bluuebunny/crossref_metadata_2025 huggingface dataset:
Motor unit behavior in canine motor neuron disease
Authors: Pinter MJ , Waldeck RF , Wallace N , Cork LC
Published: April 2018

Hereditary canine spinal muscular atrophy (HCSMA) is an autosomally dominant disease of motor neurons that shares many pathological features with human motor neuron disease. A particularly striking feature of the affected, accelerated phenotype (homozygous HCSMA) is that profound weakness develops before appreciable motor neuron cell death occurs (Cork et al., 1989a), implying that motor unit functional defects occur initially. The purpose of this study was to identify the site of these defects and characterize their nature. In most young homozygotes (2–3 months postnatal), motor neurons were encountered that could support orthodromic action potential propagation to the muscle but did not activate muscle fibers. The tetanic forces of innervated motor units in young homozygotes tended to be smaller than those in closely age-matched clinically normal animals. In older homozygotes (approximately 4.5 months, postnatal), all motor neurons sampled were capable of activating muscle fibers, but many motor units displayed abnormal behavior including an inability to sustain force output during high frequency activation. Motor units exhibiting tetanic failure also showed proportionately greater twitch potentiation than nonfailing units of similar unpotentiated twitch amplitude. Tetanic failure and large potentiation tended to occur in motor units that possessed the slowest contraction speeds. These results indicate that motor neuron functional defects in HCSMA appear initially in the most distal parts of the motor axon and involve defective neurotransmission. The possible roles of distal nerve degeneration, motor terminal sprouting, and synaptic transmission in causing these deficits are considered.
Modulation of inspiratory drive to phrenic motoneurons by presynaptic adenosine A1 receptors
Authors: Dong XW , Feldman JL
Published: April 2018

The involvement and mechanisms of adenosine A1 receptors in regulating bulbospinal synaptic transmission of inspiratory drive to phrenic motoneurons were investigated. The adenosine analog N6- cyclopentyladenosine (CPA) induced a dose-dependent decrease of both inspiratory-modulated activity of C4 ventral roots and synaptic currents of phrenic motoneurons in an in vitro brainstem/spinal cord preparation from neonatal rats. No significant changes were observed in steady-state membrane current (during the expiratory phase). The depressant action of CPA on inspiratory drive was blocked by the selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT). The adenosine receptor antagonist 3-isobutyl-1-methylxanthine (IBMX) induced varying degrees of enhancement of inspiratory-modulated synaptic current, as did CPT. This suggests a role of endogenous adenosine in synaptic transmission of respiratory drive to phrenic motoneurons. The relative contribution of pre- and postsynaptic adenosine receptors was examined by looking at the effects of CPA on postsynaptic membrane properties and on spontaneous or miniature excitatory postsynaptic currents (EPSCs). CPA had no detectable effect on the input resistance of phrenic moto-neurons. Moreover, the inward currents of phrenic moto-neurons in response to exogenously applied glutamate were not affected by adenosine-related compounds. On the other hand, CPA produced a significant decrease in the frequency of spontaneous and of miniature EPSCs. We conclude that adenosine can modulate transmission of inspiratory drive from bulbospinal neurons to phrenic motoneurons via presynaptic A1 receptors.
The effects of a lesion or a peripheral nerve graft on GAP-43 upregulation in the adult rat brain: an in situ hybridization and immunocytochemical study
Authors: Vaudano E , Campbell G , Anderson PN , Davies AP , Woolhead C , Schreyer DJ , Lieberman AR
Published: April 2018

We have sought to determine (1) if thalamic neurons upregulate the growth associated protein GAP-43 as a response to injury, or if a peripheral nerve graft is required to induce, enhance or sustain such a response, and (2) if thalamic neurons with different regenerative potentials also display different GAP-43 responses. Levels of GAP-43 protein (detected by LM and EM immunohistochemistry) and of GAP-43 mRNA (detected by in situ hybridization) were compared in the thalamus of adult rats between 1 d and 180 d after making a stab lesion or after implanting a peripheral nerve autograft. Stab injury is a sufficient stimulus to cause a transient upregulation in GAP-43 expression by neurons in the thalamus (both around the graft tip and in particular in the thalamic reticular nucleus) in the first week after injury but this response is both prolonged, and enhanced in the presence of a peripheral nerve graft. In addition, we demonstrate directly, by double labelling, that neurons of the thalamic reticular nucleus displaying high levels of the mRNA for GAP-43, have axons regenerating in the distal portion of the graft. These findings lend direct support to the hypothesis that upregulation of the GAP-43 gene is essential for prolonged regenerative axonal growth. We also demonstrate GAP-43 protein in graft Schwann cells and in brain astrocytes close to the site of graft implantation.
Synaptic interactions among excitatory afferents to nucleus accumbens neurons: hippocampal gating of prefrontal cortical input
Authors: O'Donnell P , Grace AA
Published: April 2018

