Biomedical subjects

D A Turner

Publications and source records attributed to D A Turner.

At least 55 records · Page 3Linked to original sources

PubMed1997

Interneurons of the dentate-hilus border of the rat dentate gyrus: morphological and electrophysiological heterogeneity.

Interneurons located near the border of the dentate granule cell layer and the hilus were studied in hippocampal slices using whole-cell current clamp and biocytin staining. Because these interneurons exhibit both morphological and electrophysiological diversity, we asked whether passive electrotonic parameters or repetitive firing behavior correlated with axonal distribution. Each interneuron was distinguished by a preferred axonal distribution in the molecular layer or granule cell layer, and four groups could be discerned, the axons of which arborized in (1) the granule cell layer, (2) the inner molecular layer, (3) the outer molecular layer, and (4) diffusely in the molecular layer. In our sample, interneurons with axons arborizing diffusely in the molecular layer were most frequent, and those with axons restricted to the granule cell layer were least frequent. Resting potential, input resistance, time constant, electrotonic length, and spike frequency adaptation (SFA) were not significantly different among the four groups, and the variability in SFA between cells with similar axonal distributions was striking. Clear differences in action potential morphology and afterhyperpolarizations, however, emerged when nonadapting interneurons were compared with those exhibiting SFA. Interneurons exhibiting SFA had characteristically broader spikes, progressive slowing of action potential repolarization during repetitive firing, and slow afterhyperpolarizations that distinguished them from nonadapting interneurons. We propose that the variability in repetitive firing behavior and morphology exhibited by each of these interneurons makes each interneuron unique and may provide a high level of fine tuning of inhibitory control critical to information processing in the dentate.

Action Potentials↗

PubMed1997

Excitatory synaptic site heterogeneity during paired pulse plasticity in CA1 pyramidal cells in rat hippocampus in vitro.

1. The properties of individual excitatory synaptic sites onto adult CA1 hippocampal neurons were investigated using paired pulse minimal stimulation and low noise whole-cell recordings. Non-NMDA receptor-mediated synaptic responses were isolated using a pharmacological blockade of NMDA and GABAA receptors. Amongst the twenty-five stationary ensembles there were twelve showing paired pulse potentiation, two showing paired pulse depression and eleven with no significant net change. The signal-to-noise ratio averaged 4.5:1. There was no correlation between the amplitude of the first and second responses after separation of failures: the percentage of failures averaged 33.6% for the conditioning pulse and 31.7% for the test pulse. 2. Site-directed Bayesian statistical analysis was developed to predict the likely number of activated synapses, synaptic response amplitudes, probability of release and intrinsic variation at each individual synaptic site. Extensive simulations showed the usefulness of this model and defined appropriate parameters. These simulations demonstrated only small errors in estimating parameters of data sets with a small number of sites (< 10) and similar characteristics to the physiological data sets. 3. Physiological ensembles showed between one and three synaptic sites, which exhibited a wide range of values for release probability (0.03-0.99), synaptic amplitudes (1.46-16.8 pA; approximately 62% coefficient of variation between sites) and intrinsic variation over time (approximately 36%). Paired pulse plasticity occurred primarily from alterations in the release probabilities but a few ensembles also showed small changes in site amplitude. Initial release probability correlated negatively with the degree of paired pulse potentiation. Whilst it was possible to use simple assumptions regarding site homogeneity (such as required for a binomial process) for 48% (12 out of 25) of the data sets, the Bayesian analysis was necessary to reveal the complex changes and heterogeneity that occurred in the other 52% of the data sets. The Bayesian site analysis robustly indicated the presence of considerable site heterogeneity, significant intrinsic site variation over time and changes in parameters at individual synaptic sites with plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1997

Fetal hippocampal cells grafted to kainate-lesioned CA3 region of adult hippocampus suppress aberrant supragranular sprouting of host mossy fibers.

