bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde transport of radiolabeled cytoskeletal proteins in transected nerves
Authors: Glass JD , Griffin JW
Published: April 2018

Slow axonal transport is the mechanism by which cytoskeletal proteins are distributed within the axon. This function has traditionally been considered an exclusively unidirectional, anterograde process. Previous observations of cytoskeletal redistribution in surviving, transected axons of the C57BL/Ola mouse led us to hypothesize a retrograde component of cytoskeletal transport. To test this hypothesis against previous methods of measuring slow transport of cytoskeleton, we radioactively pulse-labeled proteins in sensory neurons of C57BL/Ola mice and followed their redistribution by gel fluorography in ligated and unligated sciatic nerves. Slow axonal transport of cytoskeletal proteins proceeded with the same characteristics in C57BL/Ola as in standard C57BL/6 mice. In comparison to the transport profiles from unligated control nerves, in ligated nerves there was redistribution of radiolabeled neurofilament and tubulin proteins back toward the cell body during the 14 d experimental period. These observations demonstrate that pulse-labeled cytoskeletal proteins move bidirectionally in this experimental system, and may provide insight into the normal mechanisms of cytoskeletal maintenance.
Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain
Authors: Roberts-Lewis JM , Savage MJ , Marcy VR , Pinsker LR , Siman R
Published: April 2018

Transient ischemia-induced perturbations in calcium homeostasis have been proposed to lead to pathological activation of the cysteine protease calpain I and subsequent delayed neuronal death in the CA1 region of hippocampus. We report here on the design and characterization of antibodies selective for calpain-generated fragments of brain spectrin, and their use for immunoblot and immunohistochemical analyses of calpain activation following cerebral ischemia in the gerbil. Although spectrin was susceptible to degradation in vitro by many mammalian proteases, only calpain degraded spectrin to generate fragments immunoreactive with the antibodies. Following 5 min of global ischemia, immunoreactivity for calpain- degraded spectrin was rapidly (within 30 min) and markedly elevated in the perikarya and dendrites of several populations of forebrain neurons. The rapid calpain activation was completely prevented by the NMDA receptor antagonist MK-801. At later times postischemia, but prior to frank neuronal necrosis, calpain-degraded spectrin was restricted to hippocampal area CA1 pyramidal neurons. Silver impregnation histochemistry confirmed that neuronal damage was confined to area CA1. The results indicate that while nonpathological NMDA receptor stimulation can activate calpain, only those neurons showing sustained calpain activation are destined to die.
Responses of tonically active neurons in the primate's striatum undergo systematic changes during behavioral sensorimotor conditioning
Authors: Aosaki T , Tsubokawa H , Ishida A , Watanabe K , Graybiel AM , Kimura M
Published: April 2018

The basal ganglia have been implicated in motor planning and motor learning. In the study reported here, we directly tested for response plasticity in striatal neurons of macaque monkeys undergoing Pavlovian conditioning. To focus the study, we recorded from the tonically active neurons (TANs) of the striatum, which are known to respond to conditioned sensory stimuli that signal reward delivery and elicit behavioral reactions. The activities of 858 TANs were recorded extracellularly from the striatum in alert behaving macaque monkeys before, during, and after the acquisition of a classical conditioning task. Two monkeys were trained to lick reward juice delivered on a spoon simultaneously with the presentation of a click. Almost no licks were triggered by the cues at the start of training, but by the fifth day more than 90% of licks were triggered, and values were near 100% for the remainder of the 3 week training period. In the striatum, only a small number of TANs responded to the clicks at the start before conditioning (about 17%). During training, the numbers of responding TANs gradually increased, so that by the end of training more than 50– 70% of the TANs recorded (51.3–73.5%) became responsive to the clicks. The responses consisted of a pause in firing that occurred approximately 90 msec after the click and that was in some cells preceded by a brief activation and in most cells was followed by a rebound excitation. Prolonged recordings from single TANs (n = 6) showed that individual TANs can acquire a conditioned response within at least as short a time as 10 min. TANs retained such responsiveness after overtraining, and also after a 4 week intermission in training. When the monkey was trained to receive rewards in relation to a new conditioning stimulus, TANs were capable of switching their sensory response to the new stimulus. Histological reconstruction showed that the TANs that became responsive were broadly distributed in the region of striatum explored, which included the dorsal half to two-thirds of the caudate nucleus and putamen over a large anteroposterior span. We conclude that, during the acquisition of a sensorimotor association, TANs widely distributed through the striatum become responsive to sensory stimuli that induce conditioned behavior. This distributed change in activity could serve to modulate the activity of surrounding projection neurons in the striatum engaged in mediating learned behavior.
Differential effects of chronic antipsychotic drug treatment on extracellular glutamate and dopamine concentrations
Authors: Yamamoto BK , Cooperman MA
Published: April 2018

