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.

Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.

Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.

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.

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.

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.

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.

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.

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.

Crossed connections of the substantia nigra in the rat.

The existence of crossed multisynaptic pathways that allow for the interdependent control of activity in one substantia nigra and its contralateral counterpart has been inferred from a number of recent biochemical and neurophysiological investigations. This prompted a reexamination of the connections of the substantia nigra with an emphasis on crossed inputs to and crossed projections from that nucleus. Male albino rats received 20-50-nl pressure injections of a 1% wheat germ agglutinin-conjugated horse-radish peroxidase (WGA-HRP) solution into the substantia nigra or into surrounding areas as controls. Following a 24-hour survival period the animals were processed according to the tetramethylbenzidine protocol for the visualization of HRP. The pattern of anterograde transport of WGA-HRP after substantia nigra injections, confirming for the most part previous reports, demonstrated ipsilateral nigral efferent projections to the striatum; globus pallidus; subthalamic nucleus; the lateral dorsal, paralamellar mediodorsal, ventromedial, and parafascicular thalamic nuclei; central gray, midbrain reticular formation; superior colliculus; and peribrachial area, including the pedunculopontine nucleus. Additionally, the nigral projections to the paralamellar mediodorsal and ventromedial thalamic nuclei and to the superior colliculus were demonstrated to be bilateral. Most of these connections were confirmed by the complementary retrograde experiment. In accordance with previous reports, intranigral WGA-HRP injections retrogradely labeled neurons located in the ipsilateral prefontal cortex, motor cortex, striatum, globus pallidus, central nucleus of the amygdala, anterior hypothalamic area, subthalamic nucleus, and dorsal raphe. Additionally, labeled perikarya were observed in the ipsilateral parafascicular thalamic nucleus, in the contralateral posterior lateral hypothalamic area, and in the ipsilateral and contralateral peribrachial-pedunculopontine area. These latter nigral afferents were confirmed with complementary WGA-HRP injections into each of the regions of origin. While bilateral peribrachial-pedunculopontine innervation of the substantia nigra has been reported in the cat there has been no previous demonstration of a crossed nigral afferent system from the contralateral posterior lateral hypothalamic area. The results are discussed with reference to the pathways that may mediate the interdependent control of the activity of neurons in the left and right substantia nigra. Additionally, the association of the substantia nigra with a variety of neuronal circuits, including the cerebellofugal, tectothalamic, thalamocortical, thalamostriatal, and basal ganglia pathways, are discussed.

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.

D1 dopamine receptor agonist-induced Fos-like immunoreactivity occurs in basal forebrain and mesopontine tegmentum cholinergic neurons and striatal neurons immunoreactive for neuropeptide Y.

The present study sought to determine whether the D1 dopamine receptor agonist CY 208-243 increases Fos-like immunoreactivity in neurochemically distinct populations of interneurons in the 6-hydroxydopamine-denervated striatum. In vivo microdialysis studies indicate that cholinergic interneurons in the striatum are excited by the administration of CY 208-243 and that this effect is potentiated by dopaminergic deafferentation. Since Fos is considered to be a marker of neuronal activity, we examined the overlap between CY-208-243-induced Fos-like immunoreactivity and striatal cholinergic interneurons labelled with the cholinergic marker enzyme choline acetyltransferase. Unexpectedly, cholinergic interneurons in the striatum were not found to express Fos-like immunoreactivity. However, D1 agonist-induced Fos-like immunoreactivity was found in neurons immunoreactive for neuropeptide Y. Consequently, the failure of cholinergic neurons in the striatum to express D1 agonist-induced Fos-like immunoreactivity does not appear to be a general property of striatal interneurons. Indeed, CY 208-243 increased Fos-like immunoreactivity in choline-acetyltransferase-positive neurons in the basal forebrain and lateral dorsal tegmental nucleus. In the case of cholinergic basal forebrain neurons, administration of CY 208-243 has been shown to enhance the release of acetylcholine from their terminals located in the frontal cortex. Thus, unlike cholinergic interneurons in the striatum, the expression of Fos-like immunoreactivity in cholinergic basal forebrain neurons is correlated with an increase in transmitter release. Choline acetyltransferase immunoreactivity was markedly reduced in cholinergic basal forebrain neurons ipsilateral to the 6-hydroxydopamine lesion. This decrease in choline acetyltransferase immunoreactivity was confined to a pocket of cortically projecting neurons located in the posterior part of the horizontal limb of the diagonal band which included the medial preoptic nucleus. Interestingly, D1 agonist-induced Fos-like immunoreactivity was located predominantly in those cholinergic neurons which displayed depressed choline acetyltransferase immunoreactivity. Since Fos is often induced as a consequence of increased activity, it is tempting to speculate that those neurons which stained weakly for choline acetyltransferase had been excited by the D1 agonist administration. Accordingly, the destruction of mesencephalic dopaminergic neurons with 6-hydroxydopamine may have deprived these cholinergic neurons of an excitatory D1 receptor-mediated drive, resulting in a reduction of choline acetyltransferase immunoreactivity. These results suggest that the degeneration of nigrostriatal dopamine neurons may contribute to the loss of cholinergic basal forebrain function in Parkinson's disease.

Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs.

The distribution of galanin-like immunoreactivity in the rat hippocampal formation (hippocampus and dentate gyrus) was studied and its origins were determined using various lesioning techniques. Special reference was made to the known cholinergic and noradrenergic hippocampal inputs from the septum-basal forebrain complex and locus coeruleus, both of which have previously been shown to co-contain galanin-like immunoreactivity at the cell body level. Galanin-immunoreactive fibers in the hippocampal formation were of at least three different morphological types: (1) Fine, slender, faintly immunoreactive fibers were seen throughout the hippocampal formation. (2) A strongly fluorescent varicose fiber population was observed mainly in the strata radiatum and oriens of the ventral CA3 region. (3) A population of fine, faint puncta was seen within the granule and pyramidal cell layers throughout the hippocampal formation. Knife cut lesions of the dorsal afferent pathways resulted in almost complete disappearance of all fiber types, except for the ventral fine fibers. Lesions of the fimbria affected mainly the coarse and punctate fiber types, while lesions of the supracallosal striae depleted mainly the fine fibers. Cuts anterior and ventral to the hippocampal formation caused a decrease in ventral fine fibers. Furthermore, lesions of the dorsal bundle caused an almost complete disappearance of the fine fibers in all regions of the hippocampal formation. Neurotoxin lesions of the diagonal band/septal complex resulted in decreases in faintly immunoreactive puncta within the granule cell layer and adjacent fine fibers. It is concluded that most fine galanin-positive fibers originate in the lower brain stem, presumably the locus coeruleus, and appear to reach the hippocampal formation primarily through the supracallosal striae and the ventral route. The fimbria seems to contain a large proportion of the fibers giving rise to the coarse strongly fluorescent innervation, which appears to originate rostral to the pons. The galanin-immunoreactive fibers originating in cholinergic somata of the diagonal band, medial septal nuclei, previously shown to project to the hippocampal formation, seem to give rise to faintly labeled puncta within the granule and pyramidal cell layers, and to a small proportion of the fine fibers bordering the cell layers, as revealed by immunohistochemistry using our antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: an immunohistochemical study of adenosine deaminase-positive neurons and fibers.

The intense immunohistochemical reaction for the enzyme adenosine deaminase displayed by neurons in the tuberomammillary nucleus in the rat was used to study the distribution and morphology of cells comprising this nucleus, their fiber fields within the posterior hypothalamus and their projection pathways from the hypothalamus. Neurons immunoreactive for adenosine deaminase were found along ventricular and basal aspects of the hypothalamus from the level of the dorsomedial nucleus to the caudal pole of the mammillary body. Approximately 4500 neurons were seen on each side of the brain. Positive neurons showed a complex distribution, largely avoiding nuclear boundaries within the posterior basal hypothalamus and mammillary body. This distribution is mapped in detail and a nomenclature based on topography is introduced so that different regions of the cell distribution may be discussed more easily. Reactive neurons showed a Golgi-like staining which allowed careful study of their morphology. In general, neurons were large, with major axes of from 22 to 30 micron, and bipolar in shape. A second, smaller cell type, 14-16 micron in diameter was also seen and, although often less intensely stained, it was considered a constituent of tuberomammillary nucleus of the hypothalamus as well. Stained dendritic arbours extended considerable distances from the parent cell bodies and branched regularly. Dendrites showed very sparse spines and had an apparently scalloped surface. Features suggestive of varicose segments of dendrites were also noted. The long, smooth dendrites of positive neurons were often seen to aggregate into bundles which avoided nuclear boundaries and tended to collect adjacent to basal and ventricular surfaces of the posterior hypothalamus. Varicose fibers immunoreactive for adenosine deaminase formed a dense network within the hypothalamus. These fibers were considered to derive from the positive neurons in the tuberomammillary nucleus and were similar to adenosine deaminase-immunoreactive fibers seen throughout much of the rest of the brain. The density of this type of positive fiber was, however, much greater within the hypothalamus. The region of the posterior basal hypothalamus also contained relatively sparse populations of adenosine deaminase-positive fibers, apparently distinct from this network. These consisted of a field of fine fibers in the median division of the medial mammillary nucleus and a few large varicosities in the dorsolateral part of the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural observations of the cholinergic neuron in the rat striatum as identified by acetylcholinesterase pharmacohistochemistry.