Selective lesion of the rat hippocampus using an intracerebroventricular administration of kainic acid (KA) represents an animal model for studying both lesion recovery and temporal lobe epilepsy. This KA lesion leads initially to loss of CA3 hippocampal neurons, the postsynaptic target of mossy fibers, and later results in aberrant mossy fiber sprouting into the dentate supragranular layer (DSGL). Because of the close association of this aberrant mossy fiber sprouting with an increase in the seizure susceptibility of the dentate gyrus, delayed therapeutic strategies capable of suppressing the sprouting of mossy fibers into the DSGL are of significant importance. We hypothesize that neural grafting can restore the disrupted hippocampal mossy fiber circuitry in this model through the establishment of appropriate mossy fiber projections onto grafted pyramidal neurons and that these appropriate projections will lead to reduced inappropriate sprouting into the DSGL. Large grafts of Embryonic Day 19 hippocampal cells were transplanted into adult hippocampus at 4 days post-KA lesion. Aberrant mossy fiber sprouting was quantified after 3-4 months survival using three different measures of Timm's staining density. Grafts located near the degenerated CA3 cell layer showed dense ingrowth of host mossy fibers compared to grafts elsewhere in the hippocampus. Aberrant mossy fiber sprouting throughout the dentate gyrus was dramatically and specifically reduced in animals with grafts near the degenerated CA3 cell layer compared to "lesion only" animals and those with ectopic grafts away from the CA3 region. These results reveal the capability of appropriately placed fetal hippocampal grafts to restore disrupted hippocampal mossy fiber circuitry by attracting sufficient host mossy fibers to suppress the development of aberrant circuitry in hippocampus. Thus, providing an appropriate postsynaptic target at early postlesion periods significantly facilitates lesion recovery. The graft-induced long-term suppression of aberrant sprouting shown here may provide a new avenue for amelioration of hyperexcitability that occurs following hippocampal lesions.

Animals↗

PubMed1997

Development of long-distance efferent projections from fetal hippocampal grafts depends upon pathway specificity and graft location in kainate-lesioned adult hippocampus.

Fetal hippocampal cells grafted into the excitotoxically lesioned hippocampus of adult rats are capable of extending axonal projections into the host brain. We hypothesize that the axonal growth of grafted fetal cells into specific host targets, and the establishment of robust long-distance efferent graft projections, require placement of fetal cells in close proximity to appropriate axon guidance pathways. Intracerebroventricular administration of kainic acid in adult rats leads to a specific loss of hippocampal CA3 pyramidal neurons. We grafted 5'-bromodeoxyuridine-labeled embryonic day 19 hippocampal cells into adult hippocampus at four days post-kainic acid lesion, and quantitatively measured the projection of grafted cells into the contralateral hippocampus and the septum after three to four months survival using Fluoro-Gold and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) tracing. Grafts located in or near the degenerated CA3 cell layer exhibited numerous neurons which established commissural projections with the contralateral hippocampus. However, such projection did not occur in intrahippocampal grafts located away from the CA3 cell layer. In contrast, neurons in all grafts established robust projections into the septum regardless of location within hippocampus although grafts located near the degenerated CA3 cell layer displayed more neurons with such projections. Location of grafted cells clearly influences the development of efferent graft projections into distant targets in the adult host brain, particularly access to axon guidance pathways to facilitate the formation of projections. The establishment of robust long-distance commissural projections of fetal hippocampal grafts is clearly dependent on their placement in or near the degenerated CA3 cell layer, suggesting that appropriate axon guidance pathways for commissural pathways are tightly focussed near this cell layer. However, the establishment of septal projections of these grafts was not dependent on specific location within the CA3 cell layer, suggesting that axonal guidance mechanisms to the septum are more diffuse and not limited to the CA3 dendritic layers. The results underscore that fetal hippocampal grafts are capable of partly restoring lesioned hippocampal circuitry in adult animals when appropriately placed in the host hippocampus.

Animals↗

PubMed1996

Development of fetal hippocampal grafts in intact and lesioned hippocampus.