Typical and atypical antipsychotic drugs have been reported to affect basal dopaminergic activity differentially in nigrostriatal and limbic structures after acute and chronic administration in animals. In addition, glutamate has been implicated in the pathophysiology of schizophrenia. The purpose of this study was to examine basal and locally stimulated glutamate and dopamine efflux in the caudate, nucleus accumbens, and medial prefrontal cortex using in vivo microdialysis after chronic clozapine and haloperidol treatment. Basal extracellular concentrations of dopamine in the caudate and nucleus accumbens were not different between the drug treatment groups; however, dopamine concentrations were higher in the medial prefrontal cortex after chronic clozapine treatment. Depolarization-induced dopamine release with 80 mM K+ in all three brain regions was attenuated by haloperidol treatment. In contrast, basal concentrations of extracellular glutamate were markedly higher in the caudate and modestly increased in the nucleus accumbens but not in the prefrontal cortex after chronic haloperidol. Chronic clozapine treatment did not have an effect in any of the brain regions examined. K(+)-stimulated glutamate efflux was unaffected by haloperidol or clozapine in the caudate or prefrontal cortex; however, stimulated glutamate release in the nucleus accumbens was enhanced by clozapine. These data are suggestive of a depolarization inactivation of dopamine nerve terminals in striatum and cortex as revealed by an attenuation of local K(+)- induced stimulation of dopamine efflux. These results also provide new evidence for a role of glutamate in discriminating the neurochemical effects of chronic treatment with antipsychotic drugs.
Distributed aspects of the response to siphon touch in Aplysia: spread of stimulus information and cross-correlation analysis
Authors: Tsau Y , Wu JY , Hopp HP , Cohen LB , Schiminovich D , Falk CX
Published: April 2018

We examined two aspects of the response to siphon stimulation in an attempt to test the hypothesis that the Aplysia CNS functions as a distributed system. First, we estimated the number of central neurons that respond to a light touch to the siphon skin. We made voltage- sensitive dye recordings from the abdominal, pleural, pedal, and cerebral ganglia. From these recordings we estimated that 220 abdominal neurons, 110 pleural neurons, and 650 pedal neurons were affected by the light touch. Thus, the information about this mild and localized stimulus is very widely distributed within the Aplysia CNS. This result allows the possibility that the Aplysia CNS functions as a distributed system. If only a small number of neurons had responded to the touch, it would have supported the conclusion that the gill-withdrawal reflex could be generated by a small, dedicated circuit. Second, we searched for correlations between the spike times of the individual abdominal ganglion neurons. Two time scales were examined: a millisecond time scale corresponding to the duration of a fast synaptic potential and a seconds time scale corresponding to the duration of the gill-withdrawal movement. Neuron pairs with highly correlated spike activity on a millisecond time scale must be connected by (or have a common input that uses) relatively powerful, fast, excitatory synapses. We expected that this kind of synaptic interaction would be relatively rare in nervous systems that functioned in a distributed manner. Indeed, only 0.3% of the neuron pairs had correlation coefficients of 0.15 or greater. These correlations accounted for approximately 2% of the action potentials generated in response to siphon stimulation. Thus, large, fast excitatory synaptic interactions appear to be relatively unimportant. This result is consistent with the hypothesis that the abdominal ganglion functions as a distributed system. When the longer time scale was used for the cross-correlograms, a large fraction of the cell pairs had correlated activity because many neurons are activated by the stimulus. It was not possible to interpret the slow correlations in terms of actual synaptic interactions between individual neurons. Our results are consistent with the possibility that the abdominal ganglion functions in a distributed manner. However, this evaluation is indirect and thus only tentative conclusions can be drawn. Evidence from several sources suggests that the neuronal interactions for generating the Aplysia gill-withdrawal reflex are complex.
Kindling induces the long-lasting expression of a novel population of NMDA receptors in hippocampal region CA3
Authors: Kraus JE , Yeh GC , Bonhaus DW , Nadler JV , McNamara JO
Published: April 2018

