bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous release of acetylcholine affects the physiological nicotinic responses of rat retinal ganglion cells in culture
Authors: Lipton SA
Published: March 2018

Nictonic cholinergic responses have been previously documented in mammalian retinal ganglion cells. In the present study, dissociated retinal cells were densely plated and grown in a specific batch of rat serum. When held at negative potentials during whole-cell recording with a patch electrode, the retinal ganglion cells located close to presumptive cholinergic amacrine cells in these cultures were found to respond to acetylcholine (ACh; 20–200 microM) with an apparent outward current in the presence of physiological salines on both sides of the membrane. Other nicotinic agonists (nicotine, carbachol) produced the same effect. Conductance measurements revealed that this apparent outward current was actually a decrease in a tonic inward current. Nicotinic antagonists such as d-turbocurarine (10 microM) and dihydro- beta-erythroidine (20 microM), when applied in dishes that had never been exposed to exogenous ACh, produced a similar decrease in a tonic inward current. The reversal potential of the tonic current suppressed by ACh was similar to the nicotinic current previously studied in these central neurons. Furthermore, purified acetylcholinesterase was capable of modulating the tonic inward current of the retinal ganglion cells. Lowering an excised patch of muscle into dense areas of the retinal cultures activated nicotinic channels in the muscle membrane, indicating the presence of endogenous ACh in the culture fluid. Divalent cations such as Co2+ blocked the tonic inward current of retinal ganglion cells in these cultures. Finally, direct biochemical measurements indicated that low levels of endogenous ACh (on the order of 0.5 microM near putative cholinergic amacrine cells) were present in the retinal cultures. Taken together, these results show that ACh was being spontaneously released into these cultures, resembling at least to some degree the tonic leakage of ACh found in the intact retina. This concentration of ACh was capable of tonically depolarizing the membrane potential of retinal ganglion cells exposed to this dosage. This culture system allows the study of trophic effects of ACh on a central mammalian neuron in a precisely controlled extracellular environment.
Different times of origin of choline acetyltransferase- and somatostatin-immunoreactive neurons in the rat striatum
Authors: Semba K , Vincent SR , Fibiger HC
Published: March 2018

Two populations of aspiny interneurons have been identified in the mammalian striatum, one cholinergic and the other using the neuropeptide somatostatin as a neurotransmitter. The times at which these 2 cell populations undergo their final mitosis were studied by injecting tritiated thymidine into timed pregnant rats and then processing the brains of the progeny as young adults for immunohistochemistry with monoclonal antibodies to choline acetyltransferase and somatostatin followed by autoradiography. Choline acetyltransferase-immunoreactive neurons became postmitotic in a caudal- to-rostral gradient; the occurrence of final mitosis was maximal on embryonic day (E) 12 at the most caudal level and on E15 at the most rostral. A more subtle lateral-to-medial gradient was also observed in the precommissural striatum. In contrast, no obvious gradients were seen with somatostatin-immunoreactive neurons; regardless of their location within the striatum, these neurons underwent their final mitosis on days E15–16, towards the end of cholinergic neurogenesis. These results indicate that although both cholinergic and somatostatin- containing cells represent interneuronal populations in the striatum, they display distinctly different spatiotemporal patterns of neurogenesis.
Monosynaptic projections of single muscle spindle afferents to neck motoneurons in the cat
Authors: Keirstead SA , Rose PK
Published: March 2018

Monosynaptic connections to dorsal neck motoneurons of the cat from single afferents supplying primary endings of neck muscle spindles were studied using spike-triggered averaging techniques. Single-fiber EPSPs were detected in only 11 of the 112 afferent-motoneuron pairs examined. The average amplitude of single-fiber EPSPs recorded in motoneurons with a membrane potential of greater than -40 mV was 49 microV. Motoneurons receiving functional contacts from a single afferent were confined to a small rostrocaudal zone within the motor nucleus. The low frequency of single-fiber EPSPs in neck motoneurons could not be attributed to the absence of projections to the ventral horn or to damage to either the afferents or motoneurons. Our results suggest, therefore, that single afferents from neck muscle spindles make functional contacts with a small fraction of neck motoneurons, unlike the arrangement seen in more commonly studied hindlimb muscle systems (Henneman and Mendell, 1981).
Activation of muscle fibers in individual motor units revealed by 2- deoxyglucose-6-phosphate
Authors: Nemeth PM , Norris BJ , Lowry OH , Gordon DA , Enoka RM , Stuart DG
Published: March 2018