Functional recovery observed in Parkinson's disease patients following grafting of fetal substantia nigra has encouraged the development of similar grafting therapy for other neurological disorders. Fetal hippocampal grafting paradigms are of considerable significance because of their potential to treat neurological disorders affecting primarily hippocampus, including temporal lobe epilepsy, cerebral ischemia, stroke, and head injury. Since many recent studies of hippocampal transplants were carried out with an aim of laying the foundation for future clinical applications, an overview of the development of fetal hippocampal transplants, and their capability for inducing functional recovery under different host conditions is timely. In this review, we will summarize recent developments in hippocampal transplants, especially the anatomical and/or functional integration of grafts within the host brain under specific host conditions, including a comparison of intact hippocampus with various types of hippocampal lesions or injury. Improvements in grafting techniques, methods for analysis of graft integration and graft function will be summarized, in addition to critical factors which enhance the survival and integration of grafted cells and alternative sources of donor cells currently being tested or considered for hippocampal transplantation. Viewed collectively, hippocampal grafting studies show that fetal hippocampal tissue/cells survive grafting, establish both afferent and efferent connections with the host brain, and are also capable of ameliorating certain learning and memory deficits in some models. However, the efficacy of intracerebral fetal hippocampal grafts varies considerably in different animal models, depending on several factors: the mode of donor tissue preparation, the method of grafting, the state of host hippocampus at the time of grafting, and the placement of grafts within the hippocampus. Functional improvement in many models appeared to be caused partially by re-establishment of damaged circuitry and partially by a trophic action of grafts. However, exact mechanisms of graft-mediated behavioral recovery remain to be clarified due to the lack of correlative analysis in the same animal between the degree of graft integration and behavioral recovery. Issues of mechanisms of action, degree of restoration of host circuitry and amelioration of host pathological conditions will need to be sorted out clearly prior to clinical use of fetal hippocampal transplants for susceptible neurological conditions.

Animals↗

PubMed1996

Adenomyosis: specificity of 5 mm as the maximum normal uterine junctional zone thickness in MR images.

OBJECTIVE: Our objective was to investigate the specificity of the criterion stating that a diagnosis of adenomyosis can be made confidently from MR images of the uterus when the junctional zone is thicker than 5 mm. SUBJECTS AND METHODS: Twenty clinically normal women volunteers (chosen by strict criteria) underwent MR imaging of the uterus. Images were independently evaluated by two experienced observers who measured the greatest thickness of the junctional zone in the anterior, posterior, right and left walls of the uterine corpus and at the top of the fundus. RESULTS: The mean value of the greatest junctional zone thickness (averaged for both observers) was 4.4 +/- 2.1 mm (mean +/- SD, range, 1-9 mm) for the anterior and posterior walls of the corpus, 4.3 +/- 2.1 mm (range, 1-10 mm) for the right and left walls, and 2.3 +/- 1.1 mm (range, 1-5 mm) for the fundus. At least one region of the junctional zone was found to be thicker than 5 mm by at least one observer in 13 (65%) of the 20 subjects; at least one region was found to be thicker than 5 mm by both observers in eight (40%) subjects. At least two regions were measured as thicker than 5 mm by both observers in six (30%) subjects. Serial studies of three volunteers showed transient irregular focal or diffuse thickening (up to 12 mm) of the junctional zone and focal myometrial bulging, probably due to myometrial contractions. CONCLUSION: Our results suggest that if a diagnosis of adenomyosis is based solely on junctional zone thickness in MR images, 5 mm should not be assumed as the upper limit of normal, because this assumption may result in a high false-positive rate.

Adolescent↗

PubMed1995

Intracerebroventricular kainic acid administration in adult rat alters hippocampal calbindin and non-phosphorylated neurofilament expression.

Calbindin and non-phosphorylated neurofilament proteins were assessed in hippocampus following a unilateral intracerebroventricular kainic acid injection at 4, 26, and 60 days post-lesion, using immunocytochemical expression. The density of calbindin-positive non-pyramidal neurons throughout the hippocampus showed no significant alteration at 4 days post-lesion, a significant decrease at 26 days post-lesion, and a partial recovery at 60 days post-lesion. In addition, calbindin immunoreactivity was dramatically reduced at 26 days post-lesion in the CA1 pyramidal and dentate granule cell layers and the mossy fibers, bilaterally. Although not significant statistically, most of these reductions showed signs of reversal at 60 days post-lesion except the CA1 pyramidal cell layer where the dramatic reductions persisted. Neurofilaments were also altered throughout the post-lesion period, particularly in abnormal expression of non-phosphorylated neurofilament proteins in mossy fibers. The apparent return of calbindin immunoreactivity in non-pyramidal neurons by 60 days post-lesion suggests that recovery from the lesion may involve remaining neuronal elements which either become reactivated with time or have the capability to express normal levels of calbindin with re-innervation. On the other hand, prolonged calbindin reductions in superficial CA1 pyramidal cells suggest sustained down-regulation of calbindin expression owing to persistent reductions in the activity of these neurons. The temporal correlation of the expression of non-phosphorylated neurofilaments in mossy fibers with their sprouting response following target loss suggests a potential role for non-phosphorylated neurofilaments in neuronal plasticity involving axonal sprouting. Alternatively, it may also suggest that injury-induced neurofilament modifications are either conducive or permissive for axonal sprouting.

Animals↗

PubMed1995

Optical mapping of translucence changes in rat hippocampal slices during hypoxia.

We have evaluated the effects of hypoxia on changes in light transmittance (delta T/T) in rat hippocampal interface slices at 36 degrees C, using a digital imaging system. Slice translucence increased only slightly (delta T/T = 4.65% in CA1; control 2.27%) during brief hypoxia in which hypoxic spreading depression-like depolarization was not induced. If hypoxia duration was sufficient to trigger spreading depression in CA1, slice translucence increased rapidly in CA1 (delta T/T = 27.5%) and smaller, delayed changes were noted in other regions. These spreading depression-induced changes reversed slowly to control levels upon reoxygenation. Measurement of intrinsic optical signals revealed both spatial and temporal patterns of cell volume changes associated with hypoxia and spreading depression in hippocampus.

Animals↗

PubMed1995

Morphometric and electrical properties of reconstructed hippocampal CA3 neurons recorded in vivo.

CA3 pyramidal neurons were stained with biocytin during intracellular recording in rat hippocampus in vivo and reconstructed using a computer-based system. The in vivo CA3 neurons were characterized primarily according to their proximity to the hilus and secondarily with respect to the septotemporal location. Neurons measured in CA3a (n = 4), in CA3b (n = 4), and in posterior/ventral locations (n = 3) had the greatest dendritic lengths (19.8, 19.1, and 26.8 mm on average, respectively). Cells closer to the hilus showed much shorter dendritic lengths, averaging 10.4 mm for CA3c neurons (n = 4) and 11.6 mm for zone 3 neurons (n = 2). Half of the cells showed more than one major apical dendrite, and dendritic trees were highly variable even within CA3 subregions. The mean electronic length for these cell groups averaged between 0.30 lambda (CA3c) and 0.45 lambda (posterior/ventral), assuming a constant specific-membrane resistivity of 60 K omega-cm2. These CA3 neurons form a database of reconstructed neurons for further morphometric and electrical modelling studies. The large degree of variability between individual CA3 neurons indicates that both dendritic and electrical properties should be specifically calculated for each cell rather than assuming a "typical" morphology.

Animals↗

PubMed1995

Non-phosphorylated neurofilament protein immunoreactivity in adult and developing rat hippocampus: specificity and application in grafting studies.

Neurofilament proteins are critical to the development and maintenance of neuronal shape in the nervous system. These proteins are developmentally regulated and several transition forms are expressed, prior to full neuronal stabilization. We have studied the spatial distribution and time course of expression of non-phosphorylated neurofilament protein (NPNFP) immunoreactivity in several preparations of rat hippocampus, using a mixture (SMI 311) of several monoclonal antibodies directed against NPNFP epitopes. Differential staining was observed in young and adult hippocampus. Large pyramidal neurons in CA3 and CA4 subfields were strongly immunoreactive in adult hippocampus whereas the smaller CA1 pyramidal neurons, most interneurons and dentate granule cells were immunonegative. SMI 311 staining initially appeared at postnatal day (P) 5 with positive staining in apical dendrites and soma in a few pyramidal neurons in CA3, but almost reached the adult pattern by P10. Compared to adult hippocampus, the number of immunoreactive interneurons in all subfields appeared increased at P10 and P15. In cultures of embryonic hippocampus, all neurons, regardless of their morphology, were SMI 311 positive, suggesting loss of differential expression in tissue culture conditions. However, SMI 311 expression in fetal hippocampal neurons grafted to adult hippocampus was similar to hippocampal neurons which had developed in situ. These results suggest that SMI 311 antibody identifies a distinct group of primarily CA3 and CA4 pyramidal cells in adult hippocampus. The application of SMI 311 immunostaining appears suitable for identification of large CA3 and CA4 pyramidal neurons within hippocampal transplants grafted to adult CNS but not in tissue culture.