Kindling refers to a phenomenon in which repeated application of initially subconvulsive electrical stimulations produces limbic and clonic motor seizures of progressively increasing severity. Once established, the increased excitability is lifelong. Enhanced function of synapses using the NMDA subtype of glutamate receptor could contribute to the expression of the increased excitability. We previously found that CA3 pyramidal cells of hippocampus of kindled animals exhibit a selective and long-lasting (1 month) increased sensitivity to NMDA-evoked depolarization. The goal of this study was to develop a molecular explanation of the enhanced sensitivity to NMDA. We used radioligand binding studies of membranes isolated from microdissected regions of hippocampus including fascia dentata, CA3, and CA1. We also used quantitative in situ hybridization with subtype- specific riboprobes or oligonucleotides to determine whether increased expression of one or more of the genes encoding NMDA receptors was present in hippocampal granule and pyramidal cells of kindled animals. When studied 28 d after the last evoked seizure, we found that kindling induced a 2.8-fold increase in the number of binding sites for the competitive NMDA receptor antagonist 3-[(+/-)-2-(carboxypiperazine-4- yl)][1,2–3H-]propyl-1-phosphonic acid (3H-CPP). This increase was confined to region CA3 within the hippocampus. Similar, though much smaller, changes were detected 24 hr after the last evoked seizure. Surprisingly, no changes in the binding of another competitive NMDA receptor antagonist, cis-4-(phosphonomethyl)-2–3H-piperidinecarboxylate (3H-CGS-19755), were detected at either time point in any hippocampal region. Transcript levels of the NMDA receptor genes NMDAR1, NR2A, NR2B, NR2C, and NR2D and a glutamate-binding protein (GBP) were not altered by kindling. These findings demonstrate that kindling induces the expression of an NMDA receptor that is novel in that it is recognized by 3H-CPP but not by 3H-CGS-19755. The molecular basis of this novel NMDA receptor is not determined by differential expression of mRNA transcripts of known NMDA receptor genes. The direction, time course, and location of the kindling-induced increase in 3H-CPP binding suggest that this novel receptor may underlie the increased sensitivity of CA3 neurons to NMDA observed in kindled animals.
Embryonic neural cell adhesion molecule (N-CAM) is elevated in the denervated rat dentate gyrus
Authors: Miller PD , Styren SD , Lagenaur CF , DeKosky ST
Published: April 2018

We evaluated the immunohistological changes in neural cell adhesion molecule (N-CAM) expression in the adult rat dentate gyrus during the period of synaptic degeneration, axonal sprouting, and synaptogenesis following ipsilateral entorhinal cortex (ERC) lesion. This lesion denervates the outer two-thirds of the dentate granule cells dendrites and induces compensatory sprouting from the subjacent inner one-third into the denervated zone, as well as reactive synaptogenesis in the denervated outer molecular layer. In unlesioned adult hippocampus, antibodies to total N-CAM stained the inner molecular layer intensely, and the outer molecular layer (ML) more lightly. After ERC lesion the intense staining of the inner layer widened, the expansion following the known temporal sequence of commissural and associational (C/A) axon sprouting into the denervated zone. In normal unlesioned controls there was very light, uniform staining of the ML with antibodies directed against embryonic N-CAM (eN-CAM). By 2 d post-ERC lesion, the outer two- thirds of the ML stained robustly with antibody to eN-CAM. This area of intense staining receded as the C/A axon collaterals from the inner one- third entered the denervated zone, so that by 30 d the intense eN-CAM staining only occupied the outer half of the ML. The increased expression of eN-CAM remained present at 60 d post-ERC lesion, past the point that synaptic volume density has returned to normal levels in the denervated zone. Ultrastructural studies showed that the newly expressed eN-CAM was located on the surface of dendrites in the denervated zone, but was not found at the synaptic contacts.(ABSTRACT TRUNCATED AT 250 WORDS)
Modulation of gap junctional mechanisms during calcium-free induced field burst activity: a possible role for electrotonic coupling in epileptogenesis
Authors: Perez-Velazquez JL , Valiante TA , Carlen PL
Published: April 2018

To date, there is little experimental evidence supporting or refuting electrotonic interactions through gap junctions in the generation and/or spread of seizure activity in the mammalian brain. We have studied gap junctional mechanisms in the in vitro calcium-free induced model of epilepsy using electrophysiological and staining techniques in the CA1 area of the hippocampus. Lucifer yellow staining of CA1 pyramidal neurons revealed that dye coupling was increased 2.3 times in hippocampal slices made hyperexcitable by perfusion with calcium-free artificial cerebrospinal fluid (aCSF). Furthermore, multiple neuronal dye coupling (triplets, quintuplets) was observed in these conditions but never in control (standard aCSF). Under conditions that reduce gap junctional conductance (intracellular acidification, octanol, halothane), seizure-like activity was suppressed in the CA1 area in this epilepsy model, whereas increasing gap junctional conductance by intracellular alkalinization increased the frequency and duration of field burst events. Intracellular acidification also reduced dye coupling as well as the frequency of fast prepotentials (electrotonic potentials) without altering neuronal firing frequency. Simultaneous extracellular field and single whole-cell recordings revealed suppression of synchronization between neuronal firing and spontaneous field burst activity during acidification. These observations indicate an apparent increase in electrotonic coupling during calcium-free induced spontaneous rhythmic field burst activity in the CA1 area of the hippocampus and that electrotonic coupling may contribute substantially to the synchronization of neuronal firing underlying seizure-like events.
Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats
Authors: Berkelaar M , Clarke DB , Wang YC , Bray GM , Aguayo AJ
Published: April 2018

