Hajnalka Bokor1 and Laszlo Acsady1 1 Dept. Cellular and Network Neurobiology , Institute of Experimental Medicine, Hungary In humans intralaminar thalamus is critical to maintain normal conscious states. Recently these nuclei were shown to receive a selective and dense glycinergic input in transgenic mice expressing EGFP linked to the glycine-transporter-2 protein (GlyT2). The aims of the present study were to determine the origin of the Glyt2-positive thalamic fibers, to characterize the morphology and co-transmitter of the glycinergic axon terminals and to examine the firing properties of the projecting neurons in vivo. Following the injections of retrograde tracer fluorogold into the intralaminar thalamus, the largest proportion (51.5%) of the labeled glycinergic neurons were localized within the the ipsilateral oral and caudal pontine nuclei (PnO-PnC). Sixty-one percent of the projecting cells in this region were EGFP-positive, whereas most contralateral neurons were GlyT2-negative. Injections of anterograde tracer Phaseolus vulgaris leucoagglutine into the PnO-PnC resulted in a dense labeling of large terminals in the ipsilateral intralaminar nuclei. Out of these terminals, 48.9% were EGFP-positive, while this ratio was only 9.6% in the contralateral hemisphere. The GlyT2-positive boutons were GABA-immunopositive and established multiple synapses on the proximal dendrites of relay cells. Juxtacellular recording of a GlyT2-positive PnO cell projecting to the thalamus disclosed a firing pattern consisting of rhythmic clusters of spikes at 40-50Hz in synchrony with the EEG upstates. We demonstrated that beside the well-known excitatory ascending activating system a novel inhibitory pathway reaches the thalamus from the brainstem. The glycine- and GABAergic ponto-thalamic pathway raises the question how an inhibitory arousal system modulates the thalamic activity and the conscious states. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Giber K, Plattner V, Bokor H and Acsady L (2009). A Glycinergic input into the intralaminar thalamic nuclei. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.056 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Feb 2009; Published Online: 27 Feb 2009. * Correspondence: Kristof Giber, Dept. Cellular and Network Neurobiology , Institute of Experimental Medicine, Budapest, Hungary, giber@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kristof Giber Viktor Plattner Hajnalka Bokor Laszlo Acsady Google Kristof Giber Viktor Plattner Hajnalka Bokor Laszlo Acsady Google Scholar Kristof Giber Viktor Plattner Hajnalka Bokor Laszlo Acsady PubMed Kristof Giber Viktor Plattner Hajnalka Bokor Laszlo Acsady Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cells Expressing RFamide-Related Peptide-1/3, the Mammalian Gonadotropin-Inhibitory Hormone Orthologs, Are Not Hypophysiotropic Neuroendocrine Neurons in the Rat

An RFamide peptide named gonadotropin-inhibitory hormone, which directly inhibits gonadotropin synthesis and secretion from the anterior pituitary gland, has recently been discovered in the avian hypothalamus. It is not known whether the mammalian orthologs of gonadotropin-inhibitory hormone and RFamide-related peptide (RFRP)-1 and -3 act in the same way. We used a newly generated antibody against the rat RFRP precursor combined with retrograde tract tracing to characterize the cell body distribution and fiber projections of RFRP-1 and -3 neurons in rats. RFRP-1/3-immunoreactive cell bodies were found exclusively within the dorsomedial hypothalamus. Immunoreactive fibers were observed in the septal-preoptic area, hypothalamus, midbrain, brainstem, and hippocampus but not in the external zone of the median eminence. Intraperitoneal injection of the retrograde tracer Fluoro-Gold in rats resulted in the labeling of the majority of GnRH neurons but essentially no RFRP-1/3 neurons. In contrast, intracerebral injections of Fluoro-Gold into the rostral preoptic area and CA2/CA3 hippocampus resulted in the labeling of 75 +/- 5% and 21 +/- 8% of RFRP-1/3 cell bodies, respectively. To assess actions at the pituitary in vivo, RFRP-3 was administered as an iv bolus to ovariectomized rats and plasma LH concentration measured at 0, 2.5, 5, 10, and 30 min. RFRP-3 had no effects on basal secretion, but GnRH-stimulated LH release was reduced by about 25% at 5 min. Together these observations suggest that RFRP-3 is not a hypophysiotropic neuroendocrine hormone in rats.