Animals↗

PubMed1995

Enhanced cell survival in fetal hippocampal suspension transplants grafted to adult rat hippocampus following kainate lesions: a three-dimensional graft reconstruction study.

The success of fetal neural transplantation in alleviating neurological dysfunction depends significantly on the degree of graft cellular survival and dispersion within the host. We hypothesize that various lesion-induced host factors, such as trophic support and denervation, enhance these graft factors differentially following unilateral intracerebroventricular kainic acid lesions. We have performed quantitative graft reconstructions of embryonic day 19 fetal hippocampal cells transplanted at different post-lesion delays (four, 11, 26 and 60 days) into adult hippocampus. We have used a permanent graft prelabel (5'-bromodeoxyuridine) which allows unambiguous identification of graft cell location in the host. Cellular integration of grafted cells was rigorously assessed by calculating both absolute cell survival (cells recovered/cells injected) and quantitative cell dispersion from the graft injection site. Graft cell survival and graft volume were dramatically enhanced in transplants performed ipsilateral to the kainic acid lesion, to a maximum of 77% cell recovery at a post-lesion graft delay of four days. Cell survival decreased over time after the lesion to the level of the contralateral grafts by 60 days post-lesion (33% cell survival), though cell survival on either side remained significantly greater than grafts into normal hosts (18% survival). The time-course of post-lesion enhanced survival (four to 26 days) in hippocampus ipsilateral to the lesion strongly correlated with reported peak neurotrophic activity (four to 30 days). Graft cell dispersion was limited in this model, averaging less than 500-microns-cell movement; there were no differences compared to transplants grafted into normal hippocampus. Timm's staining demonstrated host mossy fiber innervation of transplants to be denser ipsilateral to the kainic acid lesion, resulting in a partial decrease in dentate supragranular sprouting near appropriate grafts placed at early post-lesion time points. These results suggest that lesion-induced trophic support and denervation lead to improved graft cell survival but not graft cell dispersion. The improved survival of grafts transplanted into hippocampus contralateral to the lesion, compared to transplants in normal hippocampus, suggests that denervation alone exerts a significant effect on graft cell survival. However, this denervation effect on graft cell survival is significantly less than the combination of both enhanced neurotrophic factors and denervation observed ipsilateral to the lesion.

Animals↗

PubMed1995

Selective laser-activated lesioning of prelabeled fetal hippocampal grafts by intracellular photolytic chromophore.

Selective removal of grafted tissue is critical to assess the functional role of that tissue in the host, yet is technically difficult for well-dispersed neural grafts. We labeled fetal hippocampal cells with both a nuclear marker (5'-bromodeoxyuridine) and a cytoplasmic marker (latex microspheres) before grafting into normal adult hippocampus. A nontoxic chromophore, chlorin e6, was conjugated on to the surface of latex microspheres of the treatment group. Chlorin e6 produces cytotoxic singlet oxygen only during photoactivation. Grafted animals received transcranial exposure to various intensities of near-infrared laser light. Following laser exposure, grafts were observed in all groups except in transplants prelabeled with chlorin e6 latex microspheres. An optimal laser exposure of 50-100 J/cm2 (4-8 min) was found to selectively remove only the chlorin e6-containing grafted cells. With increasing doses of laser illumination, non-specific lesions of the host tissue surrounding the graft were induced. Quantitative graft analysis, in the absence of laser exposure, indicated that the survival of grafted cells was similar between control transplants labeled with latex microspheres alone and grafts labeled with latex microspheres plus chlorin e6. This is further evidence that chlorin e6 by itself was not toxic without laser exposure. The results clearly demonstrate that singlet oxygen-induced cell photolysis can result in selective, non-invasive removal of dispersed grafted cells located in adult hippocampus. This technique may facilitate defining specific mechanisms of action of grafted cells which mediate functional recovery in different host conditions.

Animals↗

PubMed1995

Computer simulations of morphologically reconstructed CA3 hippocampal neurons.