Using quantitative anatomical techniques, we show that after intraorbital optic nerve transection in adult rats, virtually all retinal ganglion cells (RGCs) survive for 5 d and then die abruptly in large numbers, reducing the RGC population to approximately 50% of normal by day 7 and to less than 10% on day 14. During this period of rapid cell loss, some RGCs show cytochemical alterations indicative of apoptosis (“programmed cell death”), a change not previously categorized after axotomy in adult mammals. With intracranial lesions 8– 9 mm from the eye, the onset of cell death is delayed until day 8 and is greater with cut than crush. The demonstration that axotomy results in apoptosis, the long interval between axonal injury and RGC death, and the different time of onset of the massive RGC loss with optic nerve lesions near or far from the eye suggest that axonal interruption triggers a cascade of molecular events whose outcome may be critically dependent on the availability of neuronal trophic support from endogenous or exogenous sources. The role of such molecules in RGC survival and the reversible nature of these injury-induced changes is underscored by the temporary rescue of most RGCs by a single intravitreal injection of brain-derived neurotrophic factor during the first 5 d after intraorbital optic nerve injury (Mansour-Robaey et al., 1994). The delayed pattern of RGC loss observed in the present experiments likely explains such a critical period for effective neurotrophin administration.
GAP-43 expression in primary sensory neurons following central axotomy
Authors: Chong MS , Reynolds ML , Irwin N , Coggeshall RE , Emson PC , Benowitz LI , Woolf CJ
Published: April 2018

Primary sensory neurons are capable of successful regenerative growth in response to peripheral nerve but not dorsal root injury. The present study is concerned with the differential expression of the mRNA for GAP- 43, a growth-associated protein, in these sensory neurons, in response to injury of their central or peripheral axonal branches. Peripheral axotomy resulted in an elevation in message detectable within 24 hr, using Northern blot and in situ hybridization, which was maintained for 30 d, whereas dorsal root section produced no change except a transient and small increase if the axotomy was immediately adjacent to the dorsal root ganglia (DRG). Dorsal root section had no effect on GAP-43 mRNA levels in the dorsal horn or in neighboring intact DRG. It also failed to alter the laminar boundaries of the GAP-43 central terminal labeling produced by peripheral nerve section, even though vacant synaptic sites were produced in unstained laminae by this procedure. This indicates that the location of GAP-43 immunolabeling in the central terminals of primed sensory cells may not depend only on the location of vacant synaptic sites. We conclude that distinct control mechanisms regulate the response of DRG neurons to peripheral nerve and dorsal root injury, and these may be related both to the glial environment and the particular target influences exerted on the central and peripheral branches of the primary sensory neuron. Central denervation alone is insufficient to upregulate GAP-43 levels, and this may explain the relative absence of collateral sprouting after the production of central vacant synaptic sites. The failure of dorsal root section to increase GAP-43 expression may contribute to the poor regenerative response initiated by such lesions.
Activation of K current in the accessory radula closer muscle of Aplysia californica by neuromodulators that depress its contractions
Authors: Brezina V , Evans CG , Weiss KR
Published: April 2018

The neural and cellular mechanisms of plasticity apparent in the feeding behavior of the mollusk Aplysia californica have been extensively studied in a simple neuromuscular circuit consisting of the accessory radula closer (ARC) muscle and its innervating motor and modulatory neurons. In this circuit, the plasticity is largely due to modulation of the amplitude and duration of the contractions of the muscle by a variety of modulatory neurotransmitters and peptide cotransmitters, among them the small cardioactive peptides (SCPs), myomodulins (MMs), and serotonin (5-HT). We have studied dissociated but functionally intact ARC muscle fibers to determine whether modulation of membrane ion currents in the muscle might underlie these effects. Using voltage-clamp techniques, we found that two currents were indeed modulated. In the preceding article, we proposed that enhancement of “L”-type Ca current is the mechanism by which the modulators potentiate the amplitude of ARC-muscle contractions. Here, we report that the modulators also activate a distinctive K current. Large K currents were activated, in particular, by MMA, while MMB, the SCPs, and 5-HT activated much smaller currents most likely of the same kind. Buccalins, modulators that do not act directly on the ARC muscle, were ineffective. The modulator-induced K current was strongly enhanced by depolarization, but relatively slowly so that its amplitude continued to increase for several hundred milliseconds following a depolarizing voltage step. The current was Ca2+ independent, not readily blocked by extracellular Cs+ or Ba2+ and only by high concentrations of tetraethylammonium. However, it was almost completely blocked by as little as 10 microM 4-aminopyridine. In contrast to the modulator-induced enhancement of Ca current, activation of the K current was not significantly mimicked by elevation of cAMP. In the intact as well as the dissociated ARC muscle, although low levels of all of the modulators potentiate contractions, even moderate levels of MMA strongly depress them, whereas the other modulators depress them weakly only at high concentrations. The modulator-induced K current appears well suited to counteract depolarization of the muscle and thus limit activation of the “L”-type Ca current that provides Ca2+ essential for contraction. We therefore propose that the modulators depress ARC-muscle contractions in large part by activating the K current. This occurs simultaneously with the enhancement of the Ca current; net potentiation or depression then depends on the balance between the relative strengths of the modulation of the two ion currents.
Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task
Authors: Aston-Jones G , Rajkowski J , Kubiak P , Alexinsky T
Published: April 2018