In order to examine the ultrastructural features of the cholinergic neuron in the striatum (caudatoputamen) of the rat, cytochemistry for acetylcholinesterase was conducted 2-12 h after intramuscular injection of the irreversible acetylcholinesterase inhibitor diisopropylphosphorofluoridate. Light microscopic examination of Epon sections reacted from acetylcholinesterase showed that only large-sized cells in the striatum (25-35 microns in the long axis) were stained intensely. In the case of longer survival periods (10-12 h), some lightly stained cells (medium-sized) were seen dispersed amongst the large acetylcholinesterase-rich cells. Electron microscopic observations were made on ultrathin sections of selected large acetylcholinesterase-rich neurons that were first studied by light microscopy. The nucleus of these cells has an eccentric position and possesses several indentations of the nuclear envelope. The cytoplasm contains abundant organelles, many exhibiting features unique to this cell type. Many stacks of granular endoplasmic reticulum, arranged in a parallel manner and forming typical Nissl bodies, were observed in the periphery of the perikarya, and many distinct golgi complexes were seen in the perinuclear zone. At all post-diisopropylphosphorofluoridate survival times, heavy deposits of acetylcholinesterase reaction product were found within the perikarya of this cell type, for the most part within the cisternae of the granular endoplasmic reticulum. At the longer post-diisopropylphosphorofluoridate survival times, reaction product within the cytoplasm was very dense and appeared to have reached a maximum level. At these times reaction product also appeared in the secondary and tertiary dendritic branches of the large-sized neurons. Of the other cell types in the striatum, two types of medium-sized cells displayed a light deposit of reaction product in their perikarya, but this was observed only at longer recovery times (8-12 h). The majority of cells in the striatum lacked reaction particles. Throughout the early post-diisopropylphosphorofluoridate period, the recovery of enzyme activity in the neuropil was moderate compared to that seen within cell bodies. These findings indicate that the large-sized neuron is the only striatal structure that shows rapid regeneration of acetylcholinesterase activity during the early recovery phase after diisopropylphosphorofluoridate administration. Previous studies have indicated that this type of neuron represents the cholinergic interneuron of the striatum. (ABSTRACT TRUNCATED AT 400 WORDS)

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.

Quantitative histochemical analysis of cytochrome oxidase in rat dorsal root ganglia and its co-localization with carbonic anhydrase.

A quantitative histochemical method was developed and standardized and then used to characterize the heterogeneity of cytochrome oxidase activity among primary afferent neuronal cell bodies in dorsal root ganglia of rat. In addition, the relationship between cytochrome oxidase and carbonic anhydrase activities in these neurons was determine. In tests of the procedure, the density of cytochrome oxidase reaction product evaluated repeatedly in individual neurons within sections of ganglia was found to increase linearly over incubation periods of up to 6 h. The heterogeneity in cytochrome oxidase activity in ganglia was not simply a reflection of the heterogeneity in ganglion cell sizes. On the whole, each class of ganglion cell exhibited the full range of staining densities encountered but intense staining was observed in many more large type A cells than small type B cells. The latter, together with their termination fields within the substantia gelatinosa of the spinal cord, were lightly stained. A significant positive correlation was found between neuronal size and staining density (r = 0.43). However, the large scatter in the plot of these two variables suggests that the expression of cytochrome oxidase in sensory neurons is governed to a considerable extent by properties of these neurons that are unrelated to their size. Analysis of cytochrome oxidase and carbonic anhydrase activities in the same ganglion cells revealed that all neurons with dense staining for the oxidase were anhydrase positive. Conversely, however, some intensely anhydrase-positive cells exhibited only light staining for cytochrome oxidase. The heterogeneity of cytochrome oxidase activity among neurons in dorsal root ganglia may be related to the steady state electrophysiological activity of distinct populations of sensory neurons which in turn may be related to the specific sensory modalities these populations transmit. The observation that some neurons with the greatest abundance of carbonic anhydrase do not contain high or even moderate levels of cytochrome oxidase suggests some degree of dissociation between the functional requirement for carbonic anhydrase in sensory neurons and the rate of energy expenditure in these cells.

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.

Neurons derived from P19 embryonal carcinoma cells have varied morphologies and neurotransmitters.

Treatment of P19 embryonal carcinoma cells with retinoic acid induces their differentiation into a population of cells consisting of neurons and other cell types normally derived from neuroectoderm. We used immunohistological and histochemical techniques to identify some of the neurotransmitters in the P19-derived neurons. The majority of neurons contained GABA, glutamic acid decarboxylase, and GABA-transaminase. Neuropeptide Y and somatostatin were less frequently found and both were partially co-expressed with GABA and with one another. Smaller numbers of cells were positive for tyrosine hydroxylase, DOPA decarboxylase, serotonin, calcitonin gene-related peptide, galanin and substance P. The variety and proportions of cells with different transmitter types were reproducible from one experiment to the next and varied very little over 40 days in culture except for cells containing enkephalin, which were abundant only in mature cultures of 32 days or more. Synapses formed between neurons and some contained both small clear and large dense-core vesicles within the presynaptic bouton. Because GABA, neuropeptide Y and somatostatin are abundant in P19-derived neurons as well as in embryonic neurons in rostral regions of the mammalian CNS, we suggest that the developmental events occurring in P19 cell cultures closely resemble those of the embryonic neuroectoderm.

Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum.

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons. These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.