Motor units of the cat tibialis posterior muscle were selectively activated by prolonged electrical stimulation of functionally isolated motor axons in situ. During the activation, the glucose analog 2- deoxyglucose (DG) was administered systemically. Single muscle fibers were subsequently examined for accumulation of the metabolite 2- deoxyglucose-6-phosphate (DG6P) by an analytical assay and for depletion of glycogen by a PAS glycogen-specific staining reaction (periodic acid Schiff; PAS). In general, levels of DG6P were 20 times greater in unstained (PAS-negative) fibers compared with stained (PAS- positive) fibers. However, some glycogen-depleted fibers, particularly in putative ischemic fascicles of the muscle, did not have elevated DG6P, suggesting that depletion of glycogen is not always a reliable indicator of fiber activation. Furthermore, the PAS-staining reaction was not necessarily indicative of quantitative glycogen levels in single fibers. Thus, this report shows that DG6P accumulation enhances the identification of motor-unit fibers selectively activated via their common motor-nerve axon. Evidence is also presented for differential glucose uptake in muscle fibers of different phenotype, thereby indicating that the DG6P measurement in muscle has broad applicability to the investigation of cellular glucose utilization.
A persistent, TTX-sensitive sodium current in an invertebrate neuron with neurosecretory ultrastructure
Authors: Davis RE , Stuart AE
Published: March 2018

In the CNS of the giant barnacle (Balanus nubilus) a single pair of large neuronal somata (cross-commissural, or CC, cells), located near the entry of the median ocellar nerve, occasionally displays a prominent whitish luster. These somata have ultrastructure typical of neurosecretory cells: numerous Golgi complexes and abundant, large dense-cored vesicles (DCVs; size range, 75–275 nm). Injection of a CC cell with cobalt tracer shows that it arborizes over a 7 mm length of the contralateral peripheral nerve out of which it projects. The processes of the arbor are profuse and varicose; the varicosities are packed with DCVs similar to those in the soma. Stimulation of a single CC cell causes a substantial decrease in the number of DCVs and increases the incidence of clusters of small electron-lucent vesicles, as well as the occurrence of large electron-lucent vesicles and membrane-bound cisternae. We studied ionic currents flowing across this cell's somatic membrane with a single-electrode voltage clamp. Unusual among these currents is an inward current that is blocked by TTX but is essentially noninactivating. In current clamp, this “persistent” current causes the action potential to be prolonged (seconds) if opposing outward current is blocked with 4-aminopyridine. The inward current is carried by Na. Its amplitude depends on the external Na concentration, it is blockable by TTX, and it persists when the cell is bathed in Ca-free saline and/or Co. Other currents present in this cell include an outward current similar to molluscan A-current and a Ca current that contributes to the action potential (Stockbridge and Ross, 1986). The persistent Na current is partially activated at the cell's resting potential and, thus, may participate in determining the frequency of its impulse activity.
Regulation of calcium homeostasis in sensory neurons by bradykinin
Authors: Thayer SA , Perney TM , Miller RJ
Published: March 2018

The nonapeptide bradykinin (BK) activates sensory neurons and stimulates the transmission of nociceptive information into the CNS. We investigated the effect of this peptide on rat dorsal root ganglion neurons (DRG) grown in vitro. BK stimulated the synthesis of inositol trisphosphate (IP3) and the breakdown of phosphatidylinositol bisphosphate, the synthesis of diacylglycerol, and the release of arachidonic acid from DRG cells. The release of IP3 and arachidonic acid was not inhibited by pretreatment of the cells with pertussis toxin. BK also mobilized intracellular Ca2+ stores in DRG cells as assessed by fura-2-based microfluorimetry. Two types of Ca2+ stores appeared to exist in DRG neurons. One type could be mobilized by caffeine (10(-2) M), and this effect could be blocked by ryanodine in a use-dependent manner. These stores occurred primarily in the cell soma and were virtually absent from cell processes. A second type of store could be mobilized by BK, presumably through the mediation of IP3. These latter stores were distributed equally between the cell soma and processes. Experiments with combinations of caffeine and BK suggested that the stores mobilized by these 2 agents may be separate entities. Both the caffeine and BK sensitive Ca2+ storage sites appeared to participate in buffering a Ca2+ load induced in DRG neurons by cell depolarization. The relevance of these observations to the mechanism of action of BK on sensory neurons is discussed.
Corticotropin-releasing factor in olivocerebellar climbing-fiber system of monkey (Saimiri sciureus and Macaca fascicularis): parasagittal and regional organization visualized by immunohistochemistry
Authors: Cha CI , Foote SL
Published: March 2018