Impulse activity was recorded extracellularly from noradrenergic neurons in the nucleus locus coeruleus (LC; 47 single-cell and 126 multicell recordings) of four cynomolgus monkeys performing an oddball visual discrimination task. For juice reward, the subjects were required to release a lever rapidly in response to an infrequent (10– 20% of trials) target cue (CS+) that was randomly intermixed with nontarget (CS-) stimuli presented on a video display. All LC neurons examined were phasically and selectively activated by target cues in this task. Other task events elicited no consistent response from these neurons (juice reward, lever release, fix-spot stimuli, nontarget stimuli). In one animal, nontarget cues phasically inhibited LC neurons. Phasic LC excitatory responses to target cues in this task occurred at a relatively short latency (mean = 90.7 msec), approximately 200 msec prior to the behavioral response (lever release). In addition, LC response magnitudes varied with behavioral performance, being substantially attenuated during epochs of poor performance (high false alarm rate). There was a positive correlation (r = 0.30, p < 0.0001) between the latency of LC responses and the latency of behavioral responses to same target cues, consistent with the possibility that LC responses may have a role in selective attention by facilitating responses to the CS+ stimulus. Analyses of behavioral response latencies to pairs of stimuli indicated that LC responses may facilitate behavioral responses to subsequent sensory cues, consistent with a role of this system in sustained attention/vigilance. Moreover, responses became reduced in magnitude over time during prolonged task performance (> 90 min), in parallel with a behavioral performance decrement. These results show that LC neurons are activated selectively by attended stimuli that demand a rapid response in this task, and that such LC responses may contribute to conditioned behavioral responses.
D1 agonist-induced excitation of substantia nigra pars reticulata neurons: mediation by D1 receptors on striatonigral terminals via a pertussis toxin-sensitive coupling pathway
Authors: Martin LP , Waszczak BL
Published: April 2018

Iontophoresis of dopamine or the D1 agonist SKF 38393 has been shown to elicit current-dependent increases in the firing of rat substantia nigra pars reticulata neurons, suggesting a discrete physiological role for the D1 dopamine receptor population in the substantia nigra. The effects of SKF 38393 differed from those of dopamine, however, in that the D1 agonist also augmented inhibitory responses to applied GABA, whereas dopamine and D2-like agonists were previously found to attenuate responses to GABA. The present studies involved various manipulations of the nigral D1 receptors in order to examine the pharmacological specificity, receptor localization, and second messenger coupling underlying the D1 agonist response. The excitatory and GABA-potentiating effects of SKF 38393 were found to be attributable to D1 receptor stimulation, rather than a nonspecific action, since (1) the effect was mimicked by iontophoresis of A-68930, a D1 agonist of a different structural class than SKF 38393, and (2) the response to SKF 38393 was prevented by intranigral injection of the receptor inactivator N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 50 nmol/0.5 microliter) 1 d before, or the D1 antagonist SCH 23390 (1 microgram/microliter) 1 hr before electrophysiological testing. Additional studies revealed that the involved D1 receptors were located presynaptically on striatonigral terminals. For instance, in rats given ipsilateral striatal kainic acid lesions 1 week earlier, application of SKF 38393 failed to elicit the usual increases in cell firing, but loss of the response was observed only among the group of pars reticulata neurons that were shown to be unresponsive to striatal stimulation (i.e., those whose striatonigral inputs had been terminated by the lesion). Finally, to examine the second messenger coupling characteristics of the involved D1 receptors, several membrane- permeable analogs of cAMP were tested iontophoretically in place of SKF 38393. Surprisingly, none of these compounds gave a pattern of response typical of the D1 agonist, raising questions about the involvement of cAMP. Even more suggestive of an unconventional D1 coupling pathway, the excitatory and GABA-potentiating effects of applied SKF 38393 were completely abolished by prior intranigral injection of the G(i)/G(o) protein inactivator, pertussis toxin. Collectively, these results suggest that stimulation of D1 receptors on striatonigral terminals causes an excitation of substantia nigra pars reticulata neurons with an exaggerated responsiveness to GABA, and the effects appear to be mediated by a pertussis toxin-sensitive (i.e., a non-G-like) G-protein and possibly a second messenger other than cAMP.
Formation of transient inappropriate sensorimotor synapses in developing rat spinal cords
Authors: Seebach BS , Ziskind-Conhaim L
Published: April 2018

