Activity and MeCP2-dependent regulation of nNOS levels in enteric neurons.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder characterized by severe cognitive impairment, motor dyspraxia, and seizures. Rett syndrome arises predominantly from mutations in MECP2, the gene coding for methyl-CpG-binding protein 2 (MeCP2). MeCP2 is an important mediator of synaptic development and is essential in regulating homeostatic synaptic plasticity (HSP) in the brain. In addition to demonstrating central nervous system impairment, RTT patients also suffer from gastrointestinal (GI) dysmotility. We hypothesize that this is due to a similar impairment of plasticity-dependent synaptic function in the enteric nervous system (ENS). We recently reported that MeCP2 is expressed in the ENS, providing evidence that neuronal dysfunction may mediate the GI pathology. METHODS: Baseline measures of MeCP2-KO vs wild-type (WT) GI neuronal nitric oxide synthase (nNOS) were assessed in tissue samples and in vitro. Experiments were carried out to measure nNOS in baseline vs activated plasticity states in vitro. Functional in vivo studies were carried out to determine whether MeCP2-KO mice reproduced the RTT GI hypomotility. KEY RESULTS: Methyl-CpG-binding protein 2-KO mice reproduced the GI hypomotility seen in RTT. MeCP2-KO GI tissue demonstrated elevated nNOS levels. Cultured WT enteric neurons showed upregulation of nNOS following moderate, prolonged stimulation by hyperkalemia; neurons from MeCP2-KO mice failed to show this nNOS upregulation. CONCLUSIONS & INFERENCES: MeCP2 is required for proper GI motility and normal nNOS levels. Neuronal nitric oxide synthase imbalances could mediate the GI dysmotility seen in RTT. Disruption of MeCP2-dependent HSP may be the basis for aberrant nNOS levels and hence GI dysmotility in MeCP2-KO and RTT.

MeCP2 in the enteric nervous system.

BACKGROUND: Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6-18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression at various developmental stages. METHODS: Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and beta-actin antibodies. KEY RESULTS: MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. CONCLUSIONS & INFERENCES: The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation.

Striatal interneurons in dissociated cell culture.

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague-Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.

Food restriction induces long-lasting recovery of spatial memory deficits following global ischemia in delayed matching and non-matching-to-sample radial arm maze tasks.

Food restriction has been shown to be beneficial for a number of brain processes. In the current study, we characterized the impact of food restriction on hippocampal damage 70 days following ischemia. We assessed memory and cognitive flexibility of ad libitum fed (AL) and food-restricted (FR) animals using complex delayed non-matching- and matching-to-sample tasks in the radial arm maze. Our findings demonstrate that food restriction led to significant improvement of ischemia-induced memory impairments. FR ischemic animals rapidly reached comparable performance as both AL and FR sham animals in delayed-non-matching (win-shift) and matching (win-stay) radial arm maze tasks. They also made considerably fewer microchoices in the retention trials than AL ischemic animals. In contrast, AL ischemic rats showed persistent spatial memory impairments in the same paradigms. Assessment of basal and stress-induced corticosterone (CORT) secretion revealed no significant differences in baseline levels in AL and FR rats prior to or following global ischemia. However, FR animals showed a more pronounced attenuation of CORT secretion 45 min following restraint. Both FR and AL ischemic rats had comparable cell loss within CA1 and CA3 subfields of Ammon's horn (CA1 and CA3) at 70 days following reperfusion, although a trend toward increased CA3 cell survival was observed in FR ischemic rats. The functional sparing in the FR ischemic animals in the face of equivalent hippocampal cell loss suggests that food restriction somehow enhanced the efficacy of remaining hippocampal or extrahippocampal neurons following ischemia. In the current study, this phenomenon was not associated with diet- and or ischemia-related alterations of vesicular glutamate transporter 1 expression in various hippocampal regions although lower vesicular GABA transporter immunostaining was present in the CA1 stratum oriens and the CA3 stratum radiatum in FR sham and ischemic rats.

Fast evaluation of intraoperative biopsies for ganglia in Hirschsprung's disease.

