bluuebunny/crossref_metadata_2025 huggingface dataset:
Multifunctional Laryngeal Motoneurons: an Intracellular Study in the Cat
Authors: Shiba Keisuke , Satoh Isamu , Kobayashi Nobuhiro , Hayashi Fumiaki
Published: April 2018

We studied the patterns of membrane potential changes in laryngeal motoneurons (LMs) during vocalization, coughing, swallowing, sneezing, and the aspiration reflex in decerebrate paralyzed cats. LMs, identified by antidromic activation from the recurrent laryngeal nerve, were expiratory (ELMs) or inspiratory (ILMs) cells that depolarized during their respective phases in eupnea. During vocalization, most ELMs depolarized and most ILMs hyperpolarized. Some ILMs depolarized slightly during vocalization. During coughing, ELMs depolarized abruptly at the transition from the inspiratory to the expiratory phase. In one-third of ELMs, this depolarization persisted throughout the abdominal burst. In the remainder (“type A”), it was interrupted by a transient repolarization. ILMs exhibited a membrane potential trajectory opposite to that of type A ELMs during coughing. During swallowing, the membrane potential of ELMs decreased transiently at the onset of the hypoglossal burst and then depolarized strongly during the burst. ILMs hyperpolarized sharply at the onset of the burst and depolarized as hypoglossal activity ceased. During sneezing, ELMs and ILMs exhibited membrane potential changes similar to those of type A ELMs and ILMs during coughing. During the aspiration reflex, ELMs and ILMs exhibited bell-shaped hyperpolarization and depolarization trajectories, respectively. We conclude that central drives to LMs, consisting of complex combinations of excitation and inhibition, vary during vocalization and upper airway defensive reflexes. This study provides data for analysis of the neuronal networks that produce these various behaviors and analysis of network reorganization caused by changes in dynamic connections between the respiratory and nonrespiratory neuronal networks.
Immediate-Early Gene Expression in the Inferior Mesenteric Ganglion and Colonic Myenteric Plexus of the Guinea Pig
Authors: Sharkey Keith A. , Parr Edward J. , Keenan Catherine M.
Published: April 2018

Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus.
An Extraretinally Expressed Insect Cryptochrome with Similarity to the Blue Light Photoreceptors of Mammals and Plants
Authors: Egan Elizabeth S. , Franklin Tina M. , Hilderbrand-Chae Marla J. , McNeil Gerard P. , Roberts Mary A. , Schroeder Andrew J. , Zhang Xiaolan , Jackson F. Rob
Published: April 2018

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene inDrosophila(DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. TheDCrygene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded byDCryis ∼50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment,DCrymRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissuein situhybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with theDrosophilaclock neurons. Interestingly,DCrymRNA abundance oscillates in a circadian manner inDrosophilahead RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouseCry1mRNA, which is expressed in clock tissues. Finally, we show that changes inDCrygene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.
Nicotinic Acetylcholine Receptors Containing α7 Subunits Are Required for Reliable Synaptic TransmissionIn Situ
Authors: Chang Karen T. , Berg Darwin K.
Published: April 2018

Nicotinic acetylcholine receptors containing α7 subunits are widely expressed in the nervous system. The receptors are cation-selective, relatively permeable to calcium, and avid binders of α-bungarotoxin. Although the receptors can act both pre- and postsynaptically, their physiological significance is unclear. Using whole-cell patch-clamp analysis of chick ciliary ganglion neuronsin situ, we show that the receptors are required for reliable synaptic transmission early in development. Stimulation of the presynaptic nerve root elicited a biphasic synaptic current, including a large rapidly decaying component generated by α7-containing receptors. Selective blockade of α7-containing receptors by perfusing the ganglion with α-bungarotoxin induced failures in synaptic transmission. One-half of the ciliary neurons that were tested failed when stimulated synaptically at 1 Hz, and two-thirds failed at 25 Hz. Failing cells missed, on average, 80% of the trials during a test train of stimuli. The ability to fire synaptically evoked action potentials after toxin treatment was correlated positively with the amplitude of the remaining synaptic current, suggesting that α7-containing receptors were needed to augment synaptic responses. Consistent with patch-clamp analysis, toxin blockade reduced the amplitude of the synaptically evoked compound action potential in the postganglionic nerve; it also desynchronized the firing of the remaining units. Methyllycaconitine, another antagonist of α7-containing receptors, mimicked α-bungarotoxin blockade. Toxin blockade had less impact on transmission in ganglia at the end of embryogenesis. The ability of the receptors to synchronize and sustain population firing, together with their ability to deliver calcium, may influence early developmental events such as target innervation and neuronal survival.
The Ankle-Link Antigen: an Epitope Sensitive to Calcium Chelation Associated with the Hair-Cell Surface and the Calycal Processes of Photoreceptors
Authors: Goodyear Richard , Richardson Guy
Published: April 2018