The specificity of the convergence of primary afferent projections from ankle muscles onto motoneurons that innervate these muscles was studied in lumbar spinal cords of embryonic and neonatal rats. The connectivity pattern was determined for each motoneuron by stimulating nerves from ankle flexor and extensor muscles and recording the synaptic potentials in identified motoneurons. In mature mammals, muscle spindle afferents make direct excitatory connections with motoneurons that innervate homonymous and synergistic muscles, and with interneurons that inhibit motoneurons innervating antagonistic muscles. Therefore, appropriate primary afferent-motoneuron connections were identified when stimulation of homonymous and synergistic muscle nerves evoked monosynaptic EPSPs. Two criteria were used for identification of EPSPs as monosynaptic potentials: (1) the monosynaptic potentials were evoked at the shortest latency, and (2) they were more resistant to fatigue by repetitive nerve stimulation than the longer-latency, polysynaptic potentials. Functionally inappropriate primary afferent-motoneuron contacts were identified when stimulation of an antagonistic muscle nerve produced monosynaptic EPSPs instead of polysynaptic IPSPs in homonymous motoneurons. At days 18–21 of gestation, about 30% of motoneurons were innervated by primary afferents of antagonist muscles. Such functionally inappropriate synapses persisted at birth, but their percentage was significantly reduced within 3–5 d after birth. The findings suggested that in the developing spinal cord of the rat, a significant percentage of motoneurons were initially innervated by inappropriate primary afferents of antagonistic muscles. The decrease in percentage of such inappropriate connections was correlated temporally with the increase in the frequency of spontaneous activity and the onset of myelination.
Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain
Authors: Wang H , Kunkel DD , Schwartzkroin PA , Tempel BL
Published: April 2018

Multiple voltage-gated potassium (K) channel gene products are likely to be involved in regulating neuronal excitability of any single neuron in the mammalian brain. Here we show that two closely related voltage- gated K channel proteins, mKv1.1 and mKv1.2, are present in multiple subcellular locations including cell somata, juxta-paranodal regions of myelinated axons, synaptic terminals, unmyelinated axons, specialized junctions among axons, and proximal dendrites. Staining patterns of the two channel polypeptides overlap in some areas of the brain, yet each has a unique pattern of expression. For example, in the hippocampus, both mKv1.1 and mKv1.2 proteins are present in axons, often near or at synaptic terminals in the middle molecular layer of the dentate gyrus, while only mKv1.1 is detected in axons and synaptic terminals in the hilar/CA3 region. In the cerebellum, both channel proteins are localized to axon terminals and specialized junctions among axons in the plexus region of basket cells. Strong differential staining is observed in the olfactory bulb, where mKv1.2 is localized to cell somata and axons, as well as to proximal dendrites of the mitral cells. This overlapping yet differential pattern of expression and specific subcellular localization may contribute to the unique profile of excitability displayed by a particular neuron.
Ionic currents of Kenyon cells from the mushroom body of the honeybee
Authors: Schafer S , Rosenboom H , Menzel R
Published: April 2018

The mushroom bodies have been suggested to be essentially involved in learning and memory in insects. In the honeybee Apis mellifera they are composed of about 340,000 intrinsic elements, called Kenyon cells, which can be easily separated from all other neurons of the brain. Here we describe a preparation in which we studied ionic currents in the isolated Kenyon cell somata, using tight-seal whole-cell recording. Several outward and inward currents were identified and investigated by the use of pharmacological agents and in ion substitution experiments: a rapidly inactivating A-type potassium current that is completely blocked with 5 mM 4-aminopyridine; a calcium-activated potassium current that is blocked by 1–100 nM charybdotoxin; a delayed rectifier- type potassium current that is only weakly sensitive to tetraethylammonium but is blocked by 100 microM quinidine; a rapidly activating and inactivating, TTX-sensitive sodium current; a persistent sodium current that is both TTX and cadmium sensitive; and a calcium current that is completely blocked at 50 microM cadmium and is affected by verapamil and nifedipine only at high concentrations (100 microM). The currents described here are very similar to currents found in other insect neurons or muscle cells. This preparation will not only facilitate studies concerning the action of transmitters and neuromodulators that are contained within neurons converging onto the Kenyon cells, but will also allow a study of the role of the adenylyl cyclase pathway, elements of which are expressed in Kenyon cells, and are known to be essential for learning in invertebrates.
Detailed passive cable models of whole-cell recorded CA3 pyramidal neurons in rat hippocampal slices
Authors: Major G , Larkman AU , Jonas P , Sakmann B , Jack JJ
Published: April 2018

