bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha 1-adrenoceptor augmentation of beta-stimulated cAMP formation is enhanced by estrogen and reduced by progesterone in rat hypothalamic slices
Authors: Petitti N , Etgen AM
Published: April 2018

These experiments examined the influence of estradiol and progesterone given in vivo on norepinephrine (NE) regulation of cAMP synthesis in hypothalamic and preoptic area slices in vitro. Administration of progesterone to estrogen-primed female rats attenuated NE-induced slice cAMP accumulation. This hormone-dependent reduction in NE-stimulated cAMP synthesis was observed in slices incubated with TTX and in slices prepared from hypophysectomized rats, suggesting that progesterone effects on NE receptor activation of cAMP-generating systems are not secondary to the release of neurotransmitters that inhibit adenylyl cyclase or to changes in pituitary hormone secretion. Progesterone suppression of NE-induced cAMP formation could be prevented by incubating slices in the presence of a phorbol ester. In additional studies, the activity of beta-NE receptors was assessed by measuring isoproterenol (ISO)-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor RO-20–1724, and the activity of alpha 1 receptors was evaluated by measuring phenylephrine (PHE) augmentation of the ISO response. Estradiol reduced the cAMP response to ISO in both hypothalamic and preoptic area slices, and this effect was not reversed by subsequent progesterone treatment. Estradiol also enhanced PHE augmentation of ISO-stimulated cAMP synthesis. Moreover, administration of progesterone subsequent to estradiol eliminated alpha 1-receptor augmentation of the ISO response. An alpha 1 enhancement of the ISO response is observed if the progestin receptor antagonist RU 38486 is administered before progesterone. Progesterone also abolished PHE potentiation of vasoactive intestinal polypeptide-stimulated cAMP accumulation. In contrast, neither phorbol ester nor muscarinic (carbachol) potentiation of the cAMP response to ISO was affected by progesterone. The data suggest that ovarian steroids regulate the coupling of both alpha 1 and beta receptors to the membrane effector systems that generate intracellular cAMP.
Muller cells are a preferred substrate for in vitro neurite extension by rod photoreceptor cells
Authors: Kljavin IJ , Reh TA
Published: April 2018

To define the factors important in photoreceptor cell morphogenesis, we have examined the ability of rods to extend neurites in vitro. Retinas from neonatal rats were dissociated and plated onto substrate-bound extracellular matrix (ECM) components or cell monolayers. When rods, identified with monoclonal antibodies to opsin, were in contact exclusively with purified ECM (e.g., laminin, fibronectin, type I collagen, or Matrigel), neurite outgrowth was extremely limited. By contrast, rods extended long neurites on Muller cells. Retinal or brain astrocytes, endothelial cells, 3T3 fibroblasts, or other retinal neurons were less supportive of rod process outgrowth. These data demonstrate regional specificity in the promotion of neurite outgrowth by glia and suggest that not all neurons within the retina require the same morphogenic factors.
Developmental changes in the membrane current pattern, K+ buffer capacity, and morphology of glial cells in the corpus callosum slice
Authors: Berger T , Schnitzer J , Kettenmann H
Published: April 2018

Recent studies indicated that glial cells in tissue culture can express a variety of different voltage-gated channels, while little is known about the presence of such channels in glial cells in vivo. We used a mouse corpus callosum slice preparation, in which after postnatal day 5 (P5) more than 99% of all perikarya belong to glial cells (Sturrock, 1976), to study the current patterns of glial cells during their development in situ. We combined the patch-clamp technique with intracellular labeling using Lucifer yellow (LY) and subsequent ultrastructural characterization. In slices of mice from P6 to P8, we predominantly found cells expressing delayed-rectifier K+ currents. They were similar to those described for cultured glial precursor cells (Sontheimer et al., 1989). A-type K+ currents or Na+ currents were not or only rarely observed, in contrast to cultured glial precursors. LY labeling revealed that numerous thin processes extended radially from the perikaryon of these cells, and ultrastructural observations suggested that they resemble immature glial cells. In slices of older mice (P10-13), when myelination of the corpus callosum has already commenced, many cells were characterized by an almost linear current- voltage relationship. This current pattern was similar to cultured oligodendrocytes (Sontheimer et al., 1989). Most processes of LY-filled cells with such a current profile extended parallel to each other. Electron microscopy showed that these processes surround thick, unmyelinated axons. We suggest that cells with oligodendrocyte-type electrophysiology are promyelinating oligodendrocytes. In contrast to cultured oligodendrocytes, membrane currents of promyelinating oligodendrocytes in the slice decayed during the voltage command. This decay was due not to inactivation, but to a marked change in the potassium equilibrium potential within the voltage jump. This implies that, in the more mature corpus callosum, small membrane polarizations in a physiological range can lead to extensive changes in the K+ gradient across the glial membrane within a few milliseconds.
Dopamine release from interplexiform cells in the retina: effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity
Authors: Umino O , Dowling JE
Published: April 2018