1. We tested several hypotheses with respect to the mechanisms and processes that control the firing characteristics and determine the spatial and temporal dynamics of intracellular Ca2+ in CA3 hippocampal neurons. In particular, we were interested to know 1) whether bursting and nonbursting behavior of CA3 neurons could be accounted for in a morphologically realistic model using a number of the known ionic conductances; 2) whether such a model is robust across different cell morphologies; 3) whether some particular nonuniform distribution of Ca2+ channels is required for bursting; and 4) whether such a model can reproduce the magnitude and spatial distribution of intracellular Ca2+ transients determined from fluorescence imaging studies and can predict reasonable intracellular Ca2+ concentration ([Ca2+]i) distribution for CA3 neurons. 2. For this purpose we have developed a highly detailed model of the distribution and densities of membrane ion channels in hippocampal CA3 bursting and nonbursting pyramidal neurons. This model reproduces both the experimentally observed firing modes and the dynamics of intracellular Ca2+. 3. The kinetics of the membrane ionic conductances are based on available experimental data. This model incorporates a single Na+ channel, three Ca2+ channels (CaN, CaL, and CaT), three Ca(2+)-independent K+ channels (KDR, KA, and KM), two Ca(2+)-dependent K+ channels (KC and KAHP), and intracellular Ca(2+)-related processes such as buffering, pumping, and radial diffusion. 4. To test the robustness of the model, we applied it to six different morphologically accurate reconstructions of CA3 hippocampal pyramidal neurons. In every neuron, Ca2+ channels, Ca(2+)-related processes, and Ca(2+)-dependent K+ channels were uniformly distributed over the entire cell. Ca(2+)-independent K+ channels were placed on the soma and the proximal apical dendrites. For each reconstructed cell we were able to reproduce bursting and nonbursting firing characteristics as well as Ca2+ transients and distributions for both somatic and synaptic stimulations. 5. Our simulation results suggest that CA3 pyramidal cell bursting behavior does not require any special distribution of Ca(2+)-dependent channels and mechanisms. Furthermore, a simple increase in the Ca(2+)-independent K+ conductances is sufficient to change the firing mode of our CA3 neurons from bursting to nonbursting. 6. The model also displays [Ca2+]i transients and distributions that are consistent with fluorescent imaging data. Peak [Ca2+]i distribution for synaptic stimulation of the nonbursting model is broader when compared with somatic stimulation. Somatic stimulation of the bursting model shows a broader distribution in [Ca2+]i when compared with the nonbursting model.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗

PubMed1995

Glomus jugulare tumor metastatic to the sacrum after high-dose radiation therapy: case report.

A 47-year-old woman with left ear pain and hearing loss was diagnosed with a glomus jugulare tumor for which she received radiation therapy as the primary treatment. Over a period of 20 years, she developed temporal bone necrosis, brain stem calcifications, local tumor recurrence, and eventually metastases to her lungs and sacrum. This case underscores the often indolent nature of glomus jugulare tumors, the late sequelae of radiation therapy for benign intracranial tumors, and the potential of these tumors to metastasize. This patient's history suggests that aggressive surgical resection should be considered early for such tumors, particularly because radiation treatment does not ablate the tumor. This is only the second reported case of a glomus jugulare tumor metastatic to the sacrum.

Aged↗

PubMed1994

Reactive astrocytes express the embryonic intermediate neurofilament nestin.

Nestin is a neurofilament protein expressed by the immediate precursors to neurons and glia in rats and humans. Nestin immunoreactivity in the rat CNS was studied following kainic acid (KA) hippocampal lesions. Numerous nestin positive cells within the KA lesion were confirmed to be reactive astrocytes by their immunoreactivity for glial fibrillary acidic protein (GFAP). The number of these cells decreased with time after the KA lesion and no astrocyte immunostaining for nestin was observed in control animals. A subset of nestin-positive cells in the ventricular subependymal region appeared to be radial glial cells, extending to cell body layers. Nestin is one of several embryonic markers expressed by reactive astrocytes, suggesting an embryonic reversion induced by the KA lesion, possibly to enhance functional recovery.

Animals↗

PubMed1994

Denervation-induced dendritic alterations in CA1 pyramidal cells following kainic acid hippocampal lesions in rats.