The interactions among excitatory inputs arising from the prefrontal cortex, amygdala, and hippocampus, and innervating nucleus accumbens neurons were studied using in vivo intracellular recording techniques. Neurons recorded in the accumbens displayed one of three activity states: (1) silent, (2) spontaneously firing at low, constant rates, or (3) a bistable membrane potential, characterized by alternating periods of activity and silence occurring in concert with spontaneous transitions between two steady-state membrane potentials (average, - 77.3 +/- 7.1 mV base, -63.0 +/- 7.4 mV plateau). These neurons also exhibited a high degree of convergence of responses elicited by stimulation of each of the three excitatory inputs tested. Activation of hippocampal afferents, but not cortical, amygdaloid, or thalamic afferents, induced bistable cells to switch to the depolarized (active) state. In contrast, no bistable cells were encountered in the nucleus accumbens following an acute transection of the fornix. Furthermore, microinjection of lidocaine in the vicinity of the hippocampal afferents at the level of the fornix caused a reversible elimination of the plateau phase in bistable cells. These data suggest that hippocampal input is necessary for accumbens neurons to enter a depolarized, active state. Furthermore, activation of prefrontal cortical inputs fail to evoke spike firing in accumbens neurons unless they are in this active state. Consequently, the hippocampus appears to be capable of gating prefrontal corticoaccumbens throughput.
Selective regulation of current densities underlies spontaneous changes in the activity of cultured neurons
Authors: Turrigiano G , LeMasson G , Marder E
Published: April 2018

We study the electrical activity patterns and the expression of conductances in adult stomatogastric ganglion (STG) neurons as a function of time in primary cell culture. When first plated in culture, these neurons had few active properties. After 1 d in culture they produced small action potentials that rapidly inactivated during maintained depolarization. After 2 d in culture they fired large action potentials tonically when depolarized, and their properties resembled very closely the properties of STG neurons pharmacologically isolated in the ganglion. After 3–4 d in culture, however, their electrical properties changed and they fired in bursts when depolarized. We characterized the currents expressed by these neurons in culture. They included two TTX-sensitive sodium currents, a calcium current, a delayed-rectifier-like current, a calcium-dependent potassium current, and two A-type currents. The changes in firing properties with time in culture were accompanied by an increase in inward and decrease in outward current densities. A single-compartment conductance-based model of an STG neuron was constructed by fitting the currents measured in the biological neurons. When the current densities in the model neuron were matched to those measured for the biological neurons in each activity state, the model neuron closely reproduced each state, indicating that the changes in current densities are sufficient to account for the changes in intrinsic properties. These data indicate that STG neurons isolated in culture change their intrinsic electrical properties by selectively adjusting the magnitudes of their ionic conductances.
Cloned multipotential precursors from the mouse cerebrum require FGF-2, whereas glial restricted precursors are stimulated with either FGF-2 or EGF
Authors: Kilpatrick TJ , Bartlett PF
Published: April 2018

Fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) have both been reported to stimulate precursors in the developing CNS. To clarify these effects, we used clonal analysis to determine the lineage potential of precursors stimulated with each factor, at two stages of development. It was found that in cells isolated from the cerebrum of embryonic day 17 (E17) mice, FGF-2 stimulated both a multipotential precursor, which gave rise to neurons and astrocytes, and a committed glial precursor. In contrast, EGF only stimulated the glial restricted precursor. Thus, it appears that the multipotential cell, previously identified to be present at E10, remains selectively responsive to FGF-2, and that the EGF responsiveness observed at E17 reflects the presence of a new restricted class of precursors, rather than a switch in factor specificity of the multipotential cell.
A novel cholinergic "slow effect" of efferent stimulation on cochlear potentials in the guinea pig
Authors: Sridhar TS , Liberman MC , Brown MC , Sewell WF
Published: April 2018

This report documents slow changes in cochlear responses produced by electrical stimulation of the olivocochlear bundle (OCB), which provides efferent innervation to the hair cells of the cochlea. These slow changes have time constants of 25–50 sec, three orders of magnitude slower than those reported previously. Such “slow effects” are similar to classically described “fast effects” in that (1) they comprise a suppression of the compound action potential (CAP) of the auditory nerve mirrored by an enhancement of the cochlear microphonic potential (CM) generated largely by the outer hair cells; (2) the magnitude of suppression decreases as the intensity of the acoustic stimulus increases; (3) they share the same dependence on OCB stimulation rate; (4) both are extinguished upon cutting the OCB; and (5) both are blocked with similar concentrations of a variety of cholinergic antagonists as well as with strychnine and bicuculline. These observations suggest that both fast and slow effects are mediated by the same receptor and are produced by conductance changes in outer hair cells. Slow effects differ from fast effects in that (1) fast effects are greatest for acoustic stimulus frequencies between 6 and 10 kHz, whereas slow effects peak for frequencies from 12 to 16 kHz, and (2) fast effects persist over long periods of OCB stimulation, whereas slow effects diminish after 60 sec of stimulation. The time course of the slow effects can be described mathematically by assuming that each shock-burst produces, in addition to a fast effect, a small decrease in CAP amplitude that decays exponentially with a time constant that is long relative to the intershock interval. The long time constant of the slow effect compared to the fast effect suggests that it may arise from a distinct intracellular mechanism, possibly mediated by second- messenger systems.
Arachidonic acid as a possible negative feedback inhibitor of nicotinic acetylcholine receptors on neurons
Authors: Vijayaraghavan S , Huang B , Blumenthal EM , Berg DK
Published: April 2018