A monoclonal antibody, mAb E40, that specifically recognizes hair cells and photoreceptors was derived from a mouse immunized with a membrane fraction prepared from the sensory maculae of the chick inner ear. In the mature chick inner ear, punctate labeling is observed along each stereocilium, but staining is mostly concentrated around the basal end of the sensory hair bundles, where it is closely associated with surface specializations known as ankle links. The epitope recognized by mAb E40 is therefore referred to as the ankle-link antigen (ALA). During early embryogenesis, the ALA is initially distributed evenly over the surface of the hair bundle. As development proceeds, it becomes more restricted to the base of the hair bundle, although a spot of the ALA remains associated with the bundle tip until just before hatching. In the eye, mAb E40 stains the calycal processes of photoreceptors. When maculae and retinae are treated with the calcium chelator BAPTA at room temperature, the ALA disappears. BAPTA-induced loss of the ALA from the hair-bundle surface is substantially reduced by lowering the temperature to 2°C. The ALA and ankle links reappear on the hair-bundle surface when cells are cultured for 20 hr after BAPTA treatment. BAPTA sensitivity and recovery after BAPTA-induced loss are properties similar to those described for the tip link, a surface structure thought to gate the mechanotransducer channel. However, unlike the tip link, the ALA and ankle links are sensitive to subtilisin treatment. The results define a new component of the hair-bundle surface, with properties both common to and distinct from those of the tip link.
Genesis, Neurotrophin Responsiveness, and Apoptosis of a Pronounced Direct Connection between the Two Eyes of the Chick Embryo: A Natural Error or a Meaningful Developmental Event?
Authors: Thanos Solon
Published: April 2018

Unilateral intraocular injections of either of two fluorescent carbocyanine dyes into the embryonic chick eye resulted in both retrograde staining of ganglion cells (GCs) in the eye contralateral to site of injection and anterograde labeling of axons whose cell bodies were located within the injected eye. This prominent retino-retinal projection formed by thousands of GCs having a nasal origin and temporal termination appeared at embryonic day 6 (E6), attained its maximum intensity at E13–E14, and gradually disappeared until E18. The axonal growth cones ended superficially and never penetrated deeper layers of the retina. Treatment of the projection with BDNF resulted in massive terminal branching of the axons within deeper layers of the target retina. Double injection into the eye and the isthmo-optic nucleus showed a concomitant ingrowth of axons in the contralateral retina. Individual GCs died between E9 and E13, but massive apoptotic cell death was mainly monitored at E14 and later. Disintegrated cells showed typical images of apoptosis. Because degenerating cells were prelabeled with the membranophilic fluorescent carbocyanine dye, their death allowed the concomitant visualization of phagocytosing cells, too. Radial Müller glia were the only class of cells observed to become phagocytotic between E9 and E16. These cells became replaced exclusively with microglial cells from E17 on. The results suggest that the topologically restricted retino-retinal projection may have some developmental significance rather than representing a massive erroneous projection. Most likely, the projection may serve as a “template” to guide centrifugal isthmo-optic axons into the retina.
Postsynaptic Density-93 Interacts with the δ2 Glutamate Receptor Subunit at Parallel Fiber Synapses
Authors: Roche Katherine W. , Ly C. Dune , Petralia Ronald S. , Wang Ya-Xian , McGee Aaron W. , Bredt David S. , Wenthold Robert J.
Published: April 2018