In teleost fish, dopaminergic interplexiform cells provide an intraretinal centrifugal pathway from the inner to the outer plexiform layer, where they make abundant synapses on cone-related horizontal cells. The interplexiform cells receive all their input in the inner plexiform layer from centrifugal fibers and amacrine cells. In fish, centrifugal fibers contain gonadotropin hormone-releasing hormone (GnRH)-like and FMRFamide-like peptides (Munz et al., 1982; Stell et al., 1984), whereas amacrine cells contain a variety of neuroactive substances, including a number of peptides. In this study, we examined the effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity in the white perch retina in an attempt to understand the synaptic inputs to the interplexiform cells. When the retina was superfused with Ringer's solution containing GnRH, horizontal cells depolarized (approximately 10 mV), and their responses to small spots increased, whereas their responses to full-field lights decreased. Thus, GnRH closely mimicked the effects of dopamine on horizontal cells. The GnRH antagonist [D-Phe2, Pro3, D-Phe6]-GnRH blocked the effects of GnRH, as did haloperidol. GnRH also had no effect on horizontal cells in retinas treated with 6-hydroxydopamine. The results indicate that GnRH acts by stimulating the release of dopamine from interplexiform cells. FMRFamide alone produced no changes on either the membrane potential or light responses of horizontal cells, but it did suppress the effects of GnRH on horizontal cells in some experiments. FRMFamide also reversed the effects of prolonged darkness on horizontal cell responses. When bicuculline was applied to the retina, horizontal cells also depolarized (approximately 10 mV), responses to full-field illumination decreased, and responses to small spots increased. Most of the effects of bicuculline were suppressed by haloperidol, indicating that bicuculline also stimulates the release of dopamine from interplexiform cells. Similar results were obtained when [D-Ala2]-met-enkephalinamide was applied to the retina; horizontal cells depolarized (approximately 10 mV), responses to full-field stimuli decreased, and responses to the light spots increased. On the other hand, [D-Ala2]-leuenkephalinamide and [D-Ala2, D-Leu5]-enkephalin had no effects on horizontal cells. Both haloperidol and naloxone blocked the effects of [D-Ala2]-met-enkephalinamide on horizontal cells, indicating that [D-Ala2]-met-enkephalinamide stimulates dopamine release from interplexiform cells via specific opiate receptors.
Induction of the myelin proteolipid protein (PLP) gene in C6 glioblastoma cells: functional analysis of the PLP promotor
Authors: Nave KA , Lemke G
Published: April 2018

The terminal differentiation of postmitotic oligodendrocytes is marked by the induction of myelin-specific genes. In this report, we demonstrate that culture conditions that induce oligodendrocyte differentiation of glial progenitor cells also induce differentiation of C6 glioblastoma cells, as monitored by activated transcription of the gene encoding proteolipid protein (PLP), the major myelin protein of the CNS. When assayed by transfections of hybrid reporter plasmids, the transcriptional control region of the PLP gene is preferentially active in differentiated C6 cells and contains both positive and negative cis-regulatory elements. In general, functional identification of these elements is well correlated with the binding sites of glial nuclear proteins, as visualized by the presence of DNase I-protected footprints. A sequence within one positive cis-regulatory element of the PLP gene is conserved in the control regions of three other myelin- specific genes, suggesting that their coordinate transcription may involve a common regulatory mechanism.
Reversible inhibition of voltage-dependent outer hair cell motility and capacitance
Authors: Santos-Sacchi J
Published: April 2018