Neuronal acetylcholine receptors, being highly permeable to calcium, are likely to regulate calcium-dependent events in neurons. Arachidonic acid is a membrane-permeant second messenger that can be released from membrane phospholipids by phospholipases in a calcium-dependent manner. We show here that activation of neuronal acetylcholine receptors triggers release of 3H-arachidonic acid in a calcium-dependent manner from neurons preloaded with the fatty acid. Moreover, low concentrations of arachidonic acid reversibly inhibit the receptors and act most efficiently on receptors likely to have the highest permeability to calcium, namely receptors containing alpha 7 subunits. Low concentrations of arachidonic acid also reversibly inhibit alpha 7- containing receptors expressed in Xenopus oocytes following injection of alpha 7 cRNA. The oocyte results indicate following injection of alpha 7 cRNA. The oocyte results indicate that the inhibition is a feature of the receptors rather than a consequence of neuron-specific machinery. The inhibition is not mediated by specific metabolites of arachidonic acid because the effects can be mimicked by other fatty acids; their effectiveness correlates with their content of double bonds. In contrast to arachidonic effects on calcium currents, inhibition of neuronal nicotinic receptors by the fatty acid cannot be prevented by blocking production of free radicals or by inhibiting protein kinase C. An alternative mechanism is that arachidonic acid binds directly to the receptors or perturbs the local environment in such a manner as to constrain receptor function.
Differential distribution of closely related potassium channels in rat Schwann cells
Authors: Mi H , Deerinck TJ , Ellisman MH , Schwarz TL
Published: April 2018

Closely related K+ channels can coassemble to form heteromultimers in expression systems, as well as in vivo. Whether in vivo this coassembly is random and inevitable or whether highly homologous channels can be segregated and targeted independently within a given cell has not been determined. In this study, we address these questions by characterizing and localizing voltage-dependent K+ channels in Schwann cells. Transcripts for three closely related members of the Shaker-like family of K+ channels are found in adult rat sciatic nerve: Kv1.1, Kv1.2, and Kv1.5. We have examined two of these and observed that both Kv1.1 and Kv1.5 proteins are expressed in Schwann cells but differ in their distributions. Kv1.5 is localized on the Schwann cell membrane at the nodes of Ranvier and in bands that run along the outer surface of the myelin. It is also seen intracellularly in the vicinity of the nucleus. Schwann cell staining for Kv1.1, on the other hand, was seen only in perinuclear, intracellular compartments. These results provide evidence that closely related channels from the same family need not coassemble and can be localized differentially in the same cell. In addition, Kv1.1 was highly concentrated in the axonal membrane at juxtaparanodal regions. The distributions of these K+ channels in myelinated nerve highlight the elaborate molecular specializations of these membranes.
Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models
Authors: Portera-Cailliau C , Hedreen JC , Price DL , Koliatsos VE
Published: April 2018

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by selective death of striatal medium spiny neurons. Intrastriatal injections of glutamate receptor agonists (excitotoxins) recapitulate some neuropathological features of this disorder. Although this model suggests that excitotoxic injury may be involved in HD, the exact mechanisms of cell death in HD and its models are unknown. The present study was designed to test the hypothesis that HD can develop via the activation of an apoptotic mechanism of cell death and to examine whether excitotoxic striatal lesions with quinolinic acid in rats represent accurate models of HD. To characterize cell death, we employed DNA electrophoresis, electron microscopy (EM), and the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)- biotin nick end labeling (TUNEL) method for the in situ detection of DNA strand breaks. In the neostriatum of individuals with HD, patterns of distribution of TUNEL-positive neurons and glia were reminiscent of those seen in apoptotic cell death during normal development of the nervous system; in the same areas, nonrandom DNA fragmentation was detected occasionally. Following excitotoxic injury of the rat striatum, internucleosomal DNA fragmentation (evidence of apoptosis) was seen at early time intervals and random DNA fragmentation (evidence of necrosis) at later time points. In addition, EM detected necrotic profiles of medium spiny neurons in the lesioned rats. In concert, these results suggest that apoptosis occurs in both HD and excitotoxic animal models and that apoptotic and necrotic mechanisms of neuronal death may occur simultaneously within individual dying cells in the excitotoxically injured brain. However, the distribution of dying neurons in the neostriatum, the degree of glial degeneration, and the involvement of striatofugal pathways are very different between HD and excitotoxically damaged striatum. The present study suggests that multiple methods should be employed for a proper characterization of neuronal cell death in vivo.
Effects of dopamine depletion on the morphology of medium spiny neurons in the shell and core of the rat nucleus accumbens
Authors: Meredith GE , Ypma P , Zahm DS
Published: April 2018

Nucleus accumbens receives a dense dopaminergic innervation which is important in regulating motivated states of behavior such as goal- directed actions, stimulus-reward associations and reinforcement of addictive substances. The shell and core territories of this nucleus each receive functionally and morphologically distinct dopaminergic inputs and lesions of the ascending pathways totally deprive the core but not the shell of dopaminergic fibers. Medium spiny neurons are the principal targets of dopaminergic terminals. The present study explored whether the loss of dopamine inputs can affect these neurons and whether cells in the shell and core would be equally susceptible to such a loss. Intracellular injection in fixed slices and neuronal reconstruction were used to analyze the dendritic trees of 62 neurons in the shell and core of animals that received a unilateral, chronic 6- hydroxydopamine lesion of the medial forebrain bundle. In the dopamine- depleted core, dendrites are significantly shorter (16% decrease) than in the intact core and in both the dopamine-depleted core and lateral shell, dendrites are less spiny than in respective control regions. Dopamine loss in the medial shell is associated with significantly more tortuous dendrites that are lower in spine density. However, the number of spines is not reduced which may mean that the increase recorded for segment length, although insignificant in tests, could be responsible for the change in spine density. These data suggest that the loss of dopamine can affect accumbal neuronal morphology and, moreover, can affect neuronal structures differentially in the shell and core.
A redefinition of somatosensory areas in the lateral sulcus of macaque monkeys
Authors: Krubitzer L , Clarey J , Tweedale R , Elston G , Calford M
Published: April 2018

The present investigation was designed to determine the organization of somatosensory fields in the lateral sulcus of macaque monkeys using standard microelectrode recording techniques. Our results provide evidence for two complete representations of the body surface. We term these fields the second somatosensory area (SII) and the parietal ventral area (PV) because of their similarities in position, internal organization, and relationship to anterior parietal fields, as described for SII and PV in other mammals. Areas SII and PV are mirror- symmetrical representations of the body surface, sharing a common boundary at the representations of the digits of the hand and foot, lips, and mouth. These fields are located adjacent to the face representations of anterior parietal fields (areas 3b, 1, and 2), and are bounded ventrally and caudally by other regions of cortex in which neurons are responsive to somatic or multimodal stimulation. The finding of a double representation of the body surface in the region of cortex traditionally designated as SII may explain conflicting descriptions of SII organization in macaque monkeys. In addition, the present study raises some questions regarding the designation of serial processing pathways in Old World monkeys, by suggesting that fields may have been confused in studies demonstrating such pathways. We propose that SII and PV are components of a common plan of organization, and are present in many eutherian mammals.
Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy
Authors: Fu SY , Gordon T
Published: April 2018

The contribution of prolonged motoneuron axotomy to the poor functional recovery after delayed nerve repair was determined by means of a nerve cross-anastomosis paradigm in the rat. The tibial nerve was axotomized up to 12 months before it was cross-sutured to the distal stump of the freshly cut common peroneal nerve to innervate the freshly denervated tibialis anterior muscle. Three to 17 months later, muscle and motor unit (MU) forces were measured to quantify the number of axons that had successfully regenerated and reinnervated the muscle. The extent of axonal branching was estimated by the innervation ratio (IR) (i.e., the number of muscle fibers innervated by each axon), which was obtained directly by counting muscle fibers in a single glycogen-depleted MU in each muscle and indirectly by calculation. The total number of MUs in each muscle significantly decreased with progression of axotomy and was only 35% of the control when axotomy was prolonged more than 3 months. Concurrently, MU force and IR increased exponentially, with a mean increase of threefold when axotomy was more than 3 months, which largely compensated for the reduction in the number of axons that reinnervated the muscle. Consequently, muscles reinnervated by tibial motor axons that had been axotomized up to 12 months produced as much force as those reinnervated by freshly axotomized tibial motor axons. Muscle weight, size, and muscle fiber size were similar to those after immediate nerve suture. Although prolonged axotomy does not compromise the number of muscle fibers innervated by each axon, it does reduce the capacity of motor axons to regenerate and thus is an important contributing factor to the poor functional recovery in delayed nerve repair.
Dopamine enhances glutamate-activated currents in spinal motoneurons
Authors: Smith DO , Lowe D , Temkin R , Jensen P , Hatt H
Published: April 2018