The glutamate receptor subunit δ2 has a unique distribution at the parallel fiber–Purkinje cell synapse of the cerebellum, which is developmentally regulated such that δ2 occurs at both parallel fiber synapses and climbing fiber synapses early in development but is restricted to parallel fiber synapses in adult animals. To identify proteins that might be involved in the trafficking or docking of δ2 receptors, we screened a yeast two-hybrid library with the cytosolic C terminus of δ2 and isolated a member of the postsynaptic density (PSD)-95 family of proteins, which are known to interact with the extreme C termini of NMDA receptors. We find that δ2 binds specifically to PSD-93, which is enriched in Purkinje cells. In addition, PSD-93 clusters δ2 when they are coexpressed in heterologous cells, and clustering is disrupted by point mutations of δ2 that disrupt the δ2–PSD-93 interaction. Ultrastructural localization of PSD-93 and δ2 shows they are colocalized at parallel fiber synapses; however, PSD-93 also is present at climbing fiber synapses of the adult rat, where δ2 is not found, indicating that the presence of PSD-93 alone is not sufficient for determining the synaptic expression of δ2.
Differential Regulation of the Expression of Corticotropin-Releasing Factor Receptor Type 2 (CRF2) in Hypothalamus and Amygdala of the Immature Rat by Sensory Input and Food Intake
Authors: Eghbal-Ahmadi Mariam , Avishai-Eliner Sarit , Hatalski Carolyn G. , Baram Tallie Z.
Published: April 2018

The physiological consequences of activating corticotropin-releasing factor receptor type 2 (CRF2) are not fully understood. The neuroanatomic distribution of this CRF receptor family member is consistent with roles in mediating the actions of CRF and similar ligands on food intake control and integrative aspects of stress-related behaviors. However, CRF2expression in theadultrat is not influenced by stress, corticosterone (CORT), or food intake. Inimmaturerat we have demonstrated striking downregulation of CRF2mRNA in hypothalamic ventromedial nucleus (VMH) after 24 hr of maternal deprivation, a paradigm consisting of both physiological/psychological stress and food deprivation. The current study aimed to distinguish which element or elements of maternal deprivation govern CRF2mRNA expression by isolating the effects of food intake and discrete maternal sensory cues on CRF2mRNA levels in VMH and in reciprocally communicating amygdala nuclei. In maternally deprived pups, CRF2mRNA levels in VMH and basomedial (BMA) and medial (MEA) amygdala nuclei were 62, 72, and 102% of control levels, respectively. Sensory inputs of grooming and handling as well as of the pups’ own suckling activity—but not food intake—fully restored CRF2mRNA expression in VMH. In contrast, all manipulations tended to increase CRF2mRNA levels in BMA of maternally deprived rats, and surrogate grooming increased CRF2mRNA expression significantly above that of nondeprived controls. CRF2mRNA expression was not influenced significantly by plasma adrenocorticotropic hormone (ACTH) and CORT levels. Thus, in the immature rat, (1) CRF2expression is regulated differentially in hypothalamic and amygdala regions, and (2) CRF2mRNA levels in VMH are governed primarily by maternal or suckling-derived sensory input rather than food intake or peripheral stress hormones. These findings indicate a region-specific regulation of CRF2mRNA, supporting the participation of the receptor in neurochemically defined circuits integrating sensory cues to influence specific behavioral and visceral functions.
Vasoactive Intestinal Polypeptide Excites Medial Pontine Reticular Formation Neurons in the Brainstem Rapid Eye Movement Sleep-Induction Zone
Authors: Kohlmeier Kristi A. , Reiner Peter B.
Published: April 2018

Although it has long been known that microinjection of the cholinergic agonist carbachol into the medial pontine reticular formation (mPRF) induces a state that resembles rapid eye movement (REM) sleep, it is likely that other transmitters contribute to mPRF regulation of behavioral states. A key candidate is the peptide vasoactive intestinal polypeptide (VIP), which innervates the mPRF and induces REM sleep when injected into this region of the brainstem. To begin understanding the cellular mechanisms underlying this phenomenon, we examined the effects of VIP on mPRF cells using whole-cell patch-clamp recordings in thein vitrorat brainstem slice. VIP directly depolarized cells via activation of an inward current; these effects were attenuated and potentiated in low-sodium and low-calcium medium, respectively. The depolarization induced by VIP was slower in onset and longer-lived than that evoked by carbachol. The VIP-induced depolarization was reduced in a dose-dependent manner by a competitive antagonist of VIP receptors. Effects of VIP were attenuated in the presence of guanosine 5′-O-(2-thiodiphosphate, 2′5′dideoxyadenosine, and PKI15–24and were nonadditive in the presence of 8-bromo-cAMP. We conclude that VIP excites mPRF neurons by activation of a sodium current. This effect is mediated at least in part by G-protein stimulation of adenylyl cyclase, cAMP, and protein kinase A. These data suggest that VIP may play a physiological role in REM induction by its actions on mPRF neurons.
Opposing Role of Dopamine D1 and D2 Receptors in Modulation of Rat Nucleus Accumbens Noradrenaline Release
Authors: Vanderschuren Louk J. M. J. , Wardeh George , De Vries Taco J. , Mulder Arie H. , Schoffelmeer Anton N. M.
Published: April 2018

