bluuebunny/crossref_metadata_2025 huggingface dataset:
kappa-opioid receptor agonists modulate excitatory transmission in substantia gelatinosa neurons of the rat spinal cord
Authors: Randic M , Cheng G , Kojic L
Published: April 2018

This study examined the effects of selective activation of kappa 1- opioid receptors on excitatory transmission in substantia gelatinosa (SG) using intracellular recordings from SG neurons in transverse slices of the young rat lumbar spinal cord. Monosynaptic and polysynaptic excitatory postsynaptic potentials (EPSPs) were evoked by orthodromic electrical stimulation of A delta or C primary afferent fibers in the dorsal root after blocking inhibitory inputs with bicuculline and strychnine, NMDA receptors with D-2-amino-5- phosphonovaleric acid and mu- and delta-opioid receptors with CTAP and ICI 174,864, respectively. Bath application of dynorphin A1-17 or U-69, 593 caused dual modulation of the peak amplitude of presumed monosynaptic AMPA receptor-mediated EPSPs, decreasing synaptic potentials at nanomolar concentrations in a majority of SG cells examined (dynorphin, 63%; U-69,593, 91%), and increasing EPSPs at micromolar concentrations. Only the inhibitory action of dynorphin A1- 17 was consistently and completely blocked by norbinaltorphimine (nor- BNI). Since U-69,593 and nor-BNI are selective for the kappa 1-opioid receptors, the depression of EPSPs is likely to be mediated by the kappa1-opioid receptors. Under conditions of blockade of synaptic transmission with TTX and mu-and delta-opioid receptors, dynorphin A1- 17 and U-69,593 hyperpolarize most of SG neurons and decrease their membrane input resistance, the finding suggesting that direct interaction of kappa-agonists with a postsynaptic receptor is likely explanation for the inhibition of EPSPs. However, in some SG cells, the inhibition of EPSPs appears to be of presynaptic origin since dynorphin A1-17 and U-69,593 did depress the EPSPs in the absence of changes in passive membrane properties. Rp-cAMPS, a membrane permeant potent competitive inhibitor of cAMP-activated protein kinase, prevented the depressant effect of dynorphin A 1-17. This finding suggested a possibility that dynorphin A1-17, acting through a decrease in intracellular cyclic AMP levels, can reduce the synaptic responses of SG neurons. These results provide the first electrophysiological demonstration that the activation of kappa 1-opioid receptors inhibits AMPA receptor-mediated primary afferent neurotransmission in the substantia gelatinosa of the young rat spinal cord. This effect may mediate the ability of kappa-receptor agonists to produce antinociception.
Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans
Authors: Weinshenker D , Garriga G , Thomas JH
Published: April 2018

We have investigated the neurotransmitters used to control egg-laying in C. elegans. Previous studies suggested that 5-HT released by the HSN motor neurons stimulates egg laying, and that tricyclic antidepressants potentiate egg laying by blocking reuptake of 5-HT by the HSN neurons. We report studies of the wild type and a mutant that lacks detectable 5- HT that suggest 5-HT is not required for egg-laying. Furthermore, we find that ACh is required for egg laying in response to 5-HT, suggesting that 5-HT is not sufficient to activate egg laying. The dominant egl-2(n693) mutation, which causes animals to lay eggs in response to tricyclics but not 5-HT, also conflicts with the model for egg laying. Experiments in which the HSN neurons or 5-HT are removed from egl-2 animals indicate that the action of tricyclics cannot be explained by a block of 5-HT reuptake. We find that D2 family dopamine antagonists can also induce egg laying in egl-2(n693) mutants, and that dopamine inhibits egg laying in the wild type. These results suggest that dominant egl-2 mutations activate an inhibitory dopaminergic pathway that can be blocked by tricyclics and D2 antagonists. We also find that these drugs stimulate egg laying in mutants lacking 5-HT or the HSN neurons, consistent with a target on the egg-laying muscles. In contrast to tricyclics, fluoxetine and other selective 5-HT reuptake inhibitors appear to be specific for 5-HT reuptake in C. elegans egg laying.
Cholinergic, GABAergic, and glutamate-enriched inputs from the mesopontine tegmentum to the subthalamic nucleus in the rat
Authors: Bevan MD , Bolam JP
Published: April 2018

In order to clarify the origin and to examine the neurochemistry and synaptology of the projection from the mesopontine tegmentum (MTg) to the subthalamic nucleus (STN), rats received discrete deposits of anterograde tracers in different regions of the MTg. Anterogradely labeled fibers were examined in the light and electron microscopes. The distribution of GABA or glutamate immunoreactivity was examined by post- embedding immunocytochemistry. The anterograde tracing demonstrated that the projection to the STN arises from at least three divisions of the MTg: the area defined by the cholinergic neurons of the pedunculopontine region (PPN-Ch 5), the more medial and largely noncholinergic midbrain extrapyramidal area (MEA) and to a lesser extent the laterodorsal tegmental nucleus (LDTg). Post-embedding immunocytochemistry revealed that there are GABA-immunopositive and immunonegative components to this projection and at least a proportion of the GABA-immunonegative component is enriched in glutamate immunoreactivity. The similarity of the morphology, trajectory and synaptology of the anterogradely labeled fibers and the choline acetyltransferase (ChAT)-immunopositive fibers supports the proposal that at least part of the projection is cholinergic. The terminals anterogradely labeled from the MTg and the ChAT-immunoreactive terminals form asymmetrical synapses with the dendrites and spines of subthalamic neurons. Both anterogradely labeled and ChAT-positive terminals make convergent synaptic contacts with GABA-immunoreactive terminals that form symmetrical synaptic contacts and are probably derived from the globus pallidus. Taken together these findings imply that the MTg sends cholinergic, GABAergic and glutamatergic projections to the STN where at least one of the functional roles is to modulate the indirect pathway of information flow through the basal ganglia that is carried via the pallidosubthalamic projection.
Purinergic receptors and their activation by endogenous purines at perisynaptic glial cells of the frog neuromuscular junction
Authors: Robitaille R
Published: April 2018

Glial cells are closely associated with synapses and are modulated by neurotransmitters released during synaptic transmission. At many synapses, ATP is released during synaptic transmission and is involved in cell-cell signaling. Since glial cells have purinoceptors, it is possible that ATP mediates synaptic neuron-glia signaling. This work aims at determining which types of purinoceptors are present on perisynaptic Schwann cells, the perisynaptic glial cells at the frog neuromuscular junction, and test their sensitivity to endogenous purines by monitoring the relative changes of intracellular Ca2+. Local application of ATP induced the release of Ca2+ from internal stores. Adenosine induced Ca2+ responses that were blocked by A1 receptor antagonists and mimicked by an A1 receptor agonist and were caused by the release of Ca2+ from internal stores via a pertussis toxin- sensitive G-protein. A2 receptor antagonists had no effect on Ca2+ responses induced by adenosine. Me-S-ATP, an ATP analog, triggered Ca2+ release from internal stores via a pertussis toxin-sensitive G-protein, consistent with the activation of P2Y receptors. L-AMP-PCP, another ATP analog, induced Ca2+ entry mainly through L-type Ca2+ channels by a pertussis toxin-insensitive mechanism, consistent with the activation of P2X receptors. Blockade of adenosine receptors did not affect glial Ca2+ responses induced by nerve evoked transmitter release. However, blockade of ATP receptors reduced the size and increased the delay of the responses. Hence, purinoceptors are present on the perisynaptic Schwann cells and are activated by endogenous ATP released during synaptic transmission.
Local modulation of hippocampal acetylcholine release by dopamine D1 receptors: a combined receptor autoradiography and in vivo dialysis study
Authors: Hersi AI , Richard JW , Gaudreau P , Quirion R
Published: April 2018

The modulation of in vivo hippocampal ACh release by dopaminergic D1 and D2 receptors was examined in this study. Additionally, in an attempt to ascertain the location of these receptors in relation to hippocampal cholinergic terminals, fimbriaectomy and quantitative autoradiography were used. Following unilateral fimbriaectomy, whereby at least 50% of hippocampal cholineacetyltransferase (ChAT) activity was lost, a significant ipsilateral decrease in D1/3H SCH23390 binding was observed in the molecular layer of the dentate gyrus while hippocampal D2/3H raclopride binding was unaffected. The effects of prototypical D1 and D2 receptor agonists and antagonists on hippocampal ACh release were examined next using in vivo dialysis in freely moving rats. The D1 agonist SKF 38393 (10 microM to 100 microM) administered directly into the hippocampus via the dialysis probe stimulated ACh release in a concentration dependent manner. The effect of the agonist was blocked by the coadministration of the D1 receptor antagonist SCH 23390 (1 microM), which by itself failed to modulate ACh release. In contrast, neither the D2 agonist quinpirole (1–10 microM) nor the D2 antagonist sulpiride (1–10 microM) had any direct effect on hippocampal ACh release. Additionally, the infusion of these D1 and D2 drugs in the septal area failed to affect hippocampal ACh release. Taken together, these results suggest that a proportion of hippocampal D1 receptors are located on cholinergic nerve terminals and that dopamine, acting via D1 receptors, can locally stimulate hippocampal ACh release.
Stress-induced sensitization and glucocorticoids. I. Sensitization of dopamine-dependent locomotor effects of amphetamine and morphine depends on stress-induced corticosterone secretion
Authors: Deroche V , Marinelli M , Maccari S , Le Moal M , Simon H , Piazza PV
Published: April 2018

Repeated exposures to stress sensitize motor and addictive effects of drugs of abuse. Recently, it has been shown that stress-induced behavioral sensitization depends on the secretion of glucocorticoids. We investigated if sensitization of dopamine-dependent effects of psychostimulants and opioids was influenced by glucocorticoid. Sensitization of the dopaminergic response to drugs is considered the neural substrate of behavioral sensitization and has been implicated in vulnerability to drug abuse. Dopamine-dependent effects of psychostimulants and opioids were evaluated by injecting either amphetamine into the nucleus accumbens (10 micrograms/side) or morphine into the ventral tegmental area (VTA) (1 microgram/side). The locomotor response to psychostimulants and opioids injected in these brain areas depends on the mesencephalic dopaminergic transmission. Drug-induced locomotion was compared in male rats in which corticosterone secretion was either in +tct or experimentally suppressed by an adrenalectomy associated with a substitutive treatment reproducing basal levels of the hormone. Eight days of food restriction (80% of the initial body weight) were used as a stressor. Suppression of stress-induced corticosterone secretion abolished food restriction-induced sensitization of the locomotor effects of intra-accumbens amphetamine and intra-VTA morphine. This effect was corticosterone dependent since the restoration of corticosterone levels in the range of those induced by stress totally reinstates sensitization. Our results suggest that glucocorticoids control stress-induced sensitization by changing the sensitivity of the mesencephalic dopaminergic transmission to drugs of abuse. Since dopaminergic effects of drugs are related to their addictive properties, secretion of glucocorticoids may be one of the factors determining the enhanced vulnerability to drugs observed in stressed subjects.
A comparison of the effects of medial prefrontal, cingulate cortex, and cingulum bundle lesions on tests of spatial memory: evidence of a double dissociation between frontal and cingulum bundle contributions
Authors: Aggleton JP , Neave N , Nagle S , Sahgal A
Published: April 2018

Rats were trained on an automated delayed nonmatching-to-position (DNMP) task. They then received cytotoxic lesions in either the medial prefrontal cortex (n = 13) or the cingulate and retrosplenial cortices (n = 8), or radiofrequency lesions in either the fornix (n = 6) or the cingulum bundle (n = 8). Twelve animals served as surgical controls. Only the fornical and medial prefrontal lesions disrupted DNMP performance, both groups showing a loss of accuracy and an increase in bias. The rats were then trained on a lever discrimination and reversal task, the medial prefrontal and fornical groups showing evidence of an increase in bias when compared with the cingulate cortex group. Finally, the rats were trained on a forced alternation task in a T- maze. Marked deficits were observed in the fornix and cingulum bundle groups, but the medial prefrontal and cingulate groups were unimpaired. The double dissociation between the effects of the prefrontal and cingulum bundle lesions highlights the very different nature of the two spatial tasks (DNMP and T-maze alternation), even though both involved a nonmatching rule. These findings may reflect the involvement of divergent outputs from the fornix-anterior thalamic pathway. One possibility is that anterior thalamic projections to the medial prefrontal cortex are concerned with processing egocentric information, while anterior thalamic projections to temporal regions via the cingulum bundle are concerned with allocentric information. The results also indicate that the effects of conventional lesions in the cingulate cortex and medial prefrontal cortex may be compromised by additional damage to the cingulum bundle.
Age-associated impairments in a test of attention: evidence for involvement of cholinergic systems
Authors: Jones DN , Barnes JC , Kirkby DL , Higgins GA
Published: April 2018

We trained three groups of rats, young (Y; 3 months at the start of study), middle aged (MA; 15 months), and aged (AG; 22 months), in the serial five-choice serial reaction time task, a test of attention. There were clear age-related differences in task acquisition Y acquired the task quicker than MA rats, which learned faster than AG rats. A subgroup of AG rats [AG(I)] could not reach criterion (> 80% correct, < 20% omissions under standard conditions of 0.5 sec stimulus duration, 5 sec limited hold). Accordingly, they were tested under conditions of 1 sec stimulus duration. Having acquired the task, under standard conditions both MA and AG groups were slower to make a correct response but not to collect the food reward. Furthermore, parameter changes, particularly reductions in stimulus duration and intensity, revealed further age-related changes in accuracy. Following completion of these studies, animals were trained in a simpler one-choice task. Importantly, reducing stimulus duration/intensity in this task revealed no differences between Y and MA/AG groups, although AG(I) rats were impaired. This dissociation between MA/AG impairments in the one- and five-choice task suggests that these animals may show attentional deficits compared with Y rats, which are independent of changes in sensory (visual), motor function, or motivation. Finally, the MA deficit in attention was partially reversed by tacrine pretreatment (3 mg/kg). Also scopolamine (0.01–0.075 mg/kg) and mecamylamine (0.3–5 mg/kg) pretreatment impaired choice accuracy of MA but not Y rats. Taken together, the drug studies imply that the attentional deficits may at least be partially due to changes in cholinergic function.
Disruption of cerebellar maturation by an antimitotic agent impairs the ontogeny of eyeblink conditioning in rats
Authors: Freeman JH , Barone S , Stanton ME
Published: April 2018

This study represents an attempt to establish a relationship between maturation of the cerebellum and the ontogeny of eyeblink conditioning in the rat. Experiments 1 and 2 examined the effects of disrupting cerebellar maturation by neonatal exposure to the antimitotic agent methylazoxymethanol (MAM) on the ontogeny of eyeblink conditioning in infant rats. Experiment 1 demonstrated that neonatal exposure to MAM on Postnatal Day 4 (PND4) and 7 severely disrupted cerebellar maturation. This effect appeared to be specific in that there was no overt dysmorphology in other brain regions. MAM treatment also severely disrupted associative eyeblink conditioning in rats given training on PND24 and 25. However, exposure to MAM had no effect on the unconditioned response, T-maze delayed alternation, or conditioned suppression of ongoing behavior. In Experiment 2, MAM was given on PND4 and 7 and pups were tested behaviorally on PND17–18, 20–21, or 31–32. Cerebellar hypoplasia was most dramatic shortly after exposure. The cerebellar cortex continued to mature after exposure to MAM, but development of morphological endpoints examined here were static from PND19 to 33. Eyeblink conditioning was impaired at all ages, indicating that there was no functional recovery following neonatal exposure to MAM over the age range tested. These experiments suggest that normal cerebellar maturation may be important for the ontogeny of eyeblink conditioning.
Chronic cocaine exposure in the fetal rhesus monkey: consequences for early development of dopamine neurons
Authors: Ronnekleiv OK , Naylor BR
Published: April 2018

The effects of cocaine on the development of neurons containing tyrosine hydroxylase (TH) were investigated in fetal rhesus macaques. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of pregnancy until days 40 or 60. Cocaine concentration in plasma from females thus treated was approximately 800 ng/ml 10–20 min following injection. At the time of C-section, plasma levels of cocaine in fetal blood were 231 +/- 70 ng/ml. The brains from 40 and 60 d old fetuses were examined using immunocytochemistry and in situ hybridization. The appearance of neurons containing TH by day 40 of gestation was not different between control and cocaine-treated fetal monkeys. In both groups (N = 3 each) TH-IR neurons and TH mRNA were located in the mesencephalon and dorsal hypothalamus by day 40, and fiber projections extended to the developing striatum. Also in the day 60 fetuses, the TH-IR neurons were distributed similarly in both groups (N = 5 each), but the TH mRNA content, measured by quantitative in situ hybridization, was reduced in the substantia nigra (SN) and ventral tegmental area (VTA) after cocaine treatment. These data suggest that exposure to cocaine in fetal life does not affect the development of TH or the expression of its mRNA on day 40 of gestation. By day 60, however, the expression of TH mRNA was significantly reduced. This latter effect can be explained by reduced dopamine synthesis in the cocaine-treated fetuses.
In vivo actions of insulin-like growth factor-I (IGF-I) on brain myelination: studies of IGF-I and IGF binding protein-1 (IGFBP-1) transgenic mice
Authors: Ye P , Carson J , D'Ercole AJ
Published: April 2018

To study the effects and mechanisms of insulin-like growth factor I (IGF-I) on brain myelination in vivo, the morphology of myelinated axons and the expression of myelin specific protein genes have been examined in transgenic (Tg) mice that overexpress IGF-I and that those ectopically express IGF binding protein-1 (IGFBP-1), a protein that inhibits IGF-I actions when present in molar excess. Our data show that the percentage of myelinated axons and the thickness of myelin sheaths are significantly increased in IGF-I Tg and decreased in the IGFBP-1 mice. Cerebral cortical proteolipid protein (PLP) and myelin basic protein (MBP) mRNAs consistently exhibit approximately 200% increases in IGF-I Tg mice and approximately 50% decreases in IGFBP-1 Tg mice. The percentage of oligodendrocytes labeled with a PLP cRNA probe in the corpus callosum and cerebral cortex also is increased in IGF-I Tg mice and reduced in IGFBP-1 Tg mice, suggesting that IGF-I promotes oligodendrocyte survival and/or proliferation. The alterations in the number of oligodendrocytes, however, can not completely account for the changes in myelin gene expression. These results strongly indicate that IGF-I increases myelination by increasing the number of myelinated axons and the thickness of myelin sheaths, the latter by mechanisms that involve stimulation of the expression of myelin protein genes and increase of oligodendrocyte number.
A timetable of events during programmed cell death induced by trophic factor withdrawal from neuronal PC12 cells
Authors: Mesner PW , Epting CL , Hegarty JL , Green SH
Published: April 2018

We describe a timetable of events during programmed cell death (PCD) in neuronal PC12 cells, specifically, Ras signaling, immediate-early gene (IEG) expression, DNA fragmentation and commitment to PCD. Commitment occurs over a period from 10–20 hr after NGF withdrawal. Ras signaling declines rapidly after NGF removal, reaching minimal levels within 2–4 hr, well before the onset of commitment. DNA fragmentation, detected by TUNEL reaction, begins about 24 hr after NGF withdrawal, well after all cells are committed, but coincident with the onset of cell dissolution previously determined by trypan blue exclusion (Mesner et al., 1992). Among the IEGs studied here, c-jun and TIS21 are expressed within 6 hr after NGF withdrawal. Expression of c-fos, egr-1, and TIS11 does not begin until 20 hr after NGF withdrawal. IEG expression generally ends by 24 hr after NGF withdrawal. The IEGs TIS7 and nur77 are not expressed during PCD, yielding a pattern distinct from that following other stimuli. An identical pattern of IEG expression occurs in non- neuronal PC12 cells deprived of serum, although expression begins at 10– 14 hr after serum withdrawal. A similar IEG expression pattern was observed in Rat-1 fibroblasts, with various genes expressed 6–18 hr after serum withdrawal. In none of these cell types did expression of the stress-related gene Hsp70 change following trophic factor withdrawal. The distinctive pattern of IEG expression described here should facilitate identification of intracellular regulatory signals active during PCD.
Regulated secretion of beta-amyloid precursor protein in rat brain
Authors: Farber SA , Nitsch RM , Schulz JG , Wurtman RJ
Published: April 2018

The beta-amyloid precursor protein (APP) is a ubiquitous, highly conserved secretory glycoprotein that is expressed at high levels in mammalian brain by neurons, astrocytes, and activated microglia. Secreted APP (APPs) is generated by the cleavage of APP within the beta- amyloid (A beta) portion of its ectodomain. The formation and secretion of APPs can be increased by activation of particular neurotransmitter receptors and subsequent protein phosphorylation. We found that tissue slices from rat cortex, hippocampus, striatum, and cerebellum secrete APPs in vitro. APPs secretion was enhanced by electrical stimulation, but was not associated with a general increase in the release of total protein, lactate dehydrogenase (LDH) activity, or neuronal cell adhesion molecules. The pharmacological profile of stimulation-induced APPs secretion suggests complex interactions between muscarinic receptor subtypes in the tissue slices: in the unstimulated state, activation of Muscarinic M1 receptors increased APPs release while nonspecific activation of multiple muscarinic receptors had little effect on APPs release; in electrically stimulated slices, nonspecific inhibition of muscarinic receptors blunted the increase in APPs secretion. The nonspecific muscarinic agonist carbachol increased APPs secretion only in the presence of an M2 receptor antagonist, suggesting that activation of M2 receptors suppresses APPs formation. These data indicate that secretory APP processing in brain includes depolarization- enhanced cleavage of the cell-associated holoprotein within its ectodomain, and that the net effect of depolorization involves several subtypes of acetylcholine receptors.
Pharmacological characterization of the rhythmic synaptic drive onto lumbosacral motoneurons in the chick embryo spinal cord
Authors: Sernagor E , Chub N , Ritter A , O'Donovan MJ
Published: April 2018

The isolated spinal cord of the chick embryo generates episodes of rhythmic bursting in which sartorius (hip flexor) and femorotibialis (knee extensor) motoneurons exhibit characteristic patterns of activity. At the beginning of each cycle both sets of motoneurons discharge synchronously. Following this brief synchronous activation sartorius motoneurons stop firing at the time of peak femorotibialis activity, producing a period of alternation between the two sets of motoneurons. Intracellular recording from motoneurons has suggested that the pause is mediated by a synaptically induced shunt conductance. However, the pharmacological basis for this shunt and the nature of the excitatory drive to motoneurons is unknown. To address these questions we have investigated the pharmacology of the rhythmic, synaptic drive to lumbosacral motoneurons using local and bath application of several excitatory and inhibitory antagonists, and documenting their effects on motor output in E10-E12 chick embryos. Local application of bicuculline or picrotoxin over sartorius motoneurons abolished the pause in firing recorded from the sartorius muscle nerve. As a consequence, the pattern of sartorius and femorotibialis activity was similar and the motoneurons were coactive. The pause in sartorius firing was shortened following local application of the glycine antagonist strychnine the nicotinic, cholinergic antagonists mecamylamine, and dihydro-beta- erythroidine and several excitatory amino acid antagonists. Application of the GABA uptake inhibitor nipecotic acid depressed the slow potentials and discharge recorded from the sartorius muscle nerve. These findings suggest that the pause is determined primarily by synaptic inputs acting at motoneuron GABAA receptors with contributions from glycinergic, cholinergic, and glutamatergic inputs. The actions of locally applied GABA onto spinal neurons are consistent with these findings because the neurotransmitter depolarizes spinal neurons and reduces their input resistance. Local application of bicuculline, but not strychnine, onto segments containing femorotibialis motoneurons altered the amplitude and duration of femorotibialis discharge and changed the profile of the slow potentials recorded from the muscle nerve. This finding implicates GABAergic inputs in the regulation of femorotibialis discharge. The pause in sartorius firing was still present and a pause in firing appeared in each cycle of femorotibialis discharge following bath application of bicuculline or strychnine. The pause in both sets of motoneurons could be abolished by local application of the NMDA receptor antagonist AP-5 onto the motoneurons, but not by local application of bicuculline. This action of AP-5 was in contrast to its activity in normal Tyrode's solution where it shortened the pause slightly.(ABSTRACT TRUNCATED AT 400 WORDS)
The immunosuppressant FK506 increases the rate of axonal regeneration in rat sciatic nerve
Authors: Gold BG , Katoh K , Storm-Dickerson T
Published: April 2018

The axonal regenerative properties of the new immunosuppressant drug FK506 (tacrolimus) are further explored in this continuing study. In an initial report (Gold et al., 1994a), we described the ability of FK506 to reduce the time until return of function in the hind feet of rats following a sciatic nerve crush. In the present study, we examined the morphological correlate underlying this enhancement of functional recovery. In rats receiving daily subcutaneous injections of FK506 (1.0 mg/kg) for 18 d following a sciatic nerve crush the regenerating axons appeared larger in size compared to saline-injected control animals. Morphometric analysis of axonal calibers in the soleus nerve demonstrated that mean axonal areas for the largest 30% of axons were increased over axotomized control values by 93% in the FK506-treated animals. Next, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion (DRG) at 9 and 14 d following axotomy. Regression analysis of the outgrowth distances for sensory axons between 10 and 15 d revealed a 16% increase in regeneration rate. Electron microscopy of intramuscular nerve branches in the interosseus muscles confirmed that the axons in the FK506-treated animals were further advanced toward their targets; in some instances, axons were shown to reinnervate muscle spindles. The results are discussed in terms of the known ability of FK506 to inhibit the activity protein phosphatase 2B (calcineurin).
Transient versus persistent functional and structural changes associated with facilitation of Aplysia sensorimotor synapses are second messenger dependent
Authors: Wu F , Friedman L , Schacher S
Published: April 2018

Increases in activity of both protein kinase A (PKA) and protein kinase C (PKC) contribute to short-term facilitation of Aplysia sensorimotor synapses evoked by serotonin (5-HT). We report here that increasing levels of cAMP in sensory neurons evokes increases in both synaptic efficacy and in the number of sensory neuron varicosities contacting the major axons of motor cell L7 at intermediate times (3 hr) that persist for 24 hr. Treatment with phorbol esters results in a large transient increase in synaptic efficacy that is accompanied by a large transient increase in the number of sensory neuron varicosities with the newest varicosities most susceptible to elimination. The reversal of the synaptic facilitation and the structural changes does not appear to be the result of long-term inhibitory actions of persistent PKC activation by phorbol esters, since changes in synaptic efficacy can be evoked by additional applications of either phorbol esters or 5-HT. The short-lived changes in structure evoked by phorbol esters occur in preexisting sensory neurites and not by new growth, since increases in PKC activity with phorbol esters lead to reductions in neurite extension and to retractions by sensory neuron growth cones. The action of phorbol esters on growth cone extension is reversible with washout. The results suggest that increases in PKA and PKC activities by 5-HT contribute to short (minutes) and intermediate (hours) forms of facilitation of sensorimotor synapses while increases in PKA activity also mediate long-term (days) maintenance of synaptic facilitation.
Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments
Authors: Nibuya M , Morinobu S , Duman RS
Published: April 2018

The influence of chronic electroconvulsive seizure (ECS) or antidepressant drug treatments on expression of brain-derived neurotrophic factor (BDNF) and its receptor, trkB, was examined by in situ hybridization and Northern blot. In frontal cortex, acute ECS increased BDNF mRNA approximately twofold, an effect significantly augmented by a prior course of chronic ECS treatment (10 d). In the hippocampus, the influence of chronic ECS varied between the major subfields. In the dentate gyrus granule cell layer, chronic ECS decreased the acute induction of BDNF and trkB mRNA by approximately 50%, but prolonged their expression: levels remained elevated two- to threefold 18 hr later after the last chronic ECS treatment, but returned to control 18 hr after acute ECS. In CA3 and CA1 pyramidal cell layers, chronic ECS significantly elevated the acute induction of BDNF, and tended to prolong the expression of BDNF and trkB mRNA. A similar effect was observed in layer 2 of the piriform cortex, where chronic ECS significantly increased the acute induction and prolonged the expression of BDNF and trkB mRNA. Chronic (21 d), but not acute (1 d), administration of several different antidepressant drugs, including tranylcypromine, sertraline, desipramine, or mianserin, significantly increased BDNF mRNA and all but mianserin increased trkB mRNA in hippocampus. In contrast, chronic administration of nonantidepressant psychotropic drugs, including morphine, cocaine, or haloperidol, did not increase levels of BDNF mRNA. Furthermore, chronic administration of ECS or antidepressant drugs completely blocked the down-regulation of BDNF mRNA in the hippocampus in response to restraint stress. The enhanced induction and prolonged expression of BDNF in response to chronic ECS and antidepressant drug treatments could promote neuronal survival, and protect neurons from the damaging effects of stress.
Cloning and characterization of two related serotonergic receptors from the brain and the reproductive system of Aplysia that activate phospholipase C
Authors: Li XC , Giot JF , Kuhl D , Hen R , Kandel ER
Published: April 2018

Serotonin (5-HT) plays important roles in various behavioral and physiological processes in Aplysia californica. These include feeding, locomotion, circadian rhythm, learning and memory, synaptic plasticity, and synaptic growth. Serotonin modulates these various functions by interacting with different 5-HT receptor subtypes that are coupled to various second-messenger systems. We report here the isolation and characterization of the first two serotonergic receptors from Aplysia californica, Ap5-HTB1 and Ap5-HTB2, using a strategy based on the amino acid sequence homology among G-protein-coupled biogenic amine receptors. Ap5-HTB1 and Ap5-HTB2 are both intronless and highly homologous to each other, sharing 79.5% sequence identity at the amino acid level. Sequence comparison reveals that these receptors are 33.1 to 23.3% identical to the following 5-HT receptors: 5-HTdro1 > 5-HT6 > 5-HTlym > mouse 5-HT1B > 5-HTdro2A > mouse 5-HT7 > rat 5-HT2A. Both Ap5- HTB1 and Ap5-HTB2 encode functional 5-HT receptors. When expressed in cultured cells, these receptors stimulate phospholipase C in response to 5-HT in a dose-dependent manner. This stimulation can be blocked by specific 5-HT receptor antagonists. Using RT-PCR and Western blot analysis, we have detected these receptors in the CNS (Ap5-HTB2) and in the reproductive system (Ap5-HTB1). The nucleotide sequences of Ap5- HTB1 and Ap5-HTB2 were submitted to GenBank; the accession numbers areL43557andL43558, respectively.
Islet amyloid polypeptide (amylin) is expressed in sensory neurons
Authors: Mulder H , Leckstrom A , Uddman R , Ekblad E , Westermark P , Sundler F
Published: April 2018

Islet amyloid polypeptide (IAPP) or amylin is a hormone candidate predominantly expressed in insulin cells. A role for IAPP in the regulation of glucose homeostasis and the development of non-insulin- dependent diabetes mellitus has been proposed. IAPP is structurally related to the sensory neuropeptide calcitonin gene-related peptide. In the present study, using in situ hybridization, immunocytochemistry, and immunochemistry, the expression of IAPP in sensory neurons in the rat was investigated. IAPP was expressed in a population of small- to medium-sized nerve cell bodies in dorsal root ganglia from all levels and in the jugular-nodose and trigeminal ganglion; IAPP-expressing nerve cell bodies constituted a subpopulation of those expressing calcitonin gene-related peptide. In addition, IAPP-like immunoreactivity occurred in nerve cell bodies storing substance P and pituitary adenylate cyclase-activating polypeptide. IAPP-immunoreactive nerve fibers were encountered in the dorsal horns of the spinal cord, and to a lesser extent in peripheral tissues receiving sensory innervation; IAPP-immunoreactive fibers constituted a subpopulation of those containing calcitonin gene-related peptide and/or substance P. The immunochemical determinations demonstrated a low level of IAPP-like immunoreactivity in the dorsal root ganglia and spinal cord, which chromatographically coeluted with authentic rat IAPP. We conclude that IAPP is expressed in sensory neurons, thus being a novel sensory neuropeptide candidate for which a physiological role remains to be identified.
NMDA-mediated modulation of gamma-aminobutyric acid type A receptor function in cerebellar granule neurons
Authors: Zhu WJ , Vicini S , Harris BT , Grayson DR
Published: April 2018

GABAA receptors are ligand-gated CI- ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABAA receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited CI- currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 microM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated CI- currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the Bmax associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn(2+)-induced inhibition of GABA-gated CI- currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABAA receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.
Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures
Authors: Newell DW , Barth A , Papermaster V , Malouf AT
Published: April 2018

In vitro ischemia models have utilized oxygen, or oxygen and glucose deprivation to simulate ischemic neuronal injury. Combined oxygen and glucose deprivation can induce neuronal damage which is in part mediated through NMDA receptors. Severe oxygen deprivation alone however can cause neuronal injury which is not NMDA mediated. We tested the hypothesis that NMDA, or non-NMDA receptor mediated mechanisms may predominate, to induce neuronal injury following severe oxygen deprivation depending on the presence of glucose. We found that NMDA receptor blockade using dizocilpine (MK-801), DL-2-amino-5- phosphonovaleric acid (APV), or CGS 19755, was highly effective in reducing CA1 injury in organotypic hippocampal cultures, caused by complete oxygen and glucose deprivation. Complete oxygen deprivation alone however, caused CA1 neuronal injury which was not diminished using NMDA receptor blockade alone with MK-801 or APV, or in combination with AMPA/kainate receptor blockade using 6-cyano-7- dinitroquinoxalone-2,3-dione (CNQX). Neuronal protective strategies which act primarily through non-glutamate dependent mechanisms, including hypothermia, low chloride and calcium, and the free radical scavenger, alpha-phenyl-tert-butyl nitrone (PBN), provided neuronal protection against complete oxygen, as well as combined oxygen/glucose deprivation. Raising the pH using Hepes buffer during complete oxygen deprivation did not result in neuronal protection by NMDA receptor blockade. Partial oxygen deprivation alone, partial oxygen deprivation combined with glucose deprivation, glucose deprivation alone, and also glutamate exposure, all produced neuronal damage that was reduced by NMDA receptor blockade. The presence of glucose during complete oxygen deprivation appears to prevent glutamate receptor blockade from reducing neuronal injury in organotypic hippocampal cultures.
GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex
Authors: Minelli A , Brecha NC , Karschin C , DeBiasi S , Conti F
Published: April 2018

High affinity, GABA plasma membrane transporters influence the action of GABA, the main inhibitory neurotransmitter. The cellular expression of GAT-1, a prominent GABA transporter, has been investigated in the cerebral cortex of adult rats using in situ hybridization with 35S- labeled RNA probes and immunocytochemistry with affinity purified polyclonal antibodies directed to the C-terminus of rat GAT-1. GAT-1 mRNA was observed in numerous neurons and in some glial cells. Double- labeling experiments were performed to compare the pattern of GAT-1 mRNA containing and GAD67 immunoreactive cells. The majority of neurons expressing GAT-1 mRNA also contained GAD67 immunoreactivity (ir), but GAT-1 mRNA was also observed in a few pyramidal neurons. GAT-1-ir was localized to numerous puncta and fibers and to astrocytic processes, was not observed in sections incubated in GAT-1 antibodies preadsorbed with rat GAT-1 C-terminal peptide, and was observed in sections incubated in GAT-1 antibodies preadsorbed with the C-terminal portion of the related peptides rat GAT-3(607–627) or rat glycine transporter- 1(625–633). The highest number of GAT-1-ir puncta was in layer IV, followed by layers II-III. GAT-1 positive puncta appeared to have a preferential relationship to the soma and proximal dendrites of unlabeled pyramidal cells. All GAT-1 positive axon terminals formed symmetric synapses. This study demonstrates that (1) GAT-1 is expressed by both neurons and astrocytes, (2) the majority of GAT-1 expressing neurons contain GAD67, and (3) GAT-1 uptake system is more extensive than the GABA synthetizing system. These observations support the hypothesis that, in addition to its role in terminating GABA action by uptake into GABAergic axon terminals, GAT-1 influences both excitatory and inhibitory transmission by modulating the “paracrine” spread of GABA (Isaacson et al., 1993), and suggest that astrocytes may play an important role in this process.
Excitotoxic cell death and delayed rescue in human neurons derived from NT2 cells
Authors: Munir M , Lu L , Mcgonigle P
Published: April 2018

The excitotoxic response of NT2-N cells, a clonal line of human teratocarcinoma cells that are terminally differentiated into neuron- like cells, was examined using several endpoints. A 15 min exposure to glutamate produced a dose-dependent toxicity with a maximal cell loss of 80–90% in 6 week old cells. The rapidly triggered excitotoxicity induced by glutamate was blocked by NMDA selective antagonists, was calcium dependent and pH sensitive and could be mimicked by NMDA but not by non-NMDA agonists, AMPA, kainate or quisqualate. The non-NMDA agonists however caused toxicity on prolonged exposure. The NMDA receptor modulators glycine and spermidine enhanced glutamate-mediated toxicity whereas ifenprodil potently and completely inhibited toxicity suggesting that the toxic response is mediated by the NR1/NR2B combination of NMDA subunits. These cells can be rescued from death up to 1 hr after removal of glutamate by NMDA receptor blockade, removal of extracellular Ca2+ or lowering of pH. The extent of rescue is directly related to the time elapsed before intervention. Blockage of NMDA receptor activity for 1 hr immediately after removal of glutamate is both necessary and sufficient for complete rescue. Glutamate- mediated toxicity was not prevented by nitric oxide synthase inhibitors nor was nitric oxide synthase detected in NT2-N cells indicating that nitric oxide is not required for glutamate-mediated excitotoxicity. In summary, NT2-N cells exhibit a robust excitotoxic response and represent a novel model system in which to study the molecular basis of excitotoxic cell death.
Nerve injury enhances rat neuronal glutamate transporter expression: identification by differential display PCR
Authors: Kiryu S , Yao GL , Morita N , Kato H , Kiyama H
Published: April 2018

An increase in neuronal glutamate transporter expression after nerve injury was demonstrated by means of differential display PCR (DD-PCR) coupled with in situ hybridization. DD-PCR was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the rat. The expression of several gene fragments were found to be increased following nerve injury; the full length cDNA corresponding to one fragment was cloned by subsequent rat cDNA library screening. The close homology of glutamate transporters with our rat cDNA led us to conclude that this clone corresponds to the rat neuronal glutamate transporter (rat EAAC1). We speculate that the upregulation of this glutamate uptake system may increase the resistance of these cells against neurotoxic glutamate accumulation during the process of nerve regeneration.
Laminin alpha 2 chain (M chain) is found within the pathway of avian and murine retinal projections
Authors: Morissette N , Carbonetto S
Published: April 2018

Laminin-1 is found at the end-feet of neuroepithelial cells along the outer margin of the optic pathway during early stages of development. Prior to the establishment of most retinal projections in vivo, laminin- 1 expression becomes restricted to basement membranes associated with the eye and optic pathway. We report that, in contrast to the alpha 1, beta 1, and gamma 1 chains of laminin-1, laminin alpha 2 chain (formerly laminin M chain) is expressed within the pathway of avian and murine retinal ganglion cell (RGC) growth cones as they extend into the optic nerve, across the optic chiasm and into the brain. Expression of laminin alpha 2 chain is reduced soon after formation of the visual projections but nevertheless maintained at non-basal lamina sites within the adult optic nerve. Laminin alpha 2 chain, in contrast to laminin-1 chains, is also highly expressed in the developing avian tectobulbar pathway. Chick optic nerve derived type-1 astrocytes in culture express laminin alpha 2 chains as extracellular fibrils on their surface. Laminin alpha 2 chain was also detected on the surface of cultured embryonic retinal neurons and developing RGCs. These results suggest that astrocytes and/or RGCS may synthesize laminin alpha 2 chain along the developing optic pathway, and imply that laminin alpha2--in a complex with non-beta1 and non-gamma1 laminin chains-may serve as an adhesive substrate and possibly as a guidance cue for elongating RGC growth cones in vivo.