Tight-seal whole-cell recordings were made from cleaned somata of CA3 pyramidal cells deep in hippocampal slices from 19–21-d-old rats. The cells were filled with biocytin, and their voltage responses to short current pulses were recorded. After washout of initial sag, responses scaled linearly with injected current and were stable over time. The dendritic and axonal arbors of four cells were reconstructed and measured using light microscopy. Dendritic spines and axonal boutons were counted and the additional membrane area was incorporated into the relevant segments. The morphology of each neuron was converted into a detailed branching cable model by assuming values for specific membrane capacitance Cm and resistance Rm, and cytoplasmic resistivity Ri. These parameters were optimized for each cell by directly matching the model's response to that of the real cell by means of a modified weighted least-squares fitting procedure. By comparing the deviations between model and experimental responses to control noise recordings, approximate 95% confidence intervals were established for each parameter. If a somatic shunt was allowed, a wide range of possible Rm values produced acceptable fits. With zero shunt, Cm was 0.7–0.8 microFcm-2, Ri was 170–340 omega cm, and Rm ranged between 120 and 200 k omega cm2. The electrotonic lengths of the basal and oblique dendrites were 0.2–0.3 space constants, and those of the apical tufts were 0.4–0.7 space constants. The steady-state electrical geometry of these cells was therefore compact; average dendritic tip/soma relative synaptic efficacies were > 93% for the basal and oblique dendrites, and > 81% for the tufts. With fast transient synaptic inputs, however, the models produced a wide range of postsynaptic potential shapes and marked filtering of voltage-clamp currents.
Heterosynaptic suppression of developing neuromuscular synapses in culture
Authors: Lo YJ , Poo MM
Published: April 2018

Activity-dependent competition between two spinal neurons coinnervating an embryonic myocyte was studied in Xenopus cell culture. We have characterized in detail the phenomenon of heterosynaptic suppression by which tetanic stimulation of one neuron results in functional suppression of the synapse made by the untetanized neuron (Lo and Poo, 1991). Fluorescence labeling of the neurons using two different fluorophores revealed that the coinnervating nerve terminals on the spherical myocyte were in close proximity. Heterosynaptic suppression could be induced when the postsynaptic cell was held under either current-clamp or voltage-clamp conditions during the tetanic stimulation. This finding, together with the observation that repetitive postsynaptic depolarization of the myocyte by direct current injection was much less effective in inducing synaptic depression, suggests that postsynaptic ACh receptor activation plays a dominant role in the induction of heterosynaptic suppression. The heterosynaptic suppression appears to be mediated by a rise of Ca2+ levels in the postsynaptic cell, since it was not observed when the cytosolic Ca2+ concentration of the myocyte was buffered at a low level with intracellular loading of a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetra-acetic acid (BAPTA). The dependence of heterosynaptic suppression on the pattern of tetanic stimulation was also studied. At a stimulation frequency of 2 Hz, detectable heterosynaptic suppression could be induced after 20 repetitive stimuli were applied to one of the presynaptic neurons and the suppression was more effective with increasing number of stimuli. Over the range of 0.5–5 Hz, the extent of suppression was independent of the frequency of tetanic stimulation and, in some cells, detectable suppression could be induced at a frequency as low as 0.05 Hz. Except for a few cases, heterosynaptic suppression was found to last for as long as the recording was made after tetanus (up to 1 hr). The fact that the mean amplitude of spontaneous synaptic currents remained the same before and after the suppression while the evoked synaptic currents exhibited higher fluctuation after suppression suggests that the observed synaptic suppression involves a reduction of evoked ACh release from the nerve terminal, although postsynaptic changes have not been excluded. Finally we found that spontaneous synaptic activity may also contribute in part to the synaptic competition between coinnervating nerve terminals. Taken together, these findings provide a quantitative basis for further understanding of activity-dependent competition between developing neuromuscular synapses.
Linearized models of calcium dynamics: formal equivalence to the cable equation
Authors: Zador A , Koch C
Published: April 2018

The dynamics of calcium and other diffusible second messengers play an important role in intracellular signaling. We show here the conditions under which nonlinear equations governing the diffusion, extrusion, and buffering of calcium can be linearized. Because the resulting partial differential equation is formally identical to the one-dimensional cable equation, quantities analogous to the input resistance, space constant, and time constant--familiar from the study of passive electrical propagation--can be defined. Using simulated calcium dynamics in an infinite cable and in a dendritic spine as examples, we bound the errors due to the linearization, and show that parameter uncertainty is so large that most nonlinearities can usually be ignored: robust phenomena in the nonlinear model are also present in the linear model.
Augmentation of calcium channel currents in response to G protein activation by GTP gamma S in chick sensory neurons
Authors: Zong X , Lux HD
Published: April 2018