Kainic acid (KA) lesions of the CA3 region of the hippocampus lead to denervation of ipsilateral CA1 neurons. To assess denervation-induced post-synaptic changes, intracellular physiological recordings were performed in the CA1 region in vitro, from both control and KA-treated tissue. The neurons were intracellularly stained with neurobiotin, reconstructed using a quantitative three-dimensional system and analyzed for morphometric and electrotonic parameters. Total dendritic length was slightly longer in the denervated CA1 cells and there was a selective and significant increase in both branches and terminals in the mid-stratum radiatum (300-550 microns from the soma using Sholl analysis) in the KA-treated rats compared to untreated controls, particularly for cells at 5 days post-lesion and later, which exhibited graded synaptically-evoked bursts. However, there was no significant difference in the basal dendritic arborization. Electrotonic modelling of the dendritic structure revealed specific membrane resistivity values of 33.4 k omega.cm2 for the normal CA1 cells and 29.8 K omega-cm2 for the KA-treated cells, assuming an internal resistivity of 200 omega.cm2, shrinkage correction of 1.57 and a spatial distribution of dendritic spines. The number of dendritic terminals of these denervated CA1 neurons at electrotonic distances between 0.5 lambda and 0.7 lambda also significantly increased in the cells from KA-treated animals. These findings indicate that there is a selective and specific increase in the number of apical terminals and dendritic branches following the unilateral kainic acid lesion. These apical branch changes may represent dendritic sprouting as a post-synaptic response to the denervation, which was particularly marked in neurons exhibiting graded synaptic bursting behavior.

Animals↗

PubMed1994

Quantitative graft integration of fetal hippocampal transplants labeled with 5' bromodeoxyuridine into normal adult hippocampus.

Quantitative studies of neural graft development require: (1) a cell label which is both preferential for neurons and can be measured in terms of specific labeling; (2) a serial reconstruction method for identifying labeled cells in a three-dimensional pattern in the host; and (3) measurements of cell dispersion which indicate the specificity of cell movement in the host. We have used 5' bromodeoxyuridine (BrdU) as a nuclear marker of transplanted fetal hippocampal cells, performing daily injections into the donor to label the majority of cells in the hippocampus between Gestation Days 15 and 19. After harvesting, the BrdU-labeled hippocampal cell suspensions were either cultured or grafted into normal adult hippocampus. The labeling index of these fetal hippocampal cells was calculated to be 90% in both smears of cell suspensions and 48-h cultures and these were predominantly (77%) neurons by immunostains. Quantitative studies of potential toxicity of the BrdU in culture also revealed no differences in neurite development or survival, compared to unlabeled cells. The survival and migration of grafted cells were quantitatively evaluated by three-dimensional serial reconstruction of host brain sections. Absolute graft cell survival (cells recovered/cells injected) varied according to location: grafts in close proximity to the ventricle and white matter tracts (corpus callosum or fimbria) showed improved survival (20 and 25%, respectively) compared to grafts localized entirely within the (normal) hippocampus (only 9% survival). Although the grafts expanded considerably from the initial injection site, the graft cell dispersion appeared nonspecific. Thus, this format of quantitative graft assessment indicates that under normal (nonlesioned) host conditions the location of the grafts critically influenced graft development and there was minimal specific migration of grafted cells into cell body regions.

Animals↗

PubMed1994

Quantitative estimation of cryopreservation viability in rat fetal hippocampal cells.

Cryopreserved fetal tissue may be useful for neural grafting, but quantitation of yield is crucial for estimating cell transplant requirements. We have assessed cell survival and viability following dissociation, cryopreservation, and culture of rat fetal hippocampus to estimate cell yields at all stages of preparation. Hippocampal tissue from E17 fetal rats was dissected from the CNS and cryopreserved in toto after equilibration with 10% DMSO. Freshly dissociated tissue from contralateral hippocampi yielded a significantly greater number of cells per hippocampus than the cryopreserved tissue. Cell viability after dissociation and survival in culture was also significantly increased in the fresh hippocampi compared to that in the frozen group. Our results indicate that the overall cryopreservation cell yield at 24 h in culture is approximately 12% of the initial fresh tissue, taking these three factors into account. Though cryopreservation of fetal neural tissue for transplantation offers several practical advantages over immediate use, significant additional tissue may be required in comparison to fresh tissue to achieve a suitable quantity of live cells for grafting.

Animals↗