PURPOSE: The most important diagnostic features of Hirschsprung's disease (HD) are the combination of aganglionosis and hypertrophic nerve bundles. Acetylcholinesterase staining is widely used for diagnosis of HD as it identifies hypertrophic nerves in both diagnostic and intraoperative biopsies. The main drawback of this method is in the identification of ganglia. It has been suggested that the combination of this method together with another histochemical marker would be a superior diagnostic tool. Hematoxylin and eosin is still the diagnostic measure of choice for identifying ganglia in many centers, although it presents a persistent diagnostic challenge for pathologists trying to rapidly and accurately interpret the frozen biopsies that guide intraoperative decision making. METHODS: Therefore, we sought to develop a fast, intraoperative immunohistochemical protocol for visualization of ganglia and nerves in HD specimens that can be used in conjunction with these other tools. RESULTS: With the use of acetone fixation and immunofluorescence staining with antibodies to neurofilament 68 and tubulin, ganglia in sections of full thickness and suction biopsies could be visualized in only 10 minutes. This protocol facilitated the identification of ganglia in hematoxylin and eosin-stained adjacent sections and also identified hypertrophic nerve trunks. CONCLUSION: This method should significantly enable the identification of ganglia in suction and full thickness biopsies.

The distribution of dopamine D1 receptor and mu-opioid receptor 1 receptor immunoreactivities in the amygdala and interstitial nucleus of the posterior limb of the anterior commissure: relationships to tyrosine hydroxylase and opioid peptide terminal systems.

Mismatches between dopamine innervation and dopamine D1 receptor (D1) distribution have previously been demonstrated in the intercalated cell masses of the rat amygdala. Here the distribution of enkephalin and beta-endorphin immunoreactive (IR) nerve terminals with respect to their mu-opioid receptors is examined in the intercalated cell masses, along with a further immunohistochemical analysis of the dopamine/D1 mismatches. A similar analysis is also made within the extended amygdala. A spatial mismatch in distribution patterns was found between the mu-opioid receptor-1 immunoreactivity and enkephalin IR in the main intercalated island of the amygdala. Discrete cell patches of dopamine D1 receptor and mu-opioid receptor-1 IR were also identified in a distinct region of the extended amygdala, the interstitial nucleus of the posterior limb of the anterior commissure, medial division (IPACM), which displayed sparse tyrosine hydroxylase or enkephalin/beta-endorphin IR nerve terminals. Furthermore, distinct regions of the main intercalated island that showed dopamine/D1 receptor matches (the rostral and rostrolateral parts) were associated with strong dopamine and cyclic AMP regulated phosphoprotein, 32 kDa-IR in several D1 IR neuronal cell bodies and dendrites, whereas this was not the case for the dopamine/D1 mismatch areas (the rostromedial and caudal parts) of the main intercalated island. The lack of correlation between the terminal/receptor distribution patterns suggests a role for volume transmission for mu-opioid receptor- and dopamine D1 receptor-mediated transmission in distinct regions of the amygdala and extended amygdala. This may have implications for amygdaloid function, where slow long lasting responses may develop as a result of volume transmission operating in opioid peptide and dopaminergic communication.

Cerebral glucose transporters expression and spatial learning in the K-ATP Kir6.2(-/-) knockout mice.

K-ATP channels formed of the Sur and Kir subunits are widely distributed in the brain. Sur1-Kir6.2 is the most common combination of K-ATP channel subunits in the brain and Kir6.2 plays an important role in glucose metabolism through pancreatic insulin secretion or hypothalamic glucose sensing. K-ATP channels have also been reported to play a role in memory processing. Therefore, the aim of the present experiment is to assess the gene and protein expression of GLUT1, GLUT3 and GLUT4 in various brain regions of Kir6.2(-/-) K-ATP knockout mice and to test their working memory performance. GLUT4 was measured using two antibodies, one recognizing an intracellular epitope and the other, an extracellular epitope. Relative to their corresponding wild type, semi-quantitative immunohistochemistry showed that GLUT4 protein expression as measured by a GLUT4 antibody recognizing an extracellular epitope was increased in the Kir6.2(-/-) K-ATP mice. However, there was only a small increase in GLUT4 labeling using the GLUT4 antibody recognizing the intracellular epitope. These results suggest a compensatory higher GLUT4 inclusion at the cellular neuronal membrane in the cerebral cortex, hippocampus and cerebellum of the Kir6.2(-/-) K-ATP knockout mice. However, there was no change in GLUT4 gene expression assessed by TaqMan PCR except for a decrease in the cerebellum of these mice. Working memory performance of the Kir6.2(-/-) K-ATP mice was disrupted at age of 12 weeks but not at 5 weeks. The mild glucose intolerance that is observed in the Kir6.2 knockout mice is unlikely to have created the memory deficits observed. Rather, in light of the effects of K-ATP channel modulators on memory, the memory deficits in the Kir6.2(-/-) K-ATP mice are more likely due to the absence of the Kir6.2 and possible disruption of the GLUT4 activity in the brain.