In cultured embryonic chick motoneurons, glutamate-activated currents rise quickly and then decay rapidly to relatively small steady-state current levels. Dopaminergic modulation of these receptors was studied using patch-clamp recording techniques. At concentrations > or = 10 nM, dopamine enhanced glutamate-activated currents by about 200%. This enhancement was diminished by the nonspecific dopamine receptor antagonist S(+)-apomorphine and the more specific D1 receptor antagonist SCH23390, and it was mimicked by the D1 partial agonist SKF38393. Glutamate receptor desensitization rate was not altered in the presence of dopamine. Enhancement was specific to the kainate component. Current-variance analysis indicated that in the presence of dopamine the conductances of glutamate-activated channels were not altered but that the relative fraction of kainate-type channels activated by glutamate increased. Intracellular cAMP levels increased by 33% following exposure to 100 microM dopamine. The effects of elevated cAMP or protein kinase A (PKA) were tested by including 100 microM cAMP or PKA, respectively, in the recording pipette solution. This increased the kainate-activated currents specifically. Dopaminergic enhancement was not observed when a PKA inhibitor was in the pipette. mRNA encoding D1 was detected in the spinal cord by a reverse transcription, polymerase chain-reaction amplification procedure. Thus, dopamine is acting most probably on an avian homolog of the D1 receptor family. We conclude that dopamine causes cAMP to increase, which results in increased activation of kainate-gated channels during glutamate-mediated transmission.
Long aboral projections of Dogiel type II, AH neurons within the myenteric plexus of the guinea pig small intestine
Authors: Brookes SJ , Song ZM , Ramsay GA , Costa M
Published: April 2018

Enteric AH neurons, with multipolar Dogiel type II morphology, project around the circumference of the intestine to myenteric ganglia, the submucosa and mucosa. Using retrograde labeling in vitro, intracellular recording, dye filling and immunohistochemistry, the projections of these neurons along the intestine were studied. When the retrograde tracer, Dil, was applied to the myenteric plexus, labeled nerve cell bodies were located up to 111 mm orally but only 13 mm aborally, demonstrating a marked difference in the lengths of projections up and down the small intestine. Of labeled nerve cell bodies located 2–110 mm orally, 43% had Dogiel type II morphology and of these, 70% were immunoreactive for calbindin, a calcium binding protein exclusive to Dogiel type II neurons. Intracellular filling with neurobiotin revealed several long circumferentially directed nerve fibers and short, filamentous dendrites; thus these were “dendritic” Dogiel type II neurons. This class accounts for approximately 3–4% of all myenteric neurons, and about 10% of all Dogiel type II neurons. Intracellular recordings revealed AH cell characteristics, with long afterhyperpolarizations following their action potentials, pronounced slow excitatory synaptic inputs and a lack of fast excitatory synaptic inputs. Antidromic action potentials could be evoked from the Dil application site in some cells, confirming their aboral projection. This is the first account of a major aboral projection of AH/Dogiel type II neurons and suggests an important role in aborally directed reflexes in the intestine.
A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death
Authors: Hajimohammadreza I , Probert AW , Coughenour LL , Borosky SA , Marcoux FW , Boxer PA , Wang KK
Published: April 2018

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia- induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2- thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult.
Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain
Authors: Faure MP , Alonso A , Nouel D , Gaudriault G , Dennis M , Vincent JP , Beaudet A
Published: April 2018

Polypeptide hormones and growth factors have long been known to internalize into peripheral target cells as a result of their interaction with cell surface receptors. Studies in culture have suggested that certain neuropeptides might undergo a similar type of translocation in neurons. To investigate this possibility in adult mammalian brain, we have examined by confocal laser microscopy the events that follow the binding of fluorescein-tagged derivatives of the tridecapeptide neurotensin to basal forebrain cholinergic cells. Our results demonstrate a selective time- and temperature-dependent internalization of fluo-neurotensin in these cells. This internalization is receptor mediated, proceeds from the entire somatodendritic membrane of the cells, and utilizes endosome-like organelles which are mobilized from dendrites to perikarya and from the periphery of the cell to its perinuclear region. Parallel studies carried out on Sf9 insect cells expressing the rat neurotensin receptor from a recombinant baculovirus indicated that the internalization process involves receptor-ligand complexes and not merely the fluorescent peptide itself. These data suggest that receptor internalization plays a role in neuropeptide signaling in the brain and that it can be harnessed for selective identification of neuropeptide target cells.
The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton
Authors: Kirsch J , Betz H
Published: April 2018