Lateral parabrachial afferent areas and serotonin mechanisms activated by volume expansion

Recent evidence has shown that the serotonergic mechanism of the lateral parabrachial nucleus (LPBN) participates in the regulation of renal and hormonal responses to isotonic blood volume expansion (BVE). We investigated the BVE-induced Fos activation along forebrain and hindbrain nuclei and particularly within the serotonergic clusters of the raphé system that directly project to the LPBN. We also examined whether there are changes in the concentration of serotonin (5HT) within the raphé nucleus in response to the same stimulus. With this purpose, we analyzed the cells doubly labeled for Fos and Fluorogold (FG) following BVE (NaCl 0.15 M, 2 ml/100 g b.w., 1 min) 7 days after FG injection into the LPBN. Compared with the control group, blood volume-expanded rats showed a significant greater number of Fos-FG double-labeled cells along the nucleus of the solitary tract, locus coeruleus, hypothalamic paraventricular nucleus, central extended amygdala complex, and dorsal raphé nucleus (DRN) cells. Our study also showed an increase in the number of serotonergic DRN neurons activated in response to isotonic BVE. We also observed decreased levels of 5HT and its metabolite 5-hydroxyindoleacetic acid (measured by high-pressure liquid chromatography) within the raphé nucleus 15 min after BVE. Given our previous evidence on the role of the serotonergic system in the LPBN after BVE, the present morphofunctional findings suggest the existence of a key pathway (DRN-LPBN) that may control BVE response through the modulation of 5HT release.

Inflammatory demyelination induces axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis

Optic neuritis is an inflammatory disease of the optic nerve that often occurs in patients with multiple sclerosis and leads to permanent visual loss mediated by retinal ganglion cell (RGC) damage. Optic neuritis occurs with high frequency in relapsing-remitting experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, with significant loss of RGCs. In the current study, mechanisms of RGC loss in this model were examined to determine whether inflammation-induced axonal injury mediates apoptotic death of RGCs. RGCs were retrogradely labeled by injection of fluorogold into superior colliculi of 6–7 week old female SJL/J mice. EAE was induced one week later by immunization with proteolipid protein peptide. Optic neuritis was detected by inflammatory cell infiltration on histological examination as early as 9 days after immunization, with peak incidence by day 12. Demyelination occurred 1–2 days after inflammation began. Loss of RGC axons was detected following demyelination, with significant axonal loss occurring by day 13 post-immunization. Axonal loss occurred prior to loss of RGC bodies at day 14. Apoptotic cells were also observed at day 14 in the ganglion cell layer of eyes with optic neuritis, but not in control eyes. Together these results suggest that inflammatory cell infiltration mediates demyelination and leads to direct axonal injury in this model of experimental optic neuritis. RGCs die by an apoptotic mechanism triggered by axonal injury. Potential neuroprotective therapies to prevent permanent RGC loss from optic neuritis will likely need to be initiated prior to axonal injury to preserve neuronal function.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

TNF-Alpha in Nucleus Pulposus Induces Sensory Nerve Growth

We used retrograde neurotracing with fluoro-gold to investigate the relationship between tumor necrosis factor (TNF-á) and nerve growth into the nucleus pulposus (NP) of wild-type and TNF-alpha-deficient mice. To clarify mechanisms underlying nerve growth into the NP and the role of TNF-á in this process. Degeneration of lumbar intervertebral discs is a cause of low back pain. Pathogenesis may involve sensory nerve ingrowth into the inner layers and NP of degenerating discs. We hypothesized that TNF-á in the NP is a major inducer of nerve ingrowth and investigated this hypothesis in vivo using wild-type and TNF-á-deficient mice. NP was harvested at the L4/5 level from 10 wild-type and 10 TNF-deficient mice. These 20 samples of wild-type NP or TNF-deficient NP were mixed with fluoro-gold and injected into the left quadriceps muscle of 20 other wild-type mice (1 sample per mouse). Five control mice underwent sham operations in which they received similar injections of NP-free fluoro-gold into their left quadriceps muscles to detect whether neurons innervating the muscle establish contact with injected NP. Seven and 14 days after surgery, left L4 dorsal root ganglions were removed and incubated with antibodies against growth-associated protein 43 (GAP43), a marker of axonal growth. We evaluated fluoro-gold-labeled and GAP43-immunoreactive dorsal root ganglions neurons. Within the set of fluoro-gold-labeled neurons, 10% were positive for GAP43 in sham-operated animals, 22% positive in the TNF-deficient NP group, and 38% positive in the wild-type NP group. These intergroup differences in the percentage of GAP43-positive neurons were statistically significant (sham vs. TNF-deficient NP group: P = 0.009; TNF-deficient NP group vs wild-type NP group: P = 0.026). CONCLUSION.: The percentage of fluoro-gold-labeled GAP43-immunoreactive neurons significantly increased after injections of NP harvested from both mouse types. Furthermore, the percentage of GAP43-immunoreactive neurons was significantly higher in mice receiving wild-type NP compared with mice receiving TNF-deficient NP. These findings suggest that TNF-alpha acts as an inducer of axonal growth into degenerated discs, as evidenced by decreased GAP-43 immunoreactivity in mice receiving TNF-deficient NP injections and even lower GAP-43 immunoreactivity in control mice receiving NP-free fluoro-gold injections.