The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in mammals.

The lifespan of the nematode, Caenorhabditis elegans, can be extended by mutations affecting components of the insulin-like growth factor (IGF) signaling cascade or by overexpression of SIR2, an NAD+-dependent protein deacetylase. The mammalian homologue of SIR2, Sirt1, has been shown to modulate the activity of FoxO, a transcription factor that is downstream of the IGF signaling system. These results suggest that Sirt1 ought to affect the IGF pathway. We report here evidence that this is the case in mice. The loss of Sirt1 protein in mice results in increased expression of the IGF binding protein IGFBP1, a secreted modulator of IGF function. A number of the anatomical characteristics of Sirt1-null mice closely resemble those of transgenic mice overexpressing IGFBP1. Our data suggest that Sirt1 is part of a regulatory loop that limits the production of IGFBP1 thereby modulating IGF signaling.

Vibration enhancement of slide-mounted immunofluorescence staining.

Immunofluorescence techniques allow the determination of protein and small molecule distribution within tissues and individual cells. There have been important, innovative modifications of these techniques since their introduction to the biosciences including the use of a mounting medium that prevents photo-bleaching, non-ionic detergents to permeabilize membranes, multiple immunofluorescence labeling and antigen recovery techniques for optimizing ligand-target interactions. While methods have been optimized for ligand-target accessibility in free-floating sections, little innovation has occurred to improve antibody access and epitope recognition in immunohistochemistry on slide-mounted sections or cell culture. During our studies of brain signaling pathways, we sought to improve the immunofluorescence signal to noise ratio in these specimens. We present here a minor modification of immunofluorescence procedures that significantly increases antibody access to epitopes within tissue and improves staining quality while significantly shortening incubation time. Antibody-epitope interactions are dependent on access and affinity. Our technique is based upon application of a vibration source during antibody incubation which increases epitope access, shortens incubation time and thereby minimizes background immunofluorescence. Data are presented on benefits evident with several antibodies raised against proteins and peptides localized in various subcellular compartments. Analysis of the quality of labeling was performed to show that signal intensity is enhanced and background intensity is often diminished when incubations are performed under gentle vibration. This, together with the significant saving of time, should make this procedure applicable to a wide range of neurobiological questions.

The dopamine D1 receptor-rich main and paracapsular intercalated nerve cell groups of the rat amygdala: relationship to the dopamine innervation.

The intercalated cell masses are GABAergic neurons interposed between the major input and output structures of the amygdala. Dopaminergic projections to the main and paracapsular intercalated islands were examined by determining the relationship of the dopamine nerve-terminal networks to the D1-receptor immunoreactive staining of cells within the intercalated islands, using double-fluorescence immunolabelling procedures in combination with confocal laser microscopy. The relationship of terminals positive for both tyrosine hydroxylase and dopamine beta-hydroxylase (noradrenaline and/or adrenaline) to terminals positive for tyrosine hydroxylase but negative for dopamine beta-hydroxylase (dopamine terminals) was studied in relation to the D1-receptor immunoreactivity in adjacent sections at various rostrocaudal levels. The microscopy and image analysis revealed that there was only a minor dopaminergic innervation of the D1 receptor-immunoreactive cells in the rostromedial and caudal component of the main intercalated island, suggesting volume transmission as the main communication mode for dopamine in these regions. In contrast, the D1 receptor-immunoreactive areas in the rostrolateral part of the main island and also the paracapsular intercalated islands showed a high degree of dopaminergic innervation, indicating that synaptic and perisynaptic dopamine transmission plays a dominant role in these regions. It is known that amygdala neurons are involved in the elicitation and learning of fear-related behaviors. We suggest that slow dopaminergic volume transmission in the rostromedial and caudal parts of the main intercalated island may have a role in tonic excitatory modulation in these parts of the main island, allowing GABAergic activity to develop in the central amygdaloid nucleus and thereby contributing to inhibition of fear-related behavioral and autonomic responses. In contrast, a faster synaptic and perisynaptic dopaminergic transmission in the rostrolateral part of the main intercalated island and in the paracapsular intercalated islands may have a role in allowing a more rapid elicitation of fear-related behaviors.