Outer hair cells (OHC) from the organ of Corti are capable of fast voltage-induced length changes (Santos-Sacchi and Dilger, 1988), suggesting that an associated voltage sensor should reside in the OHC plasma membrane. Voltage-dependent mechanical responses and nonlinear charge movement of isolated OHCs from the guinea pig were analyzed using the whole-cell voltage-clamp technique. Ionic currents in the cells were blocked. Nonlinear voltage-dependent charge movement or, correspondingly, voltage-dependent capacitance was measured with step or AC analysis. OHC movements were measured either from video or using a differential photodiode technique. Maximum charge movements up to 2.5 pC were measured in OHCs from the low-frequency region of the cochlea. Both AC and step analyses indicated a peak nonlinear capacitance of 16- 17 pF. The voltage dependence was fit to a Boltzmann relation with the step analysis indicating a maximum nonlinear capacitance at -23 mV step potential from a holding potential of about -120 mV, and AC analysis indicating a maximum at a holding potential near -40 mV. AC analysis probably provides a more accurate evaluation of voltage dependence. Measures of OHC motility magnitude versus voltage follow the nonlinear capacitance-voltage function obtained from AC measures. Treatment of the cells with gadolinium ions (0.5-1 mM) blocked OHC motility. This treatment also produced a shift of the nonlinear capacitance function along the voltage axis in the depolarizing direction, which can be explained by membrane surface charge screening. However, maximum capacitance was reduced as well and may correspond to the reduction or abolition of OHC motility in response to gadolinium treatment. Gadolinium effects were reversible. Nonlinear capacitance is not a function of membrane deformation due to length changes, since removal of OHC cytosol via the patch pipette abolished longitudinal movement but did not reduce nonlinear charge movement. It is interesting to note that the nonlinear capacitance will dynamically influence the time constant of the OHC during acoustically evoked receptor potential generation.
Neurofilament redistribution in transected nerves: evidence for bidirectional transport of neurofilaments
Authors: Glass JD , Griffin JW
Published: April 2018

Nerve fibers of the C57BL/6/Ola mouse exhibit very slow Wallerian degeneration following axotomy, thus allowing prolonged observation of mammalian axons separated from their cell bodies. The present study utilized teased-fiber preparations, silver histochemistry, immunocytochemistry, and electron microscopy to examine the distribution of axonal components in the distal stumps of axotomized sciatic nerves in C57BL/6/Ola mice. In examining nerve segments at varying intervals after nerve transection, we found no evidence of proximal-to-distal “emptying out” of the cytoskeleton, as would be predicted if the cytoskeleton in these transected nerves were undergoing anterograde transport as an assembled structure. Instead, we observed a gradual redistribution of cytoskeletal constituents over time, dominated by the progressive accumulation of neurofilaments at the severed ends of axons. In particular, there were massive accumulations at the proximal ends of the distal stumps. These results strongly suggest that, at least in transected nerve fibers, neurofilaments can be transported bidirectionally.
Nerve growth factor stimulation of GAP-43 phosphorylation in intact isolated growth cones
Authors: Meiri KF , Burdick D
Published: April 2018

Phosphorylation of the nervous system-specific growth cone protein GAP- 43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin- stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.
Effects of monocular visual deprivation on geniculocortical innervation of area 18 in cat
Authors: Friedlander MJ , Martin KA , Wassenhove-McCarthy D
Published: April 2018

The effects of long-term monocular visual deprivation (MD) on the structure of the terminal arborizations of individual Y-type geniculocortical axons was studied in the cat's cortical area 18. Physiologically classified axons were filled with HRP by intracellular injection, and the three-dimensional distribution of the axons'terminal arborizations was quantified. Individual boutons observed at the light microscope (LM) level were verified as sites of synaptic contact by correlated light and electron microscopy (EM). Single boutons were serially sectioned and reconstructed for subsequent three- dimensional analysis. The arborizations of 17 Y-axons [6 normal (N), 6 nondeprived (ND), and 5 deprived (D)] were analyzed at the LM level, and 372 boutons (104 N, 129 ND, and 139 D) were fully reconstructed from serial sections for analysis at the EM level. MD leads to an expansion in the size of ND arborizations and a variable reduction in the size of the D arbors, which also have a higher bouton density than ND arborizations. ND axons form ectopic synapses, contacting proportionally more dendritic shafts than N or D boutons, and form more synapses per bouton, on average, than either N or D boutons. Compared to ND and N boutons, boutons of D axons are smaller, have fewer mitochondria, generally form synapses on a single target (usually dendritic spines), and occasionally make no synaptic contacts. The structural changes in the extent of individual axon arborizations may be the basis for change in ocular dominance column size with MD. However, the higher bouton density and variable effect on the extent of D axon arborization size suggest that considerable geniculocortical innervation from the deprived eye remains intact. The change in target preference for ND axons suggests that instead of a direct competition for postsynaptic sites by the developing geniculocortical axons innervated by the two retinas, the ND axon arborizations expand to invade synaptic space not normally occupied in such high proportion in the normal cortex. The severe changes in individual boutons following MD indicate that arborization size alone is not the only structural substrate underlying the altered responses of cortical neurons; changes in synaptic distribution onto target neurons may also play a role.
Cell-specific expression from the human dopamine beta-hydroxylase promoter in transgenic mice is controlled via a combination of positive and negative regulatory elements
Authors: Hoyle GW , Mercer EH , Palmiter RD , Brinster RL
Published: April 2018