G protein-mediated downregulation of current through neuronal voltage- gated Ca2+ channels is well known. We now report that G protein activation by GTP gamma S increases the Ba2+ conductance of high- voltage-activated Ca2+ channels of chick dorsal root ganglion (DRG) cells. This occurs with a delay of minutes during which the channels are inhibited by the activated G proteins. The Ba2+ current (IBa) showed an absolute enhancement by a factor near 2, 15 min after GTP gamma S application. However, by utilizing prior observations of the voltage dependence of the inhibitory action we could demonstrate that the G protein-inhibited component of IBa, was still present. Moreover, the achieved amount of IBa disinhibition showed little variation throughout the experiments. This indicates that the increase in IBa is not due to a relief of the inhibitory action of activated G proteins but to the slow appearance of a distinct upregulating action, probably through a different pathway. Augmentation of IBa was eliminated by pertussis toxin (PTX) infusion or pretreatment, but was also prevented by intracellularly infusing protein kinase C (PKC) inhibitors together with GTP gamma S. The upregulation of neuronal Ca2+ channels thus appears to be exerted through a messenger pathway upstream of PKC activation that involves G proteins. Augmentation of Ca2+ currents (ICa) was observed only with strong intracellular [Ca2+] buffering, which suggests a control of the upregulating action by even moderate increase in intracellular [Ca2+].
Post-receptor mechanisms underlying striatal long-term depression
Authors: Calabresi P , Pisani A , Mercuri NB , Bernardi G
Published: April 2018

Extracellular and intracellular recordings were obtained from striatal neurons in a brain slice preparation in order to characterize the post- receptor mechanisms underlying striatal posttetanic long-term depression (LTD). Striatal LTD was blocked in neurons intracellularly recorded either with 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′- tetraacetic acid (BAPTA) or with EGTA, calcium (Ca2+) chelators. Intracellular injection of QX-314, a lidocaine derivative that has been shown to block voltage-dependent sodium channels, abolished action potential discharge and blocked striatal LTD. However, under this condition, striatal LTD was restored when, immediately before the delivery of the tetanus, the cell was depolarized at a membrane potential ranging between -30 mV and -20 mV by injecting continuous positive current. Nifedipine (10 microM), a blocker of voltage- dependent L-type Ca2+ channels, blocked striatal LTD. Nifedipine by itself altered neither cortically evoked EPSPs nor input resistance and firing properties of most of the recorded cells. Striatal LTD was also reduced or blocked by incubation of the slices in the presence of the following inhibitors of Ca(2+)-dependent protein kinases: staurosporine (10–50 nM), 1-(5-isoquinolinesulfonyl)-2- methylpiperazine (H-7; 10–50 microM), and calphostin C (1 microM). Our findings suggest that generation of striatal LTD requires a Ca2+ influx through voltage- dependent nifedipine-sensitive Ca2+ channels and a sufficient intracellular free Ca2+ concentration. Furthermore, this form of synaptic plasticity seems to involve the activation of Ca(2+)-dependent protein kinases. Different drugs, acting at receptor and/or post- receptor level, may affect this form of synaptic plasticity and might alter the formation of motor memory.
Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction
Authors: Usui H , Falk JD , Dopazo A , de Lecea L , Erlander MG , Sutcliffe JG
Published: April 2018

We report an improved subtractive cDNA cloning procedure, named “directional tag PCR subtraction,” for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5< and 3< ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.
The role of endogenous adenosine in a poststimulation increase in the acetylcholine content of a sympathetic ganglion
Authors: Tandon A , Collier B
Published: April 2018

Previous experiments showed that exposure of sympathetic ganglia to exogenous adenosine increased acetylcholine (ACh) content and its subsequent release. This effect was not mediated through extracellular adenosine receptors, but at an intracellular site following its uptake through nitrobenzylthioinosine (NBTI)-resistant nucleoside transporters. We postulated that endogenous adenosine may play a role in modulating synaptic transmission in the superior cervical ganglion. The present study tested whether adenosine is involved in the activation of ACh synthesis that occurs during a rest period following prolonged presynaptic tetanic activity. Conditioning of ganglia with high-frequency stimulation (15 Hz) for 45 min followed by a 15 min rest increased their ACh content by 45%. The appearance of this “rebound ACh” showed sensitivity to nucleoside transport inhibitors; it was prevented by dipyridamole, but not by NBTI or meclonazepam, and it was reduced in the presence of RO 11–3624, suggesting an involvement of NBTI-resistant transporters. The effect of dipyridamole was specific for the synthesis of rebound ACh in that it did not inhibit ACh release or ACh synthesis during stimulation. The inhibitory action of dipyridamole on the synthesis of rebound ACh was not evident if it was present only during the tetanic stimulation but it was if dipyridamole was present during the rest period following it, suggesting that adenosine's presence after tetanic stimulation is of importance. This conclusion was strengthened by experiments showing that the presence of cyclopentyltheophylline, an antagonist at inhibitory adenosine receptors, increased ACh output evoked by test stimulation immediately following tetanic activity, as if endogenous adenosine was available at that time to activate the adenosine receptors that inhibit transmitter release. ACh release from conditioned ganglia was 44% greater than that from the controls. However, the rebound ACh was not mobilized in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a vesicular ACh transporter inhibitor. These results suggest that endogenous adenosine released after tetanic stimulation activates ACh synthesis, which results in an increase of ganglionic ACh that is available for subsequent mobilization and release.