Connexin26 in adult rodent central nervous system: demonstration at astrocytic gap junctions and colocalization with connexin30 and connexin43.

The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions.

Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43.

Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive, spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43. Conversely, Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43. Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF, Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata. On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.

Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons.

BACKGROUND: Carbon monoxide (CO), like nitric oxide (NO), is a putative gaseous neurotransmitter. CO is produced by the enzyme heme oxygenase (HO) acting on a family of heme-containing compounds. Two isomers of HO have been characterized (HO-1, HO-2). In the CNS and in peripheral ganglia HO-2 occurs in a majority of neurons. NO and CO function as transmitters of enteric neurons but the relative distribution of enteric neurons utilizing these gaseous transmitters is unknown in rodent. We have studied the distribution of HO-2 immunoreactivity and NO synthase (NOS) activity within the rat ileum. METHODS: Tissue sections and primary neuronal cell cultures were incubated with a HO-2 specific antibody, and then assessed or reprocessed for NOS activity using NADPH-dependent diaphorase staining. RESULTS: HO-2 immunoreactivity was expressed in subpopulations of myenteric and submucosal neurons. Approximately 45% of the ganglion cells in tissue section were HO-2 positive. This was similar in proportion to those found to stain for NOS activity, and 10% of HO-2 positive neurons also contained NOS. HO-2 immunoreactivity was also found in epithelial cells within the villi, and in interstitial cells around the myenteric plexus and within the smooth muscle. In culture, the distribution and colocalisation of HO-2 and NOS positive neurons was similar to that in tissue sections. We identified labelled neurons as either Dogiel Type I or II; only Type II cells colocalized NOS and HO-2. CONCLUSION: Neurons, endocrine-like cells and interstitial cells with the capacity for CO production are distributed throughout the ileum and some neurons have the capacity to synthesize both NO and CO as gaseous messengers.

Quantitative analyses of neurons projecting to primary motor cortex zones controlling limb movements in the rat.

The objective was to determine if projections of single neurons to primary motor cortex preferentially terminate in several efferent zones that could form synergies for the execution of limb movements. Intracortical microstimulation was used to identify zones evoking hip flexion (HF), elbow flexion (EF), and both plantarflexion (PF) and dorsiflexion (DF) about the ankle. Histological examination showed that the zones from which some movements were evoked extended beyond the agranular cortex into granular cortex. Fluorogold, Fast blue, and propridium iodide or rhodamine-labeled dextran were injected into three of these four efferent zones in each rat. There was a virtual absence of multiple-labeled cells despite having an intermingling of different-colored cells of which 15% in frontal cortex were less than 1.2 mm away from a neighboring neuron that projected to a different efferent zone. This suggests that single neurons projecting to the motor cortex do not hard-wire specific synergies but rather project to single efferent zones in order to offer the greatest degree of freedom for the generation of movements. The distribution of ventral posterolateral and ventrolateral thalamic nucleus labeling depended on whether the injections were in granular or agranular cortex. Conversely, frontal cortex projections to motor efferent zones were made irrespective of their location in either granular or agranular cortex and thereby supporting their presumed role in the control of movements. Hindlimb motor cortex injections yielded retrograde labeling that extended into the more localised distribution of frontal cortex neurons retrogradely labeled from forelimb injections. This may allow hindlimb movements to be synchronized by forelimb movements during walking on challenging terrain.

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

The hyaluronan receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model.

The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75,000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65,000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine-beta-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with beta-halobenzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.

Molecular cloning, expression, and chromosomal assignment of sarcolemmal-associated proteins. A family of acidic amphipathic alpha-helical proteins associated with the membrane.