An antiserum directed against the human form of corticotropin-releasing factor (CRF) was utilized for immunohistochemical visualization of the distribution of this peptide in the inferior olivary nucleus and cerebellum of 2 monkey species (Saimiri sciureus, Macaca fascicularis). Colchicine pretreatment was not used. In both species, immunoreactivity was evident in the vast majority of neurons in the inferior olivary nucleus, with perikarya in the medial accessory olive exhibiting especially intense staining. In cerebellum, no labeled perikarya were present, but immunoreactive axons exhibiting the morphological characteristics of climbing fibers and their collaterals were observed in cortical and nuclear structures. In the cortex, most labeled axons were confined to the molecular and Purkinje cell layers. In the sagittal plane, individual axonal arbors originated from thick, isolated axons at the base of the molecular layer and repeatedly ramified as they extended toward the cortical surface. In coronal sections, only thin, paired profiles were present. Labeled processes also formed efflorescences in the granular layer of cortex and were evident as highly arborized axons in cerebellar nuclei. In each of these instances, the labeled elements resembled climbing fibers or their collaterals as visualized by other methods. Other labeled processes in the granular layer exhibited the morphological characteristics of mossy fiber axons. Immunoreactive, climbing-fiber- like axons were present in the molecular layer throughout the major regions of cerebellar cortex. However, the most intensely labeled of these axons were strikingly clustered within particular regions and parasagittal domains. In the vermis and intermediate zone, intensely labeled axons were present only within parasagittal zones similar in location to those defined by climbing fiber innervation from the medial accessory olive. Intensely labeled axons were also densely but uniformly distributed within the uvula, the medial region of the dorsal paraflocculus, and the dorsal region of the pyramis, areas that receive their climbing fiber input primarily from the medial accessory olive. Labeled fibers were much less dense and were not clustered in the lateral hemispheres. The present observation of CRF-like immunoreactivity in the monkey olivocerebellar pathway is compatible with the previous observation of CRF mRNA within olivary neurons of rat, baboon, and human (Young et al., 1986) and with recent immunohistochemical findings in rat (Sakanaka et al., 1987; Palkovits et al., 1987), cat (Cummings et al., 1988; Kitahama et al., 1988), sheep (Cummings et al., 1988), and human (Powers et al., 1987).(ABSTRACT TRUNCATED AT 400 WORDS)
The ontogeny of brain receptors for corticotropin-releasing factor and the development of their functional association with adenylate cyclase
Authors: Insel TR , Battaglia G , Fairbanks DW , De Souza EB
Published: March 2018

This study reports the ontogeny of corticotropin-releasing factor (CRF) receptor binding sites in rat brain, using both membrane binding assays and in vitro receptor autoradiography. CRF binding sites are evident by prenatal day 17, increase to 312% of their adult density by postnatal day 8, then decrease to reach adult values by day 21. Not only the density, but the distribution of CRF binding undergoes major modifications in development. CRF binding sites are most numerous in striatum prenatally, but postnatally, binding is more dense in the cortex, reaching the adult laminar distribution by postnatal day 14. Brain CRF receptors are linked to adenylate cyclase early in postnatal life. This contrasts with the later appearance of most of the guanine nucleotide stimulatory protein and catalytic subunit sites in the rat brain and suggests that CRF receptors may become functional earlier than several other brain receptors that are linked to adenylate cyclase.
Classification of muscarinic responses in hippocampus in terms of receptor subtypes and second-messenger systems: electrophysiological studies in vitro
Authors: Dutar P , Nicoll RA
Published: March 2018