The role of dopamine receptors in the modulation of nucleus accumbens noradrenaline release was investigated in superfused rat brain slices. At concentrations of ≤1 μm, dopamine enhanced, whereas at higher concentrations dopamine inhibited electrically evoked [3H]noradrenaline release. The D1 receptor agonist SKF-38393 increased, whereas the D2 agonist quinpirole inhibited evoked [3H]noradrenaline release. These effects were attenuated by the D1 antagonist SCH-23390 and the D2 antagonist (−)-sulpiride, respectively, indicating that accumbens noradrenaline release is regulated by stimulatory D1 and inhibitory D2 receptors. Whereas (−)-sulpiride enhanced, SCH-23390 did not reduce evoked accumbens [3H]noradrenaline release, indicating a tonic activation of D2 receptors only. Given the similar apparent affinity of dopamine for D1 and D2 receptors in striatal slices, the lack of tonic D1 receptor activation suggests that D1, unlike D2, receptors are extrasynaptically localized. No dopaminergic modulation of noradrenaline release was observed in rat medial prefrontal cortex or amygdala slices. To examine the regulation of accumbens noradrenaline release under conditions of increased dopaminergic activity, measurements were made using slices of amphetamine-pretreated rats. In these slices, the electrically evoked release of [3H]dopamine and [3H]noradrenaline was enhanced. The increasing effect of (−)-sulpiride on noradrenaline release was augmented, and SCH-23390 almost completely reversed this enhancement of [3H]noradrenaline release. These data suggest that whereas although under a moderate dopaminergic tone, accumbens noradrenaline release is mainly regulated by inhibitory D2 receptors, under circumstances of increased dopaminergic activity, recruitment of extrasynaptic stimulatory D1 receptors contributes to enhancement of noradrenaline release.
Cloning and Characterization ofAplysiaNeutral Endopeptidase, a Metallo-Endopeptidase Involved in the Extracellular Metabolism of Neuropeptides inAplysia californica
Authors: Zappulla Jacques P. , Wickham Louise , Bawab Wafa , Yang Xiao-Feng , Storozhuk Maksim V. , Castellucci Vincent F. , DesGroseillers Luc
Published: April 2018

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS ofAplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3–Phe4bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that theAplysiaand mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-AplysiaNEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that theapNEPgene codes for the RB104-binding protein.In situhybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior inAplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission inAplysiaand in the metabolism of neuropeptides close to their point of release.
Transplants of Fibroblasts Genetically Modified to Express BDNF Promote Regeneration of Adult Rat Rubrospinal Axons and Recovery of Forelimb Function
Authors: Liu Yi , Kim Duckhyun , Himes B. Timothy , Chow Stella Y. , Schallert Timothy , Murray Marion , Tessler Alan , Fischer Itzhak
Published: April 2018

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNFin vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that ∼7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.
Anatomical Correlates of Functional Plasticity in Mouse Visual Cortex
Authors: Antonini Antonella , Fagiolini Michela , Stryker Michael P.
Published: April 2018

Much of what is known about activity-dependent plasticity comes from studies of the primary visual cortex and its inputs in higher mammals, but the molecular bases remain largely unknown. Similar functional plasticity takes place during a critical period in the visual cortex of the mouse, an animal in which genetic experiments can readily be performed to investigate the underlying molecular and cellular events. The experiments of this paper were directed toward understanding whether anatomical changes accompany functional plasticity in the developing visual cortex of the mouse, as they do in higher mammals. In normal mice, transneuronal label after an eye injection clearly delineated the monocular and binocular zones of area 17. Intrinsic signal optical imaging also showed monocular and binocular zones of area 17 but revealed no finer organization of ocular dominance or orientation selectivity. In normal animals, single geniculocortical afferents serving the contralateral eye showed great heterogeneity and no clustering consistent with the presence of ocular dominance patches. Growth and elaboration of terminal arbor continues beyond postnatal day 40 (P40), after the peak of the critical period. After prolonged monocular deprivation (MD) from P20 to P60, transneuronal labeling showed that the projection serving the ipsilateral eye was severely affected, whereas the effect on the contralateral eye’s pathway was inconsistent. Optical imaging also showed profound effects of deprivation, particularly in the ipsilateral pathway, and microelectrode studies confirmed continued functional plasticity past P40. Reconstruction of single afferents showed that MD from P20 to P40 promoted the growth of the open eye’s geniculocortical connections without causing the closed eye’s contralateral projection to shrink, whereas MD from P20 to P60 caused an arrest of growth of deprived arbors. Our findings reveal numerous similarities between mouse and higher mammals in development and plasticity, along with some differences. We discuss the factors that may be responsible for these differences.
Floor Plate and Netrin-1 Are Involved in the Migration and Survival of Inferior Olivary Neurons
Authors: Bloch-Gallego Evelyne , Ezan Frédéric , Tessier-Lavigne Marc , Sotelo Constantino
Published: April 2018

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. In the present study, we report experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. First, we show that the pattern of expression of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role of netrins in directing the migration of precerebellar neurons from the rhombic lips. Second, we have studied mice deficient in netrin-1 production. In these mice, the number of inferior olivary neurons is remarkably decreased. Some of them are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection.In vitroexperiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons.
Sensory Feedback Can Coordinate the Swimming Activity of the Leech
Authors: Yu Xintian , Nguyen Binh , Friesen W. Otto
Published: April 2018

Previous studies showed that sensory feedback from the body wall is important and sometimes critical for generating normal, robust swimming activity in leeches. In this paper, we evaluate the role of sensory feedback in intersegmental coordination using both behavioral and physiological measurements. We severed the ventral nerve cord of leeches in midbody and then made video andin situextracellular recordings from swimming animals. Our electrophysiological recordings unequivocally demonstrate that active intersegmental coordination occurs in leeches with severed nerve cords, refuting Schülter’s (1933) earlier conclusions that sensory feedback cannot coordinate swimming activity. Intersegmental coordination can in fact be achieved by sensory feedback alone, without the intersegmental interactions conveyed by the nerve cord.
Increased Excitability of Afferent Neurons Innervating Rat Urinary Bladder after Chronic Bladder Inflammation
Authors: Yoshimura Naoki , de Groat William C.
Published: April 2018

The properties of bladder afferent neurons in L6and S1dorsal root ganglia of adult rats were evaluated after chronic bladder inflammation induced by 2 week treatment with cyclophosphamide (CYP; 75 mg/kg). Whole-cell patch-clamp recordings revealed that most (70%) of the dissociated bladder afferent neurons from control rats were capsaicin sensitive, with high-threshold long-duration action potentials that were not blocked by tetrodotoxin (TTX; 1 μm). These neurons exhibited membrane potential relaxations during voltage responses elicited by depolarizing current pulses and phasic firing during sustained membrane depolarization. After CYP treatment, a similar proportion (71%) of bladder afferent neurons were capsaicin sensitive with TTX-resistant spikes. However, the neurons were significantly larger in size (diameter 29.6 ± 1.0 μm vs 23.6 ± 0.8 μm in controls). TTX-resistant bladder afferent neurons from CYP-treated rats exhibited lower thresholds for spike activation (−25.4 ± 0.5 mV) than those from control rats (−21.4 ± 0.9 mV) and did not exhibit membrane potential relaxation during depolarization. Seventy percent of TTX-resistant bladder afferent neurons from CYP-treated rats exhibited tonic firing (average 12.3 ± 1.4 spikes during a 500 msec depolarizing pulse) versus phasic firing (1.2 ± 0.2 spikes) in normal bladder afferent neurons. Application of 4-aminopyridine (1 mm) to normal TTX-resistant bladder afferent neurons mimicked the changes in firing properties after CYP treatment. The peak density of an A-type K+current (IA) during depolarizations to 0 mV in TTX-resistant bladder afferent neurons from CYP-treated rats was significantly smaller (42.9 pA/pF) than that from control rats (109.4 pA/pF), and the inactivation curve of theIAcurrent was displaced to more hyperpolarized levels by ∼15 mV after CYP treatment. These data suggest that chronic inflammation induces somal hypertrophy and increases the excitability of C-fiber bladder afferent neurons by suppressingIAchannels. Similar electrical changes in sensory pathways may contribute to cystitis-induced pain and hyperactivity of the bladder.
Relationships between the Prefrontal Cortex and the Basal Ganglia in the Rat: Physiology of the Cortico-Nigral Circuits
Authors: Maurice Nicolas , Deniau Jean-Michel , Glowinski Jacques , Thierry Anne-Marie
Published: April 2018

The prelimbic/medial orbital areas (PL/MO) of the rat prefrontal cortex are connected to substantia nigra pars reticulata (SNR) through three main circuits: a direct nucleus accumbens (NAcc)–SNR pathway, an indirect NAcc–SNR pathway involving the ventral pallidum (VP) and the subthalamic nucleus (STN), and a disynaptic cortico-STN–SNR pathway. The present study was undertaken to characterize the effect of PL/MO stimulation on SNR cells and to determine the contribution of these different pathways. The major pattern of responses observed in the SNR was an inhibition preceded by an early excitation and followed or not by a late excitation. The inhibition resulted from the activation of the direct NAcc–SNR pathway because it disappeared after acute blockade of the glutamatergic cortico-striatal transmission by CNQX application into the NAcc. The late excitation resulted from the activation of the indirect NAcc–VP–STN–SNR pathway via a disinhibition of the STN because it disappeared after either CNQX application into the NAcc or blockade of the GABAergic striato-pallidal transmission by bicuculline application into the VP. The early excitation, which was markedly decreased after blockade of the cortico-STN transmission by CNQX application into the STN, resulted from the activation of the disynaptic cortico-STN–SNR pathway. Finally, the blockade of the cortico-STN–VP circuit by CNQX application into STN or VP modified the influence of the trans-striatal circuits on SNR cells. This study suggests that, in the prefrontal cortex–basal ganglia circuits, the trans-subthalamic pathways, by their excitatory effects, participate in the shaping of the inhibitory influence of the direct striato-nigral pathway on SNR neurons.
Characterization of Phosphorylation Sites on the Glutamate Receptor 4 Subunit of the AMPA Receptors
Authors: Carvalho Ana Luı́sa , Kameyama Kimihiko , Huganir Richard L.
Published: April 2018

Recent studies have suggested that protein phosphorylation of glutamate receptors may play an important role in synaptic transmission. Specifically, the phosphorylation of AMPA receptors has been implicated in cellular models of synaptic plasticity. The phosphorylation of the glutamate receptor 1 (GluR1) subunit of AMPA receptors by protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized extensively. Phosphorylation of this subunit occurs exclusively on the intracellular C-terminal domain. However, the GluR1 subunit C terminus shows low homology to the other AMPA receptor subunits. In this paper we characterized the phosphorylation of AMPA receptor subunit GluR4, using site-specific mutagenesis and biochemical techniques. We found that GluR4 is phosphorylated on serine 842 within the C-terminal domainin vitroandin vivo. Serine 842 is phosphorylated by PKA, PKC, and CaMKIIin vitroand is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC phosphorylation site. These results suggest that GluR4, which is the most rapidly desensitizing AMPA receptor subunit, may be modulated by phosphorylation.
The Supporting-Cell Antigen: A Receptor-Like Protein Tyrosine Phosphatase Expressed in the Sensory Epithelia of the Avian Inner Ear
Authors: Kruger Robert P. , Goodyear Richard J. , Legan P. Kevin , Warchol Mark E. , Raphael Yehoash , Cotanche Douglas A. , Richardson Guy P.
Published: April 2018

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noisein vivoor hair-cell loss caused by ototoxic drug treatmentin vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.
Netrin-3, a Mouse Homolog of Human NTN2L, Is Highly Expressed in Sensory Ganglia and Shows Differential Binding to Netrin Receptors
Authors: Wang Hao , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Tessier-Lavigne Marc
Published: April 2018

The netrins comprise a small phylogenetically conserved family of guidance cues important for guiding particular axonal growth cones to their targets. Two netrin genes,netrin-1andnetrin-2, have been described in chicken, but in mouse so far a single netrin gene, an ortholog of chicknetrin-1, has been reported. We report the identification of a second mouse netrin gene, which we namenetrin-3.Netrin-3does not appear to be the ortholog of chicknetrin-2but is the ortholog of a recently identified human netrin gene termedNTN2L(“netrin-2-like”), as evidenced by a high degree of sequence conservation and by chromosomal localization.Netrin-3is expressed in sensory ganglia, mesenchymal cells, and muscles during the time of peripheral nerve development but is largely excluded from the CNS at early stages of its development. The murine netrin-3 protein binds to netrin receptors of the DCC (deleted in colorectal cancer) family [DCC and neogenin] and the UNC5 family (UNC5H1, UNC5H2 and UNC5H3). Unlike chick netrin-1, however, murine netrin-3 binds to DCC with lower affinity than to the other four receptors. Consistent with this finding, although murine netrin-3 can mimic the outgrowth-promoting activity of netrin-1 on commissural axons, it has lower specific activity than netrin-1. Thus, like netrin-1, netrin-3 may also function in axon guidance during development but may function predominantly in the development of the peripheral nervous system and may act primarily through netrin receptors other than DCC.
Altered Serotonin Innervation Patterns in the Forebrain of Monkeys Treated with (±)3,4-Methylenedioxymethamphetamine Seven Years Previously: Factors Influencing Abnormal Recovery
Authors: Hatzidimitriou George , McCann Una D. , Ricaurte George A.
Published: April 2018

The recreational drug (±)3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) is a potent and selective brain serotonin (5-HT) neurotoxin in animals and, possibly, in humans. The purpose of the present study was to determine whether brain 5-HT deficits persist in squirrel monkeys beyond the 18-month period studied previously and to identify factors that influence recovery of injured 5-HT axons. Seven years after treatment, abnormal brain 5-HT innervation patterns were still evident in MDMA-treated monkeys, although 5-HT deficits in some regions were less severe than those observed at 18 months. No loss of 5-HT nerve cell bodies in the rostral raphe nuclei was found, indicating that abnormal innervation patterns in MDMA-treated monkeys are not the result of loss of a particular 5-HT nerve cell group. Factors that influence recovery of 5-HT axons after MDMA injury are (1) the distance of the affected axon terminal field from the rostral raphe nuclei, (2) the degree of initial 5-HT axonal injury, and possibly (3) the proximity of damaged 5-HT axons to myelinated fiber tracts. Additional studies are needed to better understand these and other factors that influence the response of primate 5-HT neurons to MDMA injury and to determine whether the present findings generalize to humans who use MDMA for recreational purposes.
Activation of NMDA Receptors in the Suprachiasmatic Nucleus Produces Light-Like Phase Shifts of the Circadian ClockIn Vivo
Authors: Mintz Eric M. , Marvel Cherie L. , Gillespie Charles F. , Price Kristina M. , Albers H. Elliott
Published: April 2018

Although there is substantial evidence that glutamate mimics the effects of light on the mammalian circadian clockin vitro, it has been reported that microinjection of glutamate into the suprachiasmatic nucleus of the hypothalamus (SCN) regionin vivodoes not result in a pattern of phase shifts that mimic those caused by light pulses. The present study was designed to test the hypothesis that microinjection of NMDA into the SCN would induce light-like phase shifts of the circadian clock through activation of the NMDA receptor. Hamsters housed in constant darkness received microinjections of NMDA through guide cannulas aimed at the SCN region at various times throughout the circadian cycle. Wheel running was monitored as a measure of circadian phase. Microinjection of NMDA resulted in circadian phase shifts, the size and direction of which were dependent on the time of injection. The resulting phase–response curve closely resembled that of light. The circadian response showed a clear dose-dependence at circadian time (CT) 13.5 but not at CT19. Both phase delays and advances induced by NMDA were blocked by coinjection of the NMDA antagonist 2-amino-5-phosphopentanoic acid but were slightly attenuated by the non-NMDA antagonist 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione disodium. The ability of NMDA to induce phase shifts was not altered by coinjection with tetrodotoxin. These data are consistent with the hypothesis that activation of NMDA receptors is a critical step in the transmission of photic information to the SCN.
Optical Imaging Reveals Elevated Intracellular Chloride in Hippocampal Pyramidal Neurons after Oxidative Stress
Authors: Sah Renu , Schwartz-Bloom Rochelle D.
Published: April 2018