Two overlapping cDNAs encoding a novel sarcolemmal associated protein (SLAP) were isolated from a cardiac cDNA expression library by immunoscreening with anti-sarcolemmal antibodies. Further characterization of these clones showed that they belonged to a family of related cDNAs that potentially encode polypeptides of 37, 46, and 74 kDa designated SLAP1, SLAP2, and SLAP3, respectively. The SLAP3 transcript was ubiquitously expressed, whereas SLAP1 and SLAP2 transcripts were predominantly expressed in cardiac, soleus, and smooth muscle. SLAP was encoded by a single gene that mapped to chromosome 3p14.3-21.2, and the various transcripts are likely generated by alternative splicing. The primary structure of SLAP predicted that it would have large regions of coiled-coil structure including an 11-heptad acidic amphipathic alpha-helical segment. The carboxyl-terminal region of the SLAP proteins was predicted to have a transmembrane domain, although there was no discernible signal sequence. SLAPs could only be solubilized from cardiac membrane with detergents suggesting that they were integral membrane proteins. Subcellular distribution studies showed that MYC epitope-tagged SLAP localized to regions of juxtaposition between neighboring cell membranes although an intracellular pool of the protein was also present in cells undergoing apparent cleavage. Immunohistochemical localization of SLAP in cardiac muscle revealed that SLAP associated with the sarcolemma and also displayed a reticular pattern of staining that resembled the transverse tubules and the sarcoplasmic reticulum. The SLAPs define a new family of tail-anchored membrane proteins that exhibit tissue-specific expression and are uniquely situated to serve a variety of roles through their coiled-coil motifs.

Distribution of the NPY receptor subtype Y1 within human colon: evidence for NPY targeting a subpopulation of nitrergic neurons.

Neuropeptide Y is a neurotransmitter in both the central nervous system and the enteric nervous system. Neuropeptide Y receptors have been demonstrated by in situ hybridization and ligand binding techniques to be present in both of these systems. In this study we report on the distribution of the Y1 isoform of the neuropeptide Y receptor (YY1) in human colon using an antibody raised against the Y1 receptor. This method permits greater resolution in determining the distribution of the receptor and provides the opportunity to study neurotransmitter markers in relationship to the Y1 receptor. Y1 receptor immunoreactivity was localized within ganglionic neurons and axons of the myenteric and submucosal nerve networks, axons within the muscularis mucosae, longitudinal and circular smooth muscle layers, sympathetic nerve fibers around blood vessels and within scattered cells in the mucosa and basal cells of the crypts. Neuropeptide Y/Y1 double staining showed that the peptide and its Y1 receptor subtype were often colocalized within ganglion cells of Henle's plexus in the submucosa. Thus, Y1 may act as an autoreceptor within the colonic gut wall. Nitric oxide synthase was found within most neurons of the myenteric plexus which displayed Y1-receptor immunoreactivity but this correlation was not seen in the submucosa. Instead, the colocalization of nitric oxide synthase and Y1-immunoreactivity was extremely low. These results indicate a striking difference in the Y1 Neuropeptide Y activation of nitrergic mechanisms within the myenteric and submucosal nerve networks.

Efferent projections of the anterior perirhinal cortex in the rat.

Because convulsive seizures develop very rapidly from kindling sites in the anterior perirhinal cortex, we studied perirhinal efferents by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PhAL). PhAL injections into the anterior perirhinal cortex labelled a prominent network of fibers within the frontal cortex that was most dense within layers I and II and layer VI. As individual PhAL injection sites within the perirhinal cortex were restricted to one or two adjacent laminae, we were able to determine that layer V was the main source of the perirhinofrontal projection. This was confirmed by frontal cortex injections of the retrograde tracer Fluorogold (FG). Other cortical areas with densely labelled fibers following perirhinal PhAL injections included the agranular insular, infralimbic, orbital, parietal, and entorhinal cortices. Moderate to mild fiber labelling was also noted in the posterior piriform, temporal and occipital cortices, and the claustrum. Subcortical labelling was seen in the nucleus accumbens; fundus striati; basal and lateral amygdala nuclei; the "acoustic thalamus"; and the central grey. Several of these cortical and subcortical projections were bilateral. The different laminar origin of these perirhinal efferents is discussed. These results confirmed our prediction of extensive direct projections from the anterior perirhinal cortex to the frontal cortex in the rat. The significance of this projection is discussed with special reference to the anatomical basis of convulsive limbic seizures.

Retinoic acid treated P19 embryonal carcinoma cells differentiate into oligodendrocytes capable of myelination.

Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated P19 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19-derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.