The hippocampal slice preparation was used to classify cholinergic effects in terms of muscarinic receptor subtypes (M1 or M2) and biochemical effector systems linked to these effects in CA1 pyramidal cells. Based on the action of the M1 antagonist pirenzepine and the M2 antagonist gallamine, the muscarinic-induced membrane depolarization and blockade of the afterhyperpolarization appear to result from activation of an M1 receptor, while the cholinergic depression of the EPSP and the blockade of a potassium current termed the M-current appears to involve the activation of an M2 receptor. All of the muscarinic actions could be observed in pertussis toxin-treated hippocampi, suggesting that a pertussis toxin-sensitive G-protein is not involved in these actions. Cholinergic agents that are weak agonists of phosphoinositide (PI) turnover are fully effective in all of the muscarinic actions except the blockade of the M-current on which they had little agonist activity and actually blocked the action of full agonists. These results strongly suggest that the blockade of the M-current may involve stimulation of PI turnover. In addition, we show that the blockade of the M-current is mimicked by intracellular application of inositol trisphosphate. Our results do not show any obvious relationship between the muscarinic receptor subtypes and the biochemical effector systems.
Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation
Authors: James WM , Klein WL
Published: March 2018

Significant progress has been made in determining regional expression of neurotransmitter receptors within the CNS, but little information is available at the neuronal level. In the current study, to begin characterizing cellular and subcellular aspects of receptor differentiation, we have localized ACh receptors on neurons isolated from the chicken CNS. Localization was determined autoradiographically using 2 cholinergic receptor probes: 3H-propylbenzilylcholine mustard (PrBCM) a muscarinic antagonist, and 125I-alpha-bungarotoxin (BTX), a putative nicotinic ligand in the avian CNS. To isolate neurons, we incubated embryonic chicken retinas (E13-E19) in Ca2+/Mg2+-free buffer containing 8 units/ml papain for 20 min and then gently agitated the tissue by trituration. Large numbers of dendrite-rich neurons, belonging to recognizable morphological subpopulations (e.g., multipolar neurons of various sizes, small bipolar and unipolar neurons), were collected on slides for autoradiography. Cell isolation had no effect on ligand binding levels, and a high ratio of specific to nonspecific binding allowed us to associate silver grains with receptor position. Muscarinic-receptor-positive cells comprised a recognizable subpopulation that had small rounded cell bodies (6–7 micron) and a single emergent arbor. The cells had an axial or elongated appearance. Muscarinic receptors were abundant over dendrites but absent from cell bodies. Segregation to dendrites was complete by E13, the age when synapses reportedly first begin to appear. Cells labeled with 125I-BTX were more heterogeneous in morphology. The most striking BTX-positive cells comprised neurons with large cell bodies (approximately 15 micron) and multiple processes. Dendrites were profusely labeled, but only sporadic labeling was seen on cell bodies, and often this was at sites crossed by labeled dendrites. Maximum labeling occurred in the distal, smallest-caliber ends of the dendritic arbors. All limbs of BTX- positive multipolar neurons expressed abundant receptors. Occasionally, a thin uniform-caliber process was seen branching from a primary dendrite, and such processes, which may have been axons, were never labeled. Very small neurons with bipolar morphology also showed minimal or no labeling on one process, despite dense labeling on the other. Photoreceptors and Mueller cells were never labeled with BTX or PrBCM. The data show that, within a discrete CNS region, specific subpopulations of neurons independently regulate expression of ACh receptors and that, even early in development, control mechanisms segregate receptors to physiologically appropriate regions of the cell surface.
AMPA, kainate, and quisqualate activate a common receptor-channel complex on embryonic chick motoneurons
Authors: Zorumski CF , Yang J
Published: March 2018

The actions of the putative quisqualate-selective agonist DL-alpha- amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) were examined in identified embryonic chick motoneurons using gigaseal recording techniques and compared with properties of the selective non- NMDA excitatory amino acid agonists kainate and quisqualate. Pressure application of AMPA induces an inward going current when neurons are voltage-clamped at negative membrane potentials. The current-voltage relationship for this response is linear with reversal near 0 mV. Over the range of 1 microM-10 mM, the AMPA-induced current is dose-dependent with an ED50 of 40 microM. AMPA currents are insensitive to the selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate, and the putative quisqualate selective blocker, glutamate diethyl ester, but are partially inhibited by kynurenic acid. