bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in cytoskeletal gene expression affect the composition of regenerating axonal sprouts elaborated by dorsal root ganglion neurons in vivo
Authors: Oblinger MM , Szumlas RA , Wong J , Liuzzi FJ
Published: March 2018

The effect of a change in neurofilament (NF) and tubulin gene expression on the elongation of axonal sprouts by adult rat sensory neurons was examined. Distal sciatic nerve crush axotomy was used to initiate changes in cytoskeletal gene expression in lumbar dorsal root ganglion (DRG) neurons. In situ hybridization of DRG neurons with 35S- labeled cDNA probes revealed a significant reduction in the level of mRNAs for the low-molecular weight-NF protein and a significant increase in the level of beta tubulin mRNAs by 2 weeks after axotomy. A novel modification of the axonal transport paradigm was used to examine the biochemical composition of the regenerating axons formed by primed and unprimed DRG neurons. Primed neurons (which had sustained a crush axotomy of the distal sciatic nerve 2 weeks earlier) and unprimed (normal) neurons were labeled by microinjection of 35S-methionine and then stimulated to regenerate axons by a crush located very close to the DRG. In this paradigm, axonal sprouts that formed after the proximal crush axotomy incorporated radiolabeled, slow axonally transported proteins as they elongated. Fluorographs of SDS-PAGE revealed that the regenerating axonal sprouts of primed DRG cells incorporated and conveyed significantly less labeled NF protein than did the regenerating axons of unprimed DRG neurons. Electron microscopy revealed that the regenerating axonal sprouts of primed DRG cells contained numerous microtubules but very few identifiable NFs compared with the regenerating sprouts of unprimed DRG neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
Extracellular cAMP accumulation and degradation in rat cerebral cortex in dissociated cell culture
Authors: Rosenberg PA , Dichter MA
Published: March 2018

Norepinephrine (NE) stimulated the accumulation of cAMP in embryonic rat cerebral cortex in dissociated cell culture. After exposure to NE for 10 min, the intracellular cAMP content of these cultures went from 22 +/- 12 to 202 +/- 75 pmol/mg protein. Using selective culturing techniques, evidence was obtained supporting the hypothesis that NE- stimulated production of cAMP is a property associated with the glial rather than the neuronal component of these cultures. Beta adrenergic agonist stimulation of cortical cultures also resulted in the efflux of cAMP into the medium. At the peak of extracellular accumulation of cAMP (following a 40-min exposure to isoproterenol), 180 pmol cAMP/mg protein had been transported into the extracellular medium. The fate of extracellular cAMP was investigated using thin-layer chromatography. Extracellular cAMP was degraded to AMP and adenosine; this degradation did not seem to be due to the presence of serum or serum components, suggesting the existence of an extracellular phosphodiesterase. In response to NE stimulation of glia, in particular astrocytes, cAMP or its metabolites may accumulate at high enough concentrations in the extracellular space in cerebral cortex to affect neuronal function, possibly via adenosine receptors.
Removal of the basal lamina in vivo reveals growth cone-basal lamina adhesive interactions and axonal tension in grasshopper embryos
Authors: Condic ML , Bentley D
Published: March 2018

The Ti1 afferent neurons are the first cells to undergo axonogenesis in embryonic grasshopper limbs. The Ti1 growth cones migrate between the limb epithelium and its basal lamina. We have investigated the nature of growth conebasal lamina interactions in vivo by removing the basal lamina with mild enzymatic digestion. Treatment with elastase, ficin, or papain removes the basal lamina when viewed in scanning electron microscopy. Trypsin and chymotrypsin leave the basal lamina intact. If the basal lamina is removed after the Ti1 growth cones have extended over intrasegmental epithelium but are not yet in contact with either differentiated segment boundaries or neurons, the growth cones retract to the cell somata. If the basal lamina is removed by elastase, and the Ti1 neurons are allowed to extend axons after treatment, a second elastase digestion does not cause the axons to retract. It is therefore unlikely that axon retraction is due to general proteolysis. These results suggest that if Ti1 growth cones have initially extended in the presence of an intact basal lamina, they are dependent on the lamina to remain extended over this region of the limb. The retraction of the Ti1 axons after removal of the basal lamina is inhibited by cytochalasin D, suggesting that microfilament-based cytoskeletal components underlie this event. This result indicates that the axons are under tension in vivo. The ability of the Ti1 growth cones to resist axonal tension suggests that adhesive interactions between the growth cones and the basal lamina underlie normal axon outgrowth in vivo.
Pioneer growth cone adhesion in vivo to boundary cells and neurons after enzymatic removal of basal lamina in grasshopper embryos
Authors: Condic ML , Bentley D
Published: March 2018

The Ti1 pioneer neurons of embryonic grasshopper limbs extend axons between the limb epithelium and its basal lamina. Their growth cones exhibit high affinity for both limb segment boundaries and immature neurons. We have investigated the role of the basal lamina in growth cone adhesion to neurons and segment boundaries by removing the basal lamina with mild enzymatic digestion when the Ti1 growth cones are in contact with different cellular substrates. If the basal lamina is removed when the Ti1 growth cones are in contact with other neurons, the growth cones remain in contact with the neuronal somata, and the Ti1 cell bodies in contact with the neuronal somata, and the Ti1 cell bodies reposition proximally. This suggests that the basal lamina is involved in the adhesion of the Ti1 somata to the substrate but not in growth cone-neuronal adhesion. This is the first direct evidence that growth cones establish adhesive cell-cell interactions with other neurons in vivo. Enzymatic treatments that remove the basal lamina also cause embryonic limbs to elongate. If the Ti1 axons are strongly apposed to 2 segment boundaries prior to protease treatment, their somata reposition to the nearest segment boundary, yet their axons do not retract off of the segment boundaries, despite severe stretching by the enzyme-induced limb expansion. These results indicate that the affinity of the Ti1 cells for segment boundaries is due at least in part to adhesive cell-cell interactions that are resistant to proteolytic digestion and independent of the basal lamina.
Alzheimer's disease antibodies bind specifically to a neurofilament protein in Torpedo cholinergic neurons
Authors: Chapman J , Bachar O , Korczyn AD , Wertman E , Michaelson DM
Published: March 2018

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and neuritic plaques and by the degeneration of central cholinergic neurons. Recent studies indicated the presence of antibodies in the sera and cerebrospinal fluid of AD patients which react with neuronal tissue and which recognize cholinergic neurons. In order to identify the cholinergic antigens against which the AD antibodies are directed, we have recently used the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogenous and cross- react antigenically with mammalian cholinergic neurons. This study revealed that immunoglobulins (IgG) from sera of AD patients bind specifically to an antigen in Torpedo electromotor neurons with an apparent molecular weight of 200 kDa. In the present report we attempt to characterize this antigen. The similarity in size of this protein to that of the heavy neurofilament subunit (NF-H) and the association of neurofilaments with plaques and tangles prompted us to examine the possibility that it is a neurofilament protein. Our findings show that IgG from sera of AD patients bind to the NF-H protein of Torpedo cholinergic neurons. Comparison of the binding of AD and control IgG to Torpedo cholinergic NF-H revealed that AD IgG bind to this neurofilament protein more readily than do control IgG. In contrast, AD and control IgG bind similarly to NF-H obtained from the chemically heterogenous Torpedo spinal cord and rat brain. These findings suggest that AD sera contain a repertoire of anti-NF-H IgG and that a subpopulation of these antibodies whose levels are significantly elevated in AD binds to epitopes highly enriched in Torpedo cholinergic NF-H.(ABSTRACT TRUNCATED AT 250 WORDS)
The organization of spatial coding in the hippocampus: a study of neural ensemble activity
Authors: Eichenbaum H , Wiener SI , Shapiro ML , Cohen NJ
Published: March 2018

Neural activity was recorded from local groups of hippocampal single units in rats performing a spatial-memory task. The organization of functional correlates in these neural ensembles was investigated by examining the spatial relationships among the place fields of single units in each ensemble. The distance and overlap between place fields were determined together with the tuning of cellular activity to behavioral variables, including direction, speed, and turning angle during movements within place fields. The place fields of recorded neural ensembles were significantly clustered: closer in space and considerably more overlapped than chance when compared statistically with Monte Carlo simulations. Just as single units often have significant firing in more than one distinct location in the environment (subfields), the ensembles had multiple and distinct clusters of overlapping subfields. In addition, proximity and overlap between place fields were significantly, but weakly, correlated with similarity in optimal movement tuning parameters. These results suggest that the hippocampus maintains a local organization with respect to place fields despite having no apparent large-scale isomorphism with the spatial environment. The organization of multiple, clustered place fields with correlated movement tuning properties in small neural ensembles suggests the existence of functional neural ensembles serving to encode multiple sensory and behavioral aspects of a place or event. Such an organization is similar to that observed for neocortical association areas afferent to the hippocampal system.
Stretch-activated ion channels in growth cones of snail neurons
Authors: Sigurdson WJ , Morris CE
Published: March 2018

Using single-channel recording, we show that neurons contain ion channels sensitive to membrane tension. Neurons isolated from the snail, Lymnaea stagnalis, actively rearborized in culture yielding cell bodies and growth cones suitable for patch clamping. All neurons contained, in both their soma and growth cones (at a density of approximately 1–2 micron-2), stretch-activated channels highly selective for K+. The presence of this mechanosensitive channel in the motile region of the neuron, a region characterized by insertion of new membrane--the growth cone--is of particular interest. Under physiological conditions, the channel was permeable to K+, but not to Na+ or Cl-. Its conductance to K+ under these conditions was approximately 44 pS. Channel activation was steeply dependent on membrane tension, showing thresholds at between -50 to -100 mm Hg (suction was applied through the recording pipette). Kinetic analysis indicated that the stretch-dependent increase in the channel's open probability was related to a long closed state rather than to one of the open states. Given the importance of Ca2+ in the regulation of growth cone motility, we speculate that this stretch-activated K+ channel could play a role in neurite elongation by a tension-dependent modulation of membrane voltage which in turn would act on voltage-gated Ca2+ channels.
GABA regulation of circadian responses to light. I. Involvement of GABAA-benzodiazepine and GABAB receptors
Authors: Ralph MR , Menaker M
Published: March 2018

Light-induced phase shifts of the circadian locomotor rhythm of hamsters can be blocked by agents that alter GABA neurotransmission. The GABA antagonist bicuculline blocks phase delays induced by light and the benzodiazepine diazepam, which can potentiate GABA activity, blocks light-induced phase advances. In the experiments reported here, we found that the bicuculline blockade of phase delays was reduced by agents that mimic or potentiate GABA activity. Conversely, the diazepam blockade of phase advances was reduced by both competitive and noncompetitive antagonists of GABA. This indicates that the GABA- benzodiazepine receptor-ionophore complex is the most likely site of action for the effects of these drugs on circadian rhythms. However, competitive GABA agonists did not mimic the blocking effects of benzodiazepines, nor did the antagonist picrotoxin mimic the blocking effect of bicuculline. Therefore, the classic action of GABA, increased chloride conductance, may not be the effector mechanism in this case. We also found that the GABAB agonist baclofen blocked both phase advances and delays and that the blockade of advances was reversed by the antagonist delta-aminovaleric acid. Taken together, these results indicate that GABA is involved in the regulation of circadian responses to light and that the regulation is mediated by both GABAA and GABAB receptors.
Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine
Authors: Budnik V , Wu CF , White K
Published: March 2018

The anatomy of peripheral serotonin-containing fibers (5-HT fibers) in the gut of wild-type Drosophila larvae was compared to mutants deficient in the gene that encodes the enzyme dopa decarboxylase (DfDdc mutants). The 5-HT fibers, located in the proventriculus and midgut, were visualized immunocytochemically by using a monoclonal antibody against 5-HT. Since DfDdc larvae are devoid of 5-HT and dopamine in the nervous system, the highly selective uptake capability of 5-HT neurons was used to visualize the 5-HT fibers. We found that the absence of 5- HT and dopamine in the nervous system of DfDdc animals does not prevent 5-HT fibers from reaching their appropriate targets. However, these fibers in the mutant show a 2-fold increase in the extent of branching. This effect is specific to 5-HT fibers, since glutamate-like and FMRFamide-like immunoreactive fibers of the proventriculus and midgut remain unaffected in the mutant. Low but detectable levels of dopamine and 5-HT in the CNS are sufficient to prevent the increase in arborization, as indicated by analyses of a temperature-sensitive Ddc allele (Ddcts2), which has very low dopa decarboxylase activity. The abnormally extensive branching of 5-HT fibers also can be partially rescued by feeding DfDdc larvae with dopamine. In contrast, feeding with a 5-HT-containing diet had no effect on the mutant phenotype. Hypotheses that could explain the mutant phenotype are proposed.
Synaptic organization of individual neurons in the macaque lateral geniculate nucleus
Authors: Wilson JR
Published: March 2018

Parvocellular and magnocellular neurons in the dorsal lateral geniculate nucleus of macaque monkeys were recorded electrophysiologically and then injected with HRP. The injected neurons were examined with the electron microscope. Synaptic terminals contacting the dendrites of individual neurons were classified and the synapses counted to estimate the number and distribution of each type over the entire dendritic tree. Seven parvocellular and 2 magnocellular neurons were analyzed. Two of the parvocellular neurons had presynaptic dendrites and no axons. These interneurons had electro-physiological characteristics much like those of relay neurons with the exception that their receptive field center responses had the opposite sign; i.e., they had OFF centers, while most neurons around them had ON centers. All of the relay neurons had similar types and distributions of terminal contacts. However, the distribution of each synaptic type along the dendrites of an individual neuron was not homogeneous. Retinal and F terminals were located predominantly on proximal dendrites whereas RSD terminals, either from the cortex and/or brain stem, predominated on the intermediate and distal dendrites. Parvocellular neurons were estimated to have about 500 total synapses on their dendritic trees, while magnocellular neurons had about 3000 total synapses on their dendritic trees. The retinal terminals making synaptic contacts with magnocellular neurons were also presynaptic to terminals containing flattened vesicles; these latter terminals also had synapses onto the magnocellular neuron's dendrites. Such a synaptic arrangement is called a triadic arrangement, or triad. Parvocellular neurons rarely had such triadic arrangements. In comparing these data with those of the cat, it was concluded that the major synaptic difference between relay cell types in both species (Class 1/Class 2 cells for the cat and parvo/magno cells for the monkey) was the frequent occurrence of triads for Class 2 cells and magnocellular cells versus the infrequent occurrence of triads for Class 1 cells and parvocellular cells. Although these triadic arrangements have been studied for over 2 decades, their function has yet to be determined, but probably relates to inhibition of retina signals at dendrites of magnocellular neurons in the monkey and Class 2 cells in the cat.
Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics
Authors: Harris KM , Stevens JK
Published: March 2018

Serial electron microscopy and 3-D reconstructions of dendritic spines from hippocampal area CA 1 dendrites were obtained to evaluate 2 questions about relationships between spine geometry and synaptic efficacy. First, under what biophysical conditions are the spine necks likely to reduce the magnitude of charge transferred from the synapses on the spine heads to the recipient dendrite? Simulation software provided by Charles Wilson (1984) was used to determine that if synaptic conductance is 1 nS or less, only 1% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. If synaptic conductance approaches 5 nS, however, 33% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. Second, is spine geometry associated with other anatomical indicators of synaptic efficacy, including the area of the postsynaptic density and the number of vesicles in the presynaptic axon? Reconstructed spines were graphically edited into head and neck compartments, and their dimensions were measured, the areas of the postsynaptic densities (PSD) were measured, and all of the vesicles in the presynaptic axonal varicosities were counted. The dimensions of the spine head were well correlated with the area of PSD and the number of vesicles in the presynaptic axonal varicosity. Spine neck diameter and length were not correlated with PSD area, head volume, or the number of vesicles. These results suggest that the dimensions of the spine head, but not the spine neck, reflect differences in synaptic efficacy. We suggest that the constricted necks of hippocampal dendritic spines might reduce diffusion of activated molecules to neighboring synapses, thereby attributing specificity to activated or potentiated synapses.
Type 1 astrocytes inhibit myelination by adult rat oligodendrocytes in vitro
Authors: Rosen CL , Bunge RP , Ard MD , Wood PM
Published: March 2018

We have determined the effect of Type 1 astrocytes on the myelination of dorsal root ganglion cell axons by oligodendrocytes obtained from adult animals. Experiments were initiated by the addition of oligodendrocytes [purified either by density gradient centrifugation and treatment in culture with 5-fluorodeoxyuridine (FdU) or by fluorescence-activated cell sorting after immunostaining with antigalactocerebroside antibody] to cultures of purified neurons. In control conditions, the added oligodendrocytes proliferate and, after 4 weeks, accomplish substantial myelination of the sensory axons. Type 1 astrocytes (purified from cultures of dissociated newborn rat cerebral cortex by vigorous shaking to remove less adherent cells) or fibroblasts (purified from cultures of cranial periosteum by serial replating) were added to some of these cultures after the oligodendrocytes had attached and started to proliferate. We observed that the added Type 1 astrocytes, but not the added fibroblasts, strongly inhibited myelination and caused decreased oligodendrocyte proliferation or survival. These effects of added Type 1 astrocytes were reproduced with Type 1 astrocyte-conditioned medium. We conclude that Type 1 astrocytes can release soluble factors that inhibit oligodendrocyte myelination.
Sigma receptors regulate contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation elicited by both electrical stimulation and exogenous serotonin
Authors: Campbell BG , Scherz MW , Keana JF , Weber E
Published: March 2018

Sigma receptors are specific, highly localized binding sites in limbic and sensorimotor structures of the brain that interact with many psychotropic drugs. These agents include the psychotomimetic benzomorphan opiates, the psychotomimetic drug phencyclidine and its analogs, as well as numerous typical and atypical antipsychotics such as haloperidol, chlorpromazine, and the novel drugs BMY 14802 and rimcazole. So far, no physiological function has been assigned to these binding sites. We have synthesized a number of novel sigma receptor- active drugs derived from the selective sigma ligand N,N′-di(o- tolyl)guanidine (DTG). DTG and its congeners were found to inhibit contractions of the guinea pig ileal longitudinal muscle/myenteric plexus (LMMP) preparation evoked by electrical stimulation. In addition, the sigma ligands noncompetitively antagonized contractions of the LMMP preparation evoked by serotonin (5-HT). The 5-HT-evoked contractions were found to be largely due to 5-HT's activation of 5-HT3 receptors to release ACh. The activity of DTG congeners in inhibiting electrically or 5-HT-evoked contractions of the LMMP highly correlated with their potency to inhibit binding of both 3H-DTG and (+)3H-3-PPP [3(3-OH-phenyl)-N-(1-propyl)piperidine] to sigma receptors in guinea pig brain homogenates. Two DTG congeners that did not bind to sigma receptors also showed no activity in the bioassay. Many other (but not all) sigma receptor ligands showed a high correlation between their potency to inhibit electrically evoked contractions of the LMMP and their sigma receptor binding affinity. The benzomorphans (+)SKF 10,047 and (+)cyclazocine potentiated electrically evoked contractions of the LMMP. Sigma ligands also inhibited the contractions of the LMMP in the presence of the opiate antagonist naloxone and in preparations in which opioid receptors had been inactivated by treatment with the irreversible opiate antagonist beta-chlornaltrexamine. Control experiments suggested that the sigma ligands act via a neuronal mechanism to inhibit ACh release evoked by electrical stimulation or by stimulation with 5-HT. These results suggest that there are functional sigma receptors on cholinergic nerve terminals or within the myenteric plexus and that these receptors can inhibit stimulated ACh release through an opioid receptor-independent mechanism. However, sigma receptor activation in the ileum has the same effect on ACh release as activation of naloxone-sensitive opioid receptors. The LMMP may be an in vitro bioassay system for characterizing the mechanism of action of sigma receptors and for determining the biological efficacy of drugs known to bind to sigma receptors in radioligand binding assays.
The tegmental pedunculopontine nucleus: a brain-stem output of the limbic system critical for the conditioned place preferences produced by morphine and amphetamine
Authors: Bechara A , van der Kooy D
Published: March 2018

The potent reinforcing properties of psychoactive drugs have been attributed to the activation of motivational processes localized to the limbic system. We investigated the role of 2 specific outputs of the forebrain limbic system, the tegmental pedunculopontine nucleus (TPP) and the periacqueductal gray (PAG) of the pons-midbrain, in the positive motivational effects of morphine and amphetamine. We now report that the TPP, but not the PAG nor other nearby regions, is a critical site in the neural system subserving the rewarding effects of both opiates and stimulants. Bilateral ibotenic acid lesions of the TPP blocked the positive reinforcing effects of both morphine and amphetamine in naive rats as measured in a conditioned place preference paradigm. However, TPP lesioned animals were still capable of acquiring a conditioned place preference to an environment paired with the peripheral opiate antagonist methylnaltrexone. This suggested that TPP lesions did not cause nonspecific deficits in the basic learning mechanisms underlying conditioned place preferences. Furthermore, while the TPP was critical for the acquisition of a conditioned preference to an environment paired with morphine in naive rats, rats that had acquired a morphine conditioned place preference prior to the lesions were capable of retaining and demonstrating these place preferences after lesions of the TPP. This again demonstrates that TPP lesions are producing an unconditioned deficit in motivation rather than a deficit in learning or memory. Finally, direct comparisons of the place preference data of individual animals with their correspondent TPP lesion sites indicated that the most effective lesions overlapped to a greater degree TPP perikarya with descending, rather than ascending, axons. This suggests that motivational information generated by drug stimuli acting at “upstream” neural structures flows in a descending direction through the TPP region of the brain stem. These results suggest that opiates and stimulants must ultimately activate a single brain-stem substrate in order to have a positive motivational impact. It is hypothesized that the neural circuits mediating the rewarding effects of drug stimuli acting at forebrain sites exit the limbic system in the TPP region of the brain stem, where motivation may ultimately influence or be isomorphic with the elicitation of motor responses subserving approach and exploration.
The reappearance of a developmental stage-specific antigen in adult regenerating neurons of the cockroach
Authors: Denburg JL
Published: March 2018

A monoclonal antibody has previously been described that binds to all neurons in the 15 d (50% development) cockroach embryo but to only a small subset of neurons in the adult (Denburg et al., 1989). Experiments were performed in order to determine whether the developmental stage-specific antigen recognized by this antibody would reappear in adult neurons that were induced to undergo axonal regeneration by axotomy. It is demonstrated here that after nerve crush motor, sensory and interneurons undergo axonal regeneration and regain their ability to bind this antibody. This indicates that the developing and regenerating states of these neurons selectively use the same molecules to perform apparently similar cellular functions. The increase and subsequent decrease of antibody binding as a function of time after nerve crush was determined for each of these adult neurons. Correlations between the temporal distribution of the antigen and cellular events occurring during axonal regeneration are consistent with a role for this molecule in axon growth and the elimination of inappropriate synaptic connections. The antigen was localized to the external surface of the plasma membrane, and preliminary biochemical characterization has led to the tentative identification of the antigen as a glycolipid. These characteristics distinguish this growth- associated antigen from other previously described molecules whose temporal distribution has implicated a role for them in axon growth.
The adult rat olfactory system expresses microtubule-associated proteins found in the developing brain
Authors: Viereck C , Tucker RP , Matus A
Published: March 2018

We have compared the expression and localization of neuronal microtubule-associated proteins (MAPs) MAP5, MAP2, and tau in the adult rat olfactory system and cerebral cortex. Each of these MAPS is known to exist as distinct “early” and “late” forms in the developing and adult brain, respectively. Because axonal growth and dendritic reinnervation continue in the adult olfactory system, it can serve as a test of whether expression of early MAP forms is necessary for neuronal growth and plasticity. We found that for all three MAPs, the early forms continue to be expressed in the adult olfactory system, whereas the cerebral cortex switches to the late forms during neuronal maturation (between 10 and 20 days of age in the rat). For MAP2, the 6 kb mRNA and low-molecular weight MAP2c, both of which are typically found in embryonic tissue, persist in the adult olfactory bulb. For MAP5, an early highly phosphorylated form, MAP5a, is present throughout the brain and disappears during maturation simultaneously with a several-fold drop in the overall level of MAP5. However, in the olfactory bulb, MAP5 levels do not fall, and MAP5a persists in the adult. The early form of tau is also prominent in the adult olfactory bulb and, by immunohistochemistry, is mainly confined to a subset of olfactory axons, the vomeronasal nerve. Thus, in the adult olfactory bulb, both MAP protein synthesis and phosphorylation conform to a pattern associated with the developing brain. Immunohistochemistry also showed that MAP5 is concentrated in the olfactory nerve axons and the mitral cell dendrites of the olfactory bulb, i.e., exactly those elements that are involved in the olfactory nerve innervation that takes place in the adult. These results suggest that the expression of the early MAP forms is closely associated with neurite outgrowth and plasticity.
Evidence for task-dependent memory dysfunction in the aged monkey
Authors: Rapp PR , Amaral DG
Published: March 2018

Experimentally naive adult (9–11 years old) and aged (approximately 22– 26 years old) female rhesus monkeys were evaluated on 3 neuropsychological tests of memory function. Aged monkeys were impaired in a delayed response test of visuospatial memory when the retention interval of the task was increased from 0 to 10 sec. These animals performed as well as younger subjects, however, at very short delays (0 and 1 sec), when the memory demands of the task were minimal. The same subjects were then trained in a delayed nonmatching to sample (DNMS) test of visual object recognition memory. Although they required significantly more training than the younger subjects to learn the nonmatching principle of the task, aged animals were only minimally impaired when recognition memory was tested at retention intervals ranging from 10 sec to 22 hr. In contrast to their relatively intact performance on the object recognition task, aged monkeys were dramatically impaired in a second version of DNMS that required subjects to remember the temporal order in which objects were presented. These findings support the view that certain memory functions are differentially susceptible to age-dependent deterioration. Since neuropsychological studies in young subjects demonstrate that different brain regions make relatively specific contributions to learning and memory, the task-dependent deficits observed in the aged monkey are important for determining which neural structures mediate age-dependent cognitive dysfunction. According to this perspective, aged monkeys were impaired on tasks known to be sensitive to prefrontal cortical damage, but the same animals performed well on a DNMS procedure that subjects with medial temporal lobe damage fail. These results suggest that prefrontal cortical dysfunction may mediate prominent aspects of age-dependent cognitive impairment in the monkey.
GABA-activated Cl- channels in astrocytes of hippocampal slices
Authors: MacVicar BA , Tse FW , Crichton SA , Kettenmann H
Published: March 2018

We used kainic acid-lesioned hippocampal slices to examine glial responses to the inhibitory neurotransmitter GABA in a neuron-free environment. Slices were prepared from rats which received intracerebroventricular injections of kainic acid 1 month prior to experiments. Astrocytes (membrane potential averaged 81.4 +/- 5.5 mV; n = 46; mean +/- SD) were impaled in the CA3 region of the slice, which was completely depleted of neurons. GABA (1 mM) application by bath perfusion depolarized membrane potential from 1 to 5 mV. The GABA- induced depolarization was not affected by a tetrodotoxin (1 microM)/high-Mg2+/low-Ca2+ solution. Changing the Cl- equilibrium potential by reducing extracellular Cl- greatly increased the GABA- induced depolarization. Muscimol mimicked the GABA response, while picrotoxin (0.1 mM), an antagonist of the GABA-activated Cl- channel, resulted in a 60% blockade. The barbiturate, pentobarbital (0.1 mM), and the benzodiazepine agonist, flunitrazepam (1 mM), enhanced the depolarization by 60 and 40%, respectively. A blocker of glial GABA uptake, beta-alanine (1 mM), did not affect the GABA-induced membrane depolarization, indicating that the depolarization is not caused by electrogenic uptake of the amino acid. The pharmacological properties of the GABA response of astrocytes from the hippocampal slice is similar to that previously described for cultured astrocytes from rat cerebral hemispheres. Our data suggest that GABA receptors, which are coupled to Cl- channels, are also expressed by astrocytes in an intact tissue.
Electrical coupling synchronizes subthreshold activity in locus coeruleus neurons in vitro from neonatal rats
Authors: Christie MJ , Williams JT , North RA
Published: March 2018

Locus coeruleus neurons in brain slices prepared from neonatal rats have rhythmic oscillations in membrane potential at frequencies ranging from 0.3 to 3 Hz. Recordings from pairs of neurons separated by 50–300 microns showed that this oscillatory activity was synchronized at ages less than 24 d. Slices cut from rats 24–27 d old showed rhythmic activity which was only partially synchronous between cell pairs, but full synchrony could be restored by superfusion with tetraethylammonium (2 mM) and/or barium (2 mM). No synchronous rhythmic activity was observed between neurons in slices from rats 40 d old, even in the presence of tetraethylammonium and barium. In those cells in which rhythmic potential oscillations were synchronous, action potentials occurring in one cell were not observed in the second cell. Electrotonic coupling was directly demonstrated between 41% of neurons (12 of 29 pairs) in slices from rats less than 10 d old but not in tissue from older rats (4 pairs). The input resistance of neurons from neonatal rats (less than 15 d old) was about half (81 M omega) that measured under identical conditions from neurons from adult rats (213 M omega). The electrotonic potential in cells from rats less than 15 d old was best fit by a double exponential, whereas that from adults was best fit by a single exponential. The results demonstrate that significant electrical coupling occurs among locus coeruleus neurons from neonatal rats; this appears to decline with age. The coupling serves as a low-pass filter and causes the synchronized occurrence of membrane potential oscillations. Such a rhythmic background activity within the entire nucleus may be important for the widespread trophic role of the noradrenergic neurons during development.
In situ protein phosphorylation in hippocampal tissue slices
Authors: Yip RK , Kelly PT
Published: March 2018

We have studied the subcellular distribution of phosphoproteins in intact hippocampal slices and examined factors that regulate their phosphorylation and dephosphorylation in situ. The presence of Ca2+ in slice equilibration and prelabeling buffers and high-K+-induced depolarization markedly increased 32Pi incorporation into endogenous proteins. Ca2+-stimulatory effects were significantly reduced by Ca2+- channel blockers and the calmodulin antagonist W-13. Certain proteins were dephosphorylated in situ, and their dephosphorylation was dependent on both Ca2+ and depolarization. A number of proteins phosphorylated in situ was similar to those previously characterized in synaptic fractions phosphorylated in vitro. Many phosphoproteins were identified on the basis of molecular weight, isoelectric point, immunoreactivity, and phosphopeptide mapping; these included the 87 kDa substrate of protein kinase C, synapsin I, the 50 and 60 kDa subunits of Ca2+/calmodulin-dependent protein kinase II (CKII), tubulin, B-50, the alpha-subunit of pyruvate dehydrogenase and myelin basic proteins. CKII phosphorylation in situ appeared similar but not identical to its in vitro counterpart. Phosphopeptide mapping analysis of in situ labeled substrate proteins indicated that cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases were all active in slice preparations under basal conditions. Increased 32Pi labeling of hippocampal proteins following tissue depolarization appeared to be associated with increased activity of endogenous protein kinases since depolarization did not result in 32Pi-labeling of any new phosphoproteins.
ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine- innervated brain regions. I. Amino acid sequence of ARPP-21B from bovine caudate nucleus
Authors: Williams KR , Hemmings HC , LoPresti MB , Greengard P
Published: March 2018

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS-PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of the rat CNS. It has recently been purified to homogeneity from bovine caudate nucleus and characterized (Hemmings and Greengard, 1989). ARPP-21 is isolated as 2 isoforms, ARPP-21A and ARPP-21B. The amino acid sequence of purified bovine ARPP-21B has now been determined by gas-phase sequencing. The S- 14C-carboxymethylated protein was subjected to enzymatic cleavage with trypsin, chymotrypsin, subtilisin, and endoproteinase Lys-C. The resulting peptides were purified by high-performance liquid chromatography, and selected peptides were subjected to amino acid analysis and/or amino acid sequencing by automated Edman degradation. ARPP-21B consists of a single NH2-terminal blocked polypeptide chain of 88 residues, with a calculated molecular mass of 9561 Da, including an NH2-terminal acetyl group inferred by deblocking with an acylaminopeptidase. This molecular mass is significantly lower than earlier estimates based on SDS-PAGE or hydrodynamic measurements. The seryl residue phosphorylated by cAMP-dependent protein kinase (Hemmings et al., 1989) is located at position 55. The molecule contains 1 cysteinyl residue, at position 71, and contains no methionyl, tyrosyl, phenylalanyl, tryptophanyl, or histidinyl residues. Determination of the primary structure of ARPP-21, one of several phosphoproteins localized to dopaminoceptive neurons in the basal ganglia, provides a framework for further investigations into the molecular mechanisms involved in dopaminergic neurotransmission.
Neuromuscular transmission in diabetes: response to high-frequency activation
Authors: Schiller Y , Rahamimoff R
Published: March 2018

In searching for the cellular correlates of diabetic neuropathy, we examined the response to tetanic stimulation of diabetic neuromuscular junctions and of age-matched controls. The experiments were performed on the soleus nerve muscle preparation of the rat in which diabetes was induced by streptozotocin. Tetanic potentiation was substantially lower in diabetic rats. In addition, it was found that the diabetic neuromuscular junction is more resistant to high-frequency stimulation than normal age-matched controls, in which such stimulation causes a progressively increasing number of failures in synaptic transmission. Tetanic failures cannot be predicted from the stochastic properties of transmitter release and are due to propagation block of action potentials into the nerve terminals. The resistance of diabetic nerves to tetanic stimulation is a function of the duration of diabetes; the earliest significant difference between the number of tetanic failures in diabetic and normal age-matched controls was observed after 19 d of diabetes, and this difference grew with increased duration of diabetes. The resistance to tetanic stimulation in diabetic rats is reversed by insulin in vivo but not in vitro. Elevation in extracellular [K+] increases the number of tetanic failures in both diabetic and normal preparations. Furthermore, elevating extracellular [K+] to 8.5 mM brings the number of tetanic failures into the range of tetanic failures in normal nerves. This finding is consistent with the hypothesis that differences in extracellular [K+] accumulation during high-frequency stimulation are responsible for the diabetic nerve's relative resistance to high-frequency stimulation. The lower number of failures corrects only partially the impaired neuromuscular transmission in the diabetic state, and there is an overall reduction in tetanic potentiation in diabetes.
Muscarinic receptor activation stimulates B-50/GAP43 phosphorylation in isolated nerve growth cones
Authors: Van Hooff CO , De Graan PN , Oestreicher AB , Gispen WH
Published: March 2018

A characteristic feature of neurite formation is high expression of the phosphoprotein B-50/GAP43. Previous studies with growth cone membranes have indicated that this neuron-specific protein kinase C substrate may be involved in transmembrane signal transduction at the growth cone. We monitored the degree of phosphorylation of B-50 by quantitative B-50 immunoprecipitation from intact nerve growth cones, isolated from 5-day- old rat brain and prelabeled with 32P-orthophosphate. B-50 phosphorylation in nerve growth cones is stimulated by 4 beta-phorbol 12,13-dibutyrate (PDB) and 1,2-dioctanoylglycerol (DOG) in a concentration-dependent manner, but not by 4 alpha-phorbol 12,13- didecanoate (4 alpha-PDD). These results confirm that B-50 is a substrate of PKC in intact growth cones. Depolarization induced by 30 mM K+ produces a transient increase in B-50 phosphorylation, which is maximal after 15 sec and declines to basal level within 5 min. This rise in B-50 phosphorylation can be partially blocked by atropine (10(- 3)-10(-4) M), suggesting the involvement of muscarinic receptors. Indeed, the cholinergic receptor agonist carbachol enhances B-50 phosphorylation in a concentration-dependent manner (50% at 10(-3) M). Since the effect of carbachol (10(-3) M) can be blocked by atropine (10(-7) M), we conclude that this increase in B-50 phosphorylation is mediated through activation of the muscarinic receptors on the growth cones. The carbachol-induced stimulation is further increased by concurrent K+-depolarization. The effects of carbachol and depolarization are additive. To our knowledge, this is the first report showing receptor-mediated effects on the PKC substrate B-50 in growth cones. Our data support the hypothesis that phosphorylation of B-50 by PKC is involved in signal transduction in nerve growth cones.
Elementary processes of response selection mediated by distinct regions of the striatum
Authors: Brown VJ , Robbins TW
Published: March 2018

The relative contributions of the medial and lateral caudate putamen to performance of a visual reaction time task were compared by examining the effects of unilateral ibotenate-induced lesions to these regions in the rat. Different groups of rats were trained to respond either towards (Discrimination 1) or away from (Discrimination 2) unpredictable, brief visual stimuli presented to either side of the head. Lateral striatal lesions produced a strong spatial bias towards the side of the lesion but left the latency for the initiation of responses to the visual cues unchanged. By contrast, the medial striatal lesions resulted in a smaller degree of spatial response bias but a significant slowing of initiation latency to the side contralateral to the lesion. These effects were irrespective of the side of the stimulus presentation and thus represent response-related impairments. The lesions were further dissociated in their effects on responding inappropriately to task requirements. These results demonstrate a double dissociation of behavioral effects of lateral and medial striatal damage that can be used to infer the operation of distinct elementary processes of response output within a single task. They are important not only in demonstrating functional effects of hypothetical segregated parallel corticostriatal loops but also in showing that these loops must interact to produce integrated performance.
Axotomy-induced changes in the expression of a type III neuronal intermediate filament gene
Authors: Oblinger MM , Wong J , Parysek LM
Published: March 2018

The effect of axotomy on the expression of the 57 kDa neuronal intermediate filament (IF) protein in adult rat dorsal root ganglion (DRG) neurons was examined. This IF protein is known to have an exclusively neuronal localization but is considerably more limited in its distribution in the nervous system than the neurofilament (NF) triplet proteins. The 57 kDa neuronal IF protein is similar (and perhaps identical) to the protein “peripherin” and is known to be the product of a Type III IF gene. Since the down-regulated expression of NF proteins (products of type IV IF genes) has been well established, it was of interest to determine whether the novel 57 kDa IF protein was regulated in a similar or different manner from that of the NFs in axotomized neurons. In vitro pulse-labeling of DRGs with 35S- methionine: cysteine followed by 2-dimensional gel electrophoresis/fluorography revealed that the synthesis of the 57 kDa neuronal IF protein was increased 2 weeks after sciatic nerve crush. Immunocytochemical studies using a polyclonal antibody to the 57 kDa neuronal IF protein showed that the immunodetectable levels of this protein increased in DRG neurons after peripheral axotomy. In the normal DRG, staining was localized almost exclusively to small-sized neurons. At 2 weeks after axotomy, however, large- and medium-sized neurons also became immunoreactive; in addition, the overall level of staining in the DRG was greater than normal. Quantitative analysis of in situ hybridizations of DRG neurons with a 35S-labeled cDNA probe specific for the 57 kDa neuronal IF protein revealed a significant increase in the level of 57 kDa IF mRNA in the large-sized (greater than 1000 microns2) neurons 2 weeks after axotomy; the level of 57 kDa IF mRNA in the small neurons was not different from normal at that time. Finally, using a newly developed paradigm for examining the composition of regenerating axons by axonal transport, we determined that significant amounts of the 57 kDa neuronal IF protein were conveyed into the regrowing axonal sprouts of DRG neurons. When DRG neurons were conditioned by a previous axotomy (a crush axotomy of the distal sciatic nerve 2 weeks earlier) and then stimulated to regenerate axons by a second crush axotomy located very close to the DRG, the regenerating sprouts incorporated and conveyed significantly more 57 kDa IF protein by slow axonal transport than did those elaborated by unprimed DRG neurons.(ABSTRACT TRUNCATED AT 400 WORDS)