The mechanisms underlying the postsynaptic localization of neurotransmitter receptors are poorly understood. Recently, the peripheral membrane protein gephyrin has been shown to be essential for the formation of inhibitory glycine receptor clusters in cultured rat spinal cord neurons. In vitro gephyrin binds with high affinity to polymerized tubulin. Here, the interaction of gephyrin with different components of the cytoskeleton was investigated in primary cultures of rat spinal neurons. After treatment with alkaloids affecting the cytoskeleton, the morphology of post-synaptic gephyrin clusters was analyzed by confocal immunofluorescence microscopy. Depolymerization of microtubules by demecolcine reduced both the percentage of cells with postsynaptic gephyrin clusters and the number of clusters/cell. The size of the remaining gephyrin clusters was increased whereas their gephyrin density was significantly lower than under control conditions. Depolymerization of microfilaments by cytochalasin D in contrast generated smaller clusters of increased gephyrin density. Demecolcine also dispersed postsynaptic glycine receptor clusters as revealed by immunostaining with a specific monoclonal antibody. These findings support the view that in vivo gephyrin anchors receptor polypeptides to the cytoskeleton by a complex interaction with microtubules and microfilaments.
Cytochrome-oxidase blobs in cat primary visual cortex
Authors: Murphy KM , Jones DG , Van Sluyters RC
Published: April 2018

Cytochrome-oxidase blobs are central to two of the most influential ideas in contemporary visual neuroscience--cortical modularity and parallel processing pathways. In particular, the regular 2D array of cytochrome-oxidase-rich blobs in primate visual cortex is arguably the most compelling evidence for cortical modularity and has been hypothesized to mark a separate processing stream through the visual cortex. Although previously a variety of mammals have been studied, blobs have only been demonstrated in the visual cortex of primates, which has led to the conclusion that blobs represent a primate-specific feature of visual cortical organization. Here we demonstrate the presence of cytochrome-oxidase blobs in a nonprimate species. Throughout the full tangential extent of layers II-III in cat visual cortex the cytochrome-oxidase staining pattern is distinctly patchy, with the darkly stained blobs forming a regular 2D array. In addition, the blobs in cat visual cortex are functionally related to the underlying ocular dominance columns. The presence of cytochrome-oxidase blobs in the cat clearly demonstrates that they no longer can be considered a primate-specific feature of visual cortical organization.
V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behavior in rats
Authors: Landgraf R , Gerstberger R , Montkowski A , Probst JC , Wotjak CT , Holsboer F , Engelmann M
Published: April 2018

To develop and validate a vasopressin (AVP) receptor knockdown strategy, we infused an antisense oligodeoxynucleotide to the V1 subtype mRNA into the septum of male rats with osmotic minipumps and measured behavioral, cellular and molecular parameters. Compared to vehicle and scrambled-sequence oligo controls, chronic antisense administration for up to 4 d diminished the ability of the animals to distinguish a previously exposed juvenile from a novel one and to respond to exogenous AVP (1 ng/5 microliters, intracerebroventricular) with an improved social memory. Furthermore, anxiety-related behavior was reduced. As measured in the behaviorally tested rats, antisense treatment resulted in a reduced binding of radiolabeled AVP in the septum, but not in other limbic brain areas (receptor autoradiography), and an increased amount of V1 receptor mRNA (reverse transcriptase PCR), indicating translational arrest and ongoing transcriptional activity. In sense oligo-treated rats, on the other hand, both the social and the anxiety-related behavior scores lay between levels obtained in control and antisense-treated animals. These sense-treated rats showed a slightly reduced V1 receptor density in the septum and reduced receptor mRNA levels, indicating hybridization of the sense oligo to the DNA. The data show the potential of antisense targeting to further reveal relationships between local gene expression, neuropeptide-receptor interactions in distinct brain areas, and behavioral performance.
Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin
Authors: Kraszewski K , Mundigl O , Daniell L , Verderio C , Matteoli M , De Camilli P
Published: April 2018

Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.
Potassium currents responsible for inward and outward rectification in rat neostriatal spiny projection neurons
Authors: Nisenbaum ES , Wilson CJ
Published: April 2018

Many of the nonlinear membrane properties displayed by neostriatal spiny projection neurons are conferred by their voltage-gated potassium (K+) currents, including an inwardly rectifying current (IKir), fast (IAt), and slowly (IAs)-inactivating A-currents, and a slow, noninactivating current. The relative contribution of these K+ currents to the pronounced inward and outward rectification of the current- voltage (I-V) relationship of spiny neurons was investigated in a neostriatal slice preparation. Manipulation of the equilibrium potential for K+ (EK) showed that the voltage dependence of activation of inward rectification was identical to that of IKir. In addition, application of barium (100 microM), which is known to reduce IKir in a time- and voltage-dependent manner, had equivalent effects on inward rectification. Subsequent application of cesium (3 mM) or tetraethylammonium (TEA, 25 mM) blocked inward rectification in a solely voltage-dependent fashion consistent with the action of these blockers on IKir. Administration of 4-aminopyridine (4-AP, 100 microM) at concentrations that selectively depress IAs, reduced outward rectification of spiny neurons at subthreshold membrane potentials. Higher concentrations of 4-AP (2 mM), which block both IAs and IAt, revealed an early transient overshoot in voltage deflections at potentials near spike threshold, but rectification persisted at the end of the responses. The transient overshoot and the residual rectification were eliminated by TEA (25 mM), a blocker of the slow, noninactivating K+ current. Collectively, these results indicate that all three depolarization-activated K+ currents contribute to outward rectification at different times and membrane potentials defined by their voltage dependence of activation and kinetics of inactivation. The spontaneous activity of neostriatal spiny neurons recorded in intact animals is characterized by sustained and limited shifts in membrane potential from relatively hyperpolarized potentials to depolarized potentials near spike threshold. The present data suggest that the hyperpolarized state is determined principally by IKir and the limits on the depolarized state are defined by IAf, IAs, and the noninactivating current. These outward K+ currents also are hypothesized to govern the spike discharge characteristics once the depolarized state has been reached.
On the linkage between AMPA and NMDA receptor-mediated EPSPs in homosynaptic long-term depression in the hippocampal CA1 region of young rats
Authors: Xiao MY , Karpefors M , Gustafsson B , Wigstrom H
Published: April 2018

Homosynaptic long-term depression (LTD) was studied in hippocampal slices from 12–18-d-old rats using field EPSP recording in the apical dendritic layer of CA1 pyramidal cells. Independent estimates of the alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and the N- methyl-D-aspartic acid (NMDA) receptor-mediated components of the field EPSP were obtained in parallel using early and late measurements of a dual-component EPSP in a low-magnesium solution. LTD was induced by low- frequency stimulation (LFS; 2 Hz for 10 min), resulting in equal relative changes of the AMPA and NMDA receptor-mediated components. Under conditions when the AMPA receptor-mediated component was fully blocked, a similarly sized LTD was observed for the pure NMDA receptor- mediated EPSP (measured as initial slope or peak amplitude). Equal changes in AMPA and NMDA receptor-mediated components occurred also upon application of the adenosine agonist N6-cyclohexyladenosine (CHA), known to act by decreasing transmitter release. On the other hand, LTD was found to interact in a multiplicative manner with the presynaptic release changes induced by CHA and by paired-pulse facilitation. The induction of the LTDs of both AMPA and NMDA receptor-mediated EPSPs was blocked by the NMDA receptor antagonist D(-)-2-amino-5- phosphonopentanoic acid and by the phosphatase inhibitor okadaic acid. Partial blockade of LTD by okadaic acid resulted in equal partial blockade of the LTDs of the AMPA and NMDA receptor-mediated components. On the other hand, the L-type calcium channel blocker nifedipine, the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4- carboxyphenylglycine, the nitric oxide synthase inhibitor N omega-nitro- L-arginine, and the heme oxygenase inhibitor protoporphyrin IX zinc(II) had no effect on LTD of either the AMPA or the NMDA receptor-mediated component. These results of equal changes of AMPA and NMDA receptor- mediated components of the field EPSP in association with LTD, and the consistent parallelism of effects or noneffects on these components by various receptor antagonists and enzyme inhibitors, seem more easily explained by a presynaptic locus for LTD than by a postsynaptic one.
Mechanism for modulation of nicotinic acetylcholine receptors that can influence synaptic transmission
Authors: Amador M , Dani JA
Published: April 2018

Only recently has it been appreciated that neuronal nicotinic ACh receptors (NnAChRs) are highly permeable to Ca2+ and are modulated by Ca2+ in a dose-dependent manner. These findings suggest that Ca2+ could have roles in cholinergic synaptic plasticity. We report a possible mechanism for Ca(2+)-initiated synaptic plasticity that differs from the intracellular Ca2+ cascade associated with plasticity of glutamatergic synapses. Rapid changes in external Ca2+ modulate cholinergic spontaneous synaptic currents in superior cervical ganglionic sympathetic neurons. Inhibition of cholinergic currents by chlorisondamine, which blocks only open channels and becomes trapped in the pore, showed that the modulation is not by a mechanism that activates a previously unresponsive population of NnAChRs. Rather, single-channel recordings with ganglionic NnAChRs from chromaffin cells indicated that Ca2+ directly alters the probability of the channels being open. We hypothesize from the results that activity-dependent decreases in external Ca2+, which occur throughout the nervous system, could directly underlie a rapid negative-feedback mechanism that decreases the responsiveness of NnAChRs at synapses. When external Ca2+ is decreased, presynaptic Ca2+ currents and transmitter release also are diminished. Thus, several mechanisms could combine to potently and rapidly depress synaptic nicotinic receptors until the external Ca2+ concentration recovers.
Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus
Authors: Cameron HA , McEwen BS , Gould E
Published: April 2018