Structural remodeling of nucleus ambiguus projections to cardiac ganglia following chronic intermittent hypoxia in C57BL/6J mice

The baroreflex control of heart rate (HR) is reduced following chronic intermittent hypoxia (CIH). Since the nucleus ambiguus (NA) plays a key role in baroreflex control of HR, we examined whether CIH remodels vagal efferent projections to cardiac ganglia. C57BL/6J mice (3-4 months of age) were exposed to either room air (RA) or CIH for 3 months. Confocal microscopy was used to examine NA axons and terminals in cardiac ganglia following Fluoro-Gold (FG) injections to label cardiac ganglia, and microinjections of tracer DiI into the left NA to anterogradely label vagal efferents. We found that: 1) Cardiac ganglia were widely distributed on the dorsal surface of the atria. Although the total number of cardiac ganglia did not differ between RA and CIH mice, the size of ganglia and the somatic area of cardiac principal neurons (PNs) were significantly decreased (P < 0.01), and the size of the PN nuclei was increased following CIH (P < 0.01). 2) NA axons entered cardiac ganglia and innervated PNs with dense basket endings in both RA and CIH mice, and the percentage of innervated PNs was similar (RA: 50 +/- 1.0%; CIH: 49 +/- 1.0%; P > 0.10). In CIH mice, however, swollen cardiac axons and terminals without close contacts to PNs were found. Furthermore, varicose endings around PNs appeared swollen and the axonal varicose area around PNs was almost doubled in size (CIH: 163.1 +/- 6.4 microm(2); RA: 88 +/- 3.9 microm(2), P < 0.01). Thus, CIH significantly altered the structure of cardiac ganglia and resulted in reorganized vagal efferent projections to cardiac ganglia. Such remodeling of cardiac ganglia and vagal efferent projections provides new insight into the effects of CIH on the brain-heart circuitry of C57BL/6J mice.

Visualization of the neurovascular bundles and major pelvic ganglion with fluorescent tracers after penile injection in the rat

To evaluate whether fluorescent tracers can consistently label the neurovascular bundles (NVBs) and major pelvic ganglion (MPG) after an intracavernosal penile injection, as the reported incidence of erectile dysfunction (ED) in men after radical prostatectomy (RP) is 55-65% and thus preservation of erectile function, sparing one or both of the NVBs remains one of the most vital factors. Male Sprague-Dawley rats (3 months old) received penile injections (20 microL; seven rats/group) of either deionized water (DW), Fluoro-Gold (FG), Fast-Blue (FB), Fluoro-Ruby (FR) or green fluorescent pseudorabies virus (GF-PRv). The rats were killed at 2, 3 and 14 days after injection and the NVBs and MPG were harvested and placed directly under fluorescence light. Image analysis was done by computer, coupled to a microscope equipped with a digital camera. Each NVB and MPG were analysed for its staining pattern and consistency. When compared with the FB, FR and GF-PRv rats, the FG-injected rats had better staining of the NVB at 2, 3 and 14 days after injection. Under x200, FG highlighted the axons of the cavernous nerve (CN) and cell bodies (MPG). This indicates that FG injection into the penis induced the strongest CN labelling (positive staining) at 2 and 3 days after injection as compared with FB-, FR- and GF-PRv-injected rats. FG injection into the penis has consistent retrograde staining of the NVBs and MPG after 3 days. Therefore, we predict that FG could potentially be used to improve the identification of the NVB in other models. However, further studies need to be carried out before these tracers can be used in humans.