The promoter region of the human dopamine beta-hydroxylase (DBH) gene was analyzed in transgenic mice to identify DNA sequences responsible for the tissue- and cell-specific expression of the gene. Transgenic mice were generated that carried the Escherichia coli lacZ gene under control of DBH promoter fragments between 0.6 and 5.8 kilobases (kb) in length. Sequences required for expression in adult and fetal noradrenergic neurons were located between 0.6 and 1.1 kb 5′ to the DBH transcriptional start site. Sequences in this region and farther upstream also directed expression to dopaminergic and noncatecholaminergic brain neurons that was repressed by negative elements elsewhere in the gene. The results indicate that the neuron- specific expression of the DBH gene is mediated by positive regulatory elements but that negative elements are required to restrict expression to the proper subset of neurons.
The role of oligodendrocytes and myelin on axon maturation in the developing rat retinofugal pathway
Authors: Colello RJ , Pott U , Schwab ME
Published: April 2018

In neonatal mammals, newly grown optic axons are uniformly small in diameter. In the adult, in contrast, axons within the optic nerve can be classified into distinct groups according to their diameter. Because axon diameters are also related to the thickness of the myelin sheath, which in turn determines the velocity of action potential propagation, the question of what determines the axon diameter is of critical importance. In a project aimed at determining the influence of the ensheathing cell on axon maturation, oligodendrocyte development was prevented by eliminating their precursors by unilateral x-irradiation at birth. Axon diameters in both the normal and the myelin-free optic nerves were then measured at varying stages of development. The results demonstrate that axon diameter growth remained substantially reduced in the absence of oligodendrocytes. Interestingly, by x-irradiating the optic nerve and tract on one side of the brain, fibers crossing the chiasm became larger as they went from an unmyelinated nerve to a myelinated tract; fibers on the nonirradiated side became smaller as they went from a myelinated nerve and crossed into the nonmyelinated tract. These results clearly point to a local regulation of axon diameter by oligodendrocytes. Moreover, ganglion cells measured 9 d after the initiation of myelination (postnatal day 6, P6) were of similar size within normal retinas and retinas whose axons were x- irradiated, suggesting that ganglion cell growth occurs in spite of the lack of myelin and axon diameter maturation. Finally, we showed, through both section staining with antibodies to myelin basic protein (MBP) and Northern blot analysis using a probe to MBP, that the x- irradiated nerve began a delayed myelination period (in a gradient from chiasm to eye) at P15 and reached an almost normal myelin pattern at P28. Axons from these nerves grew to seemingly normal diameter concomitant with this delayed myelination.
GABA transporters and GABAC-like receptors on catfish cone- but not rod- driven horizontal cells
Authors: Dong CJ , Picaud SA , Werblin FS
Published: April 2018

The effects of GABA and related agents were studied in solitary rod- and cone-driven horizontal cells, acutely isolated from the catfish retina using enzymatic and mechanical treatment. Both types of horizontal cells, which normally receive glutamatergic input from photoreceptors, responded to pressure ejection of the glutamate analog kainate (50 microM) with an inward current of 300–800 pA when voltage- clamped at -70 mV using the whole-cell patch-clamp technique. But pressure ejection of GABA (500 microM) elicited an inward current only in cone-driven horizontal cells. This current, ranging between 100 and 400 pA, consisted of two components: (1) a GABA receptor-gated chloride current that reversed near the chloride equilibrium potential and was blocked by bath application of picrotoxin (100–500 microM), and (2) a GABA transporter-mediated current that was picrotoxin resistant but was blocked by NO-711 (1 microM) and cis-4-hydroxynipecotic acid (250 microM), two potent GABA transporter blockers. The GABA transporter current could also be eliminated when sodium was replaced by either choline or lithium in the bathing medium. The picrotoxin-sensitive receptor-gated current could not be elicited by the GABAB receptor agonist baclofen, nor could it be blocked by the potent GABAB receptor antagonist 2-hydroxysaclofen. The picrotoxin-sensitive current could be divided into two components based on their sensitivity to the specific GABAA receptor antagonist bicuculline methiodide. The bicuculline- sensitive component was found only in some cells, whereas the bicuculline-resistant, picrotoxin-sensitive component was found in all cells tested. The bicuculline-resistant current was insensitive to pentobarbital, an allosteric modulator of GABAA receptor. To confirm the effectiveness of the specific batch of bicuculline methiodide and pentobarbital, we tested both drugs in ganglion cells in the salamander retinal slice preparation, where the GABA-elicited current is almost exclusively mediated by GABAA receptors. Bicuculline methiodide almost completely blocked, while pentobarbital significantly enhanced, the GABA current recorded in ganglion cells. Thus, in catfish cone horizontal cells the bicuculline-resistant GABA receptor current is most likely mediated by the GABAC receptor based on the above pharmacological profile. The relative effectiveness of GABA, muscimol, trans- and cis-4-aminocrotonic acid (TACA and CACA) was determined at this GABAC receptor site after cells were bathed in choline Ringer to eliminate the transporter current and in the presence of 100 microM bicuculline methiodide to block GABAA receptor current.(ABSTRACT TRUNCATED AT 400 WORDS)
Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat's infraorbital nerve
Authors: Vos BP , Strassman AM , Maciewicz RJ
Published: April 2018

Video recordings of free behavior and responses to mechanical facial stimulation were analyzed to assess whether chronic constriction injury (CCI) to the rat's infraorbital nerve (IoN) results in behavioral alterations indicative of neuropathic pain. A unilateral CCI was produced by placing loose chromic gut ligatures around the IoN. After CCI to the IoN, rats exhibited changes in both non-evoked and evoked behavior. Behavioral changes developed in two phases. Early after CCI (postoperative days 1–15), rats showed increased face-grooming activity with face-wash strokes directed to the injured nerve territory, while the responsiveness to stimulation of this area was decreased. Later after CCI (postoperative days 15–130), the prevalence of asymmetric face grooming was reduced but remained significantly increased compared to control rats. The early hyporesponsiveness was abruptly replaced by an extreme hyperresponsiveness: all stimulus intensities applied to the injured nerve territory evoked the “maximal” response (brisk head withdrawal, avoidance behavior plus directed face grooming). This response was never observed in control rats. Concurrently, IoN ligation rats showed a limited increase in the responsiveness to stimulation of the contralateral IoN territory, and around postoperative days 30–40 the responsiveness to stimulation of facial areas outside the IoN territories also increased. The hyperresponsiveness to stimulation of the ligated IoN territory slightly decreased from 60 d postoperative. Throughout the study, IoN ligation rats showed decreased exploratory behavior, displayed more freezing-like behavior, had a slower body weight gain, and a higher defecation rate, compared to control rats. The behavioral alterations observed after CCI to the IoN are indicative of severe sensory disturbances within the territory of the injured nerve: mechanical allodynia develops after a period of relative hypo- /anesthesia during which behavioral signs of recurrent spontaneous, aversive (possibly painful) sensations (paresthesias/dysesthesias) are maximal.
Functional organization of corticocortical projections from area 17 to area 18 in the cat's visual cortex
Authors: Price DJ , Ferrer JM , Blakemore C , Kato N
Published: April 2018

We used anatomical and physiological methods to study the functional organization of the association projection from area 17 to area 18 in the cat's visual cortex. Neurons in area 17 projecting to area 18 (revealed by retrograde transport of fluorescent tracer) tend to be clustered over regions of layer 4 receiving input from the ipsilateral eye (visualized by anterograde transneuronal tracing). Since the contralateral input overlaps these ipsilateral patches, the association cells lie preferentially in regions that are likely to be binocularly innervated. Indeed, almost all cells recorded electrophysiologically within the association clusters were strongly binocular, whereas between the clusters, many neurons were dominated by the contralateral eye. There is sufficient jitter in the retinotopic organization of area 17 for the discontinuous distribution of association cells to provide a continuous representation of the visual field. Cells in each association cluster in the rostral part of area 17 project divergently to innervate a zone extending up to 3 mm wide, anteroposteriorly, in the superficial layers of area 18. The receptive fields of cells at any point in area 18 are larger than for the corresponding point in area 17. Neurons recorded at two points in area 18, separated by a distance equal to the limit of anatomical divergence of the projection from area 17, have receptive fields that overlap by an amount similar to the region of visual field covered by the receptive fields of cells in a single association cluster in area 17 at a similar retinotopic position. Thus, area 18 receives a full and strongly binocular representation of the visual field not only from the lateral geniculate nucleus but also from area 17. The divergence of the area 17 to 18 projection compensates for the difference in receptive field size by ensuring that the receptive fields of each cluster of projecting neurons overlap fairly precisely those of the recipient neurons in area 18.
Changes in microtubule number and length during axon differentiation
Authors: Yu W , Baas PW
Published: April 2018

Hippocampal neurons in culture initially extend several minor processes that are approximately 20 microns in length. The first minor process to grow approximately 10 microns longer than the others will continue to grow rapidly and become the axon (Goslin and Banker, 1989). We sought to define changes in the microtubule (MT) array that occur during axon differentiation. In theory, axon differentiation could involve an increase in MT number, MT length, or some combination of both. To address this issue, we first serially reconstructed the entire MT array of a minor process from a cell whose axon had not yet differentiated. This minor process contained 182 MTs that ranged in length from 0.14 to 20.09 microns. The average MT length was 3.87 +/- 3.83 microns, and the total MT length was 704 microns. We then reconstructed the MT arrays of a minor process and the 56 microns axon from a cell that had undergone axon differentiation. The minor process contained 157 MTs that ranged in length from 0.24 to 17.95 microns. The average MT length was 3.91 +/- 4.84 microns, and the total MT length was 600 microns. The axon contained 1430 MTs that ranged in length from 0.05 to 40.14 microns. The average MT length was 4.02 +/- 5.28 microns, and the total MT length was 5750 microns. These data indicate that a shift occurs toward shorter as well as longer MTs, but that virtually no change in average MT length occurs during axon differentiation. Thus, elongation of existing MTs cannot account for the major expansion of the MT array that occurs as a minor process becomes an axon. In contrast, the number of MTs increases by approximately 10-fold as a minor process differentiates and grows into an axon of the length we analyzed. Based on these data, we conclude that the MT array of a minor process is substantially expanded as it differentiates into an axon, and that the principal mechanism by which this expansion occurs is the copious addition of new MTs.
Hippocampal reflected optical patterns during sleep and waking states in the freely behaving cat
Authors: Poe GR , Rector DM , Harper RM
Published: April 2018

We examined reflected light as a measure of neural activity from a 2 mm2 area of dorsal hippocampus and surrounding neocortex in nine freely behaving cats during sleep and waking states. Light reflectance at 660 or 700 nm was measured by a coherent fiber optic probe attached to a charge-coupled device video camera that allowed acquisition of images from subcortical structures. In the dorsal hippocampus, rapid eye movement sleep (REMS) and active waking (AW) resulted in a significant decline (-0.9% +/- 0.3 and -2.0% +/- 0.5, respectively) in overall reflected light from the dorsal hippocampus relative to quiet sleep (QS), while quiet waking (QW) resulted in an overall increase (+2.0% +/- 0.4). In the neocortical probe placement group, reflectance also decreased during AW (-1.6% +/- 0.5) and increased during QW (+1.7 +/- 0.6) as compared to QS. In contrast to the hippocampus, however, overall reflectance increased, rather than decreased, in the neocortex during REMS (+2.7% +/- 1.3). We interpret a decline in reflectance as representing increased activation of underlying neural tissue. Thus, the cat dorsal hippocampus increased overall activity during REMS as compared to QS, while neocortical structures decreased overall activity during the same state. These results concur with expected activity changes based on electrophysiologic and autoradiographic studies. The imaging procedure provided a continuous assessment of spatially organized neural activity changes in the freely behaving animal.
High levels of expression of the tumor suppressor gene APC during development of the rat central nervous system
Authors: Bhat RV , Baraban JM , Johnson RC , Eipper BA , Mains RE
Published: April 2018

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is mutated in human familial adenomatous polyposis, an autosomal dominant condition with predisposition to colorectal carcinoma and brain tumors. Although tumor suppressor genes appear to play a general role in regulating cellular proliferation, the normal biological function of the APC gene product is unknown. In the present study, we cloned fragments of the rat homolog of the APC gene and examined its tissue distribution by Northern blot analysis. These studies demonstrated particularly high levels of APC mRNA in brain. To gain clues to the role of the APC gene in brain function, we examined the neuroanatomical distribution of APC mRNA using in situ hybridization. In the adult, prominent expression of APC mRNA was observed in the olfactory bulb, hippocampus, and cerebellum, with low levels of hybridization in other regions of adult rat brain. In contrast, during embryonic and early postnatal development (1–2 weeks), high levels of APC expression were found throughout the brain and then decreased to adult levels by 6 weeks after birth, except in the olfactory bulb where the high levels of APC mRNA found in development persist in the adult. During development of cortex, cerebellum, and retina, APC mRNA expression was particularly prominent in layers containing newly formed postmitotic neurons, with lower levels observed in the proliferative zones where neurogenesis occurs. The high levels of APC expression from early neurogenesis until late stages of neuronal maturation suggest that APC may contribute to suppressing neuronal proliferation during this period of intense growth.
The minK potassium channel exists in functional and nonfunctional forms when expressed in the plasma membrane of Xenopus oocytes
Authors: Blumenthal EM , Kaczmarek LK
Published: April 2018

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N- terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte.
Identification of membrane proteins that comprise the plasmalemmal junction between migrating neurons and radial glial cells
Authors: Cameron RS , Rakic P
Published: April 2018

We have initiated studies to identify membrane polypeptides of radial glial cells that contribute to the selective cell-cell recognition and migration events in developing brain. Of several polyclonal antisera evaluated, one (D4), developed against formaldehyde fixed type 1 cerebellar glial cells, immunolabels the free surface of cortical and cerebellar astroglial and radial glial cells in a patchy pattern. In dissociated glial-neuronal cell cocultures, microdomains of immunoreactivity are detected at the site where the somal region of cells with the morphology of migrating neurons is in contact with an elongated glial cell fiber. Microdomains are absent from oligodendrocytes, process-bearing astrocytes, and neurons. The surface microdomains do not colocalize with components that compose focal adhesion plaques--integrin subunits, vinculin, or actin--and their integrity appears to require an intact microtubule rather than actin cytoskeleton. Furthermore, microdomain structure is maintained in the absence of extracellular Ca and Mg ions. Immunoblot analyses using antibodies affinity purified to individual proteins indicate that the microdomains are composed of two antigens with apparent molecular mass of approximately 48 kDa and approximately 72 kDa. The 48 kDa antigen is not observed in non-neural epithelial tissues and is detected in cortical and cerebellar tissues only at a developmental period that coincides with the stage of active neuronal cell migration. In contrast, the 72 kDa antigen is expressed in many neural and non-neural tissues at late developmental and adult stages. Our data suggest that the identified membrane proteins may contribute to the formation of the junction between migrating neurons and radial glial cell processes and that this junctional complex is linked to the microtubule cytoskeleton.
Astroglial differentiation is required for support of neurite outgrowth
Authors: Wang LC , Baird DH , Hatten ME , Mason CA
Published: April 2018

Models of astrocyte differentiation stress a lineage program that involves a progressive loss of astroglial support of neuronal differentiation. These models predict that astroglial promotion of neurite extension declines with the “age” of the astrocyte. An alternative view is that astroglial support of neurite growth is regulated by epigenetic factors that induce the cells either to differentiate and support neuronal functions or to undergo cell proliferation and fail to support neurons. To compare the contribution of astroglial cell “age” to astroglial support of neurite extension, mouse cerebellar astroglia were maintained in vitro for 3–90 d, and assayed for their ability to support neurite formation. When cultured in isolation, astroglial support of neurite extension declined with time in vitro, as assayed by quantifying outgrowth from explants of pontine nuclei, falling from a robust level just after the astroglia were harvested to negligible levels 21–90 d later. Since previous studies have shown that neurons can change the state of astroglial cells (Hatten, 1985), we tested the neurite promoting activity of astroglia that were cultured for 21–90 d in vitro and subsequently induced to differentiate by the addition of neurons. When granule neurons were added to aged astroglia and pontine explants plated 2 d later, neurite growth from the explants was exuberant, regardless of the time astroglia spent in vitro prior to the addition of neurons. The state of astroglia that were growth promoting or growth inhibiting was examined by bromodeoxyuridine staining and with antisera to glial filament protein. Aged astroglia cultured alone and thus inhibitory to axon growth, proliferated at high rates and had polygonal shapes. In contrast, aged astroglia to which neurons had been added, proliferated at low rates and developed process-bearing stellate shapes. To test further whether proliferation levels related to the growth-supporting properties of astroglia, astroglia were plated alone in medium without serum, or with the addition of transforming growth factor-beta 1, each treatment known to arrest proliferation. In both cases, promotion of neurite growth was restored in aged astroglia, but the morphology of astroglia did not correlate with the ability to support neurite growth. Finally, the growth-inhibiting properties of aged astroglia do not appear to be mediated by diffusible factors, and require close apposition with living astroglial cells. We conclude that astroglial support of neurite extension depends on the state of differentiation of astroglial cells, and that these properties can be modified by coculture with neurons or conditions that arrest of astroglial proliferation, irrespective of astroglial “age”.
Modulation of spontaneous transmitter release during depression and posttetanic potentiation of Aplysia sensory-motor neuron synapses isolated in culture
Authors: Eliot LS , Kandel ER , Hawkins RD
Published: April 2018

An Aplysia motor neuron cocultured with a single presynaptic sensory neuron exhibits spontaneous miniature EPSPs or EPSCs (“minis”) that can be used to assay the release process directly, independent of the presynaptic action potential. Sensory-motor synapses in culture undergo homosynaptic depression with low frequency stimulation (< 1 Hz) and posttetanic potentiation (PTP) with high-frequency stimulation (20 Hz) much as they do in intact ganglia, except that PTP does not occur in culture when sensory neurons are impaled. We measured spontaneous release during each of these two forms of homosynaptic plasticity as a way of testing whether they involve depletion or mobilization of synaptic vesicles (Gingrich and Byrne, 1985). We find that PTP is accompanied by an increase in mini frequency that decays with a time course parallel to the decay of evoked EPSP facilitation. In contrast, depression is not paralleled by a reduction of mini frequency, although extensive stimulation reduces mini frequency for a brief period immediately following stimulation. Neither form of plasticity altered miniature EPSP or miniature EPSC amplitude, corroborating previous evidence that both are presynaptically mediated. These findings suggest that PTP is mediated by a presynaptic mechanism independent of the action potential, such as vesicle mobilization. This presumably Ca(2+)- dependent mechanism does not involve protein kinase C, since we found that the inhibitor H7 does not specifically block PTP. In contrast to PTP, depression appears to involve changes unique to excitation- secretion coupling, such as reduced Ca2+ influx during the action potential (Klein et al., 1980).
Convergence of mechanosensory afferents from different classes of exteroceptors onto spiking local interneurons in the locust
Authors: Burrows M , Newland PL
Published: April 2018

Tactile stimuli to a leg of a locust are detected by two classes of exteroceptors: trichoid sensilla (tactile hairs), which are purely mechanoreceptors, and basiconic sensilla, which serve a dual role as mechano- and chemoreceptors. The trichoid sensilla have the longer shafts, but because the two types of receptors are intermingled over the surface of a leg, both can be excited when the leg contacts an obstacle. This article analyzes the mechanisms by which the tactile sensory information from these two classes of receptor is collated in the CNS so that spatial information is preserved. The mechanosensory afferents from both types of receptors on a hind leg make convergent connections that are excitatory and apparently direct with the same spiking local interneurons in a midline population of the metathoracic ganglion. The anatomy of the afferent projections suggests that the synapses from the two types of receptor are made onto the same region of branches of an interneuron. The slower conduction velocity of the spikes of the basiconic afferents compared to that of the trichoid afferents means that a mechanical stimulus to the tibia will first produce a depolarization caused by the trichoid afferents, followed up to 20 msec later by a depolarization caused by the basiconic sensilla. Each interneuron is excited by a contiguous and overlapping array of trichoid and basiconic receptors that form its receptive field. Different interneurons have different receptive fields such that the surface of the leg is mapped as a series of overlapping receptive fields. Within a receptive field the contribution of individual receptors can be markedly different: some basiconic receptors evoke large-amplitude EPSPs so that each afferent spike can cause a spike in the interneuron, whereas others generate small EPSPs that must sum with other inputs to evoke a spike. The amplitudes of the EPSPs generated by different receptors within a receptive field form gradients that are distributed according to the axes of the leg. The most effective basiconic and the most effective trichoid sensilla within a particular receptive field can be at the same or at different locations on the leg. Tactile sensory information from different types of receptor on one region of a leg is therefore collated initially by particular spiking local interneurons. This organization ensures summation between the inputs from the different types of receptors and should enhance sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of Functional GABAergic Synapses Formed between Rat Hypothalamic Neurons and Pituitary Intermediate Lobe Cells in Coculture: Ca2+Dependence of Spontaneous IPSCs
Authors: Poisbeau Pierrick , René Frédérique , Egles Christophe , Félix Jean-Marc , Feltz Paul , Schlichter Rémy
Published: April 2018

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the α-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 μm) and SR95531 (5 μm), two competitive antagonists of the GABAAreceptor. At a holding potential of −60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10–100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast (≤1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant, 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mminduced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8–10 GABAAchannels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.
Effects of Salicylate and Lanthanides on Outer Hair Cell Motility and Associated Gating Charge
Authors: Kakehata Seiji , Santos-Sacchi Joseph
Published: April 2018

Salicylate, one of the most widely used drugs, is known to induce reversible tinnitus and hearing loss. Salicylate interferes with outer hair cells (OHCs), which are believed to underlie normal auditory frequency selectivity and sensitivity. In the present experiments, the effects of salicylate and lanthanides on OHC motility and nonlinear capacitance were investigated by using isolated guinea-pig OHCs while attempting to avoid inadvertent intracellular pressure change, which itself can affect OHC motility and capacitance. Either extracellularly or intracellularly applied salicylate reduced nonlinear peak capacitance (Cmpk) and shifted the voltage at peak capacitance to depolarized levels. Concentration–response curves for reduction inCmpkby salicylate and GdCl3revealed a half-maximal concentration and Hill coefficient of 1.6 mmand 1.0, and 0.6 mmand 1.2, respectively. In comparable groups of OHCs, the normalCmpkvalues of which were near 40 pF, averageCmpkdecreased to 28 and 36 pF for intracellularly and extracellularly applied salicylate, respectively. Salicylate reduced, but did not completely block, the voltage-induced length change. Extracellularly, but not intracellularly, applied lanthanide blocked voltage-induced movement and capacitance almost completely. After intracellular trypsin treatment, salicylate reduced voltage-dependent capacitance reversibly, suggesting that salicylate directly acts on the sensor/motor and not via effects on intracellular structures, such as the subsurface cisternae. The results are consistent with the hypothesis that the dissociated, charged form of salicylate directly interacts with the sensor/motor on the inner aspect of the OHC plasma, whereas lanthanides interact on the outer aspect.