The accumulation of reactive oxygen species (ROS) in the brain is associated with several neurodegenerative conditions. ROS can affect ionic homeostasis leading to impaired neurotransmission. Here, we determined the ability of H2O2, a membrane permeant ROS, to alter intraneuronal Cl−, an important regulator of neuronal excitability. Real-time alterations in intracellular chloride, [Cl−]i, were measured with UV laser scanning confocal microscopy in hippocampal slices loaded with the cell-permeant form of 6-methoxy-N-ethylquinolium iodide (MEQ), a Cl−-sensitive fluorescent probe. In slices superfused with H2O2for 10 min, there was a significant decrease in MEQ fluorescence (elevation in [Cl−]i) in area CA1 pyramidal cell soma but not in interneurons located in stratum radiatum. Alterations in [Cl−]i induced by H2O2were prevented by the iron chelator deferoxamine and the vitamin E analog Trolox, suggesting the involvement of free radicals. The influx of Cl−probably occurred through the GABA-gated Cl−channel because the effects of H2O2were blocked by picrotoxin. In addition, HPLC analysis of the superfusates indicated that GABA and glutamate accumulated extracellularly after H2O2exposure. Excitatory amino acid receptor antagonists 2-amino-5-phoshopentanoic acid and 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide also attenuated the effect of H2O2on MEQ fluorescence. The changes in [Cl−]i induced by H2O2were Ca2+-dependent and Na+-independent. After exposure of slices to H2O2, the ability of the GABA agonist muscimol to increase [Cl−]i was attenuated. Thus, ROS, like H2O2, may impair transmembrane Cl−gradients and reduce inhibitory neurotransmission, further promoting neuronal damage in oxidative stress-related disease and in aging.
The CB1 Cannabinoid Receptor Can Sequester G-Proteins, Making Them Unavailable to Couple to Other Receptors
Authors: Vásquez Clemente , Lewis Deborah L.
Published: April 2018

We tested the hypothesis that human CB1 cannabinoid receptors (hCB1) can sequester Gi/o-proteins from a common pool and prevent other receptors from signaling. Human CB1 cannabinoid receptors were expressed in superior cervical ganglion (SCG) neurons by microinjection of hCB1 cDNA. Expression of hCB1 cannabinoid receptors abolished the Ca2+current inhibition by endogenous pertussis toxin-sensitive Gi/o-coupled receptors for norepinephrine (NE) and somatostatin (SOM) but not by endogenous pertussis toxin-insensitive Gs-coupled receptors for vasoactive intestinal polypeptide. Signaling by NE was rescued by expression of GαoB, Gβ1, and Gγ3. Expression of mGluR2 metabotropic glutamate receptors, another pertussis toxin-sensitive G-protein-coupled receptor, had no effect on the signaling by NE or SOM. Some hCB1 receptors were constitutively active because the cannabinoid receptor inverse agonist SR 141617A enhanced the Ca2+current. Some hCB1 receptors also appear to be precoupled to Gi/o-proteins because the cannabinoid agonist WIN 55,212–2 decreased the Ca2+current at a time when no G-proteins were available to couple to α2-adrenergic and somatostatin receptors. In SCG neurons microinjected with a lower concentration of hCB1 cDNA, the effect of SR 141716A was reduced, and the response to NE and SOM was partially restored. Subsequent to the application of SR 141716A, the Ca2+current inhibition by NE and SOM was abolished. These results suggest that both the active and inactive states of the hCB1 receptor can sequester Gi/o-proteins from a common pool. Cannabinoid receptors thus have the potential to prevent other Gi/o-coupled receptors from transducing their biological signals.
Targeted Expression of TruncatedGluedDisrupts Giant Fiber Synapse Formation inDrosophila
Authors: Allen Marcus J. , Shan Xiaoliang , Caruccio Phyllis , Froggett Stephan J. , Moffat Kevin G. , Murphey R. K.
Published: April 2018

Glued1(Gl1) mutants produce a truncated protein that acts as a poison subunit and disables the cytoplasmic retrograde motor dynein. Heterozygous mutants have axonal defects in the adult eye and the nervous system. Here we show that selective expression of the poison subunit in neurons of the giant fiber (GF) system disrupts synaptogenesis between the GF and one of its targets, the tergotrochanteral motorneuron (TTMn). Growth and pathfinding by the GF axon and the TTMn dendrite are normal, but the terminal of the GF axon fails to develop normally and becomes swollen with large vesicles. This is a presynaptic defect because expression of truncatedGluedrestricted to the GF results in the same defect. When tested electrophysiologically, the flies with abnormal axons show a weakened or absent GF–TTMn connection. InGlued1heterozygotes, GF–TTMn synapse formation appears morphologically normal, but adult flies show abnormal responses to repetitive stimuli. This physiological effect is also observed when tetanus toxin is expressed in the GFs. Because the GF–TTMn is thought to be a mixed electrochemical synapse, the results show that Glued has a role in assembling both the chemical and electrical components. We speculate that disrupting transport of a retrograde signal disrupts synapse formation and maturation.
Differentiation of Mammalian Vestibular Hair Cells from Conditionally Immortal, Postnatal Supporting Cells
Authors: Lawlor Patrick , Marcotti Walter , Rivolta Marcelo N. , Kros Corné J. , Holley Matthew C.
Published: April 2018

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2kbtsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the “T” antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of α9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuablein vitropreparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.