The effects of afferent input and N-methyl-D-aspartate (NMDA) receptor activation on neurogenesis were examined in an intact system, the rat dentate gyrus, where neurons are naturally born in the adult. In the adult dentate gyrus, activation of NMDA receptors rapidly decreased the number of cells synthesizing DNA, whereas blockade of NMDA receptors rapidly increased the number of cells in the S phase identified with 3H- thymidine. Acute treatment with NMDA receptor antagonists increased the birth of neurons and increased the overall density of neurons in the granule cell layer. Lesion of the entorhinal cortex, the main excitatory afferent population to the granule neurons, also increased the birth of cells in the dentate gyrus. These results suggest that adult neurogenesis in the dentate gyrus of the rat is altered by afferent input, via NMDA receptors, and may be regulated naturally by endogenous excitatory amino acids.
Neurotrophins promote maturation of developing neuromuscular synapses
Authors: Wang T , Xie K , Lu B
Published: April 2018

Although the effects of neurotrophins on survival and differentiation of various neuronal populations have been well studied, little is known about their role in synaptic development and function. We have investigated the long-term effects of neurotrophins in the maturation of neuromuscular synapses in Xenopus nerve-muscle cocultures. BDNF and NT-3, but not NGF, elicited significant changes in several properties of spontaneous synaptic currents (SSCs), indicative of more mature synapses. Most synapses treated by the neurotrophins exhibited a bell- shaped distribution of SSC amplitudes, which reflects mature quantal secretion. The neurotrophins also potentiated the efficacy and reliability of stimulus-induced synaptic transmission. Moreover, BDNF and NT-3 increased the levels of the synaptic vesicle proteins, synaptophysin, and synapsin 1 in the spinal neurons. The number of varicosities per neuron also showed a significant increase after neurotrophin treatment. The effects of the neurotrophins appear to be mediated by the Trk family of receptor tyrosine kinases, primarily through a presynaptic mechanism. These results suggest that BDNF and NT- 3 promote functional maturation of synapses.
The mouse mutation reeler causes increased adhesion within a subpopulation of early postmitotic cortical neurons
Authors: Hoffarth RM , Johnston JG , Krushel LA , van der Kooy D
Published: April 2018

Early postmitotic cortical neurons are mostly corticofugal projection neurons that take up positions in deep cortical laminae. Later postmitotic neurons are preferentially localized to superficial cortical laminae. In reeler mutant mice it appears that cortical laminar positions with respect to birthdate are reversed (Caviness, 1982). In a reanalysis of reeler lamination we found that early postmitotic cortical neurons labeled by embryonic day (E) 11–13 injections of a birthdate marker, or by early postnatal day (PND) 2 retrograde labeling through their output projections, appear to take up positions both in the superficial and deep cortex. Neurons born on E11 and E12 are more likely to be situated superficially in the reeler cortex and neurons born on E13 are more likely to be situated in the deep reeler cortex. Many corticofugal projection neurons in the deep (but not superficial) reeler cortex either die or retract their axons before PND 21. We hypothesize that the earliest postmitotic (E11-E12) of the early postmitotic reeler cortical neurons are overly adhesive and act as a barrier to later postmitotic migrating neurons. In vitro cortical aggregation cultures confirmed that early postmitotic (E12) reeler neurons are more adhesive than early postmitotic (E12) wild-type neurons or late postmitotic (E16) reeler or wild-type cortical neurons. We suggest that the moderate wild-type preferential adhesion of early postmitotic cortical neurons to each other helps deep and superficially fated lineages to form cortical laminae.
Differential properties of cells in the feline primary visual cortex providing the corticofugal feedback to the lateral geniculate nucleus and visual claustrum
Authors: Grieve KL , Sillito AM
Published: April 2018

We have examined the responses of 141 layer VI cells in the feline visual cortex. Within this group we compared the responses of a subpopulation of cells checked for connectivity by electrical stimulation in the dLGN and the visual claustrum. The antidromically identified corticogeniculate projecting cells had relatively short receptive fields, as judged from length response curves, measured quantitatively, and were located at the “short” end of the receptive field length spectrum seen in the general population. Of the 17 corticogeniculate projecting cells, 71% were S type cells, which were typically monocular and directionally selective, with relatively long latencies following electrical stimulation. The remaining 29% were C type cells, also directionally selective, but with a wider spread of ocular dominance preferences and shorter latencies following electrical stimulation. S and C type subpopulations did not differ in their receptive field lengths. The mean receptive field length for this subpopulation was 2.2 degrees +/- 0.27, the shortest field being 1 degrees and the longest 5 degrees. The five layer VI cells activated by electrical stimulation from electrodes within the dorsocaudal (visual) claustrum all had much longer receptive field lengths than the corticogeniculate population, often 10 degrees or longer and were monocular and directionally selective S type cells. These data indicate that the information carried in the corticogeniculate stream (and that from layer VI directly to layer IV carried by axon collaterals) is relatively tightly focused in spatial terms whilst the less spatially focused, long receptive field output from layer VI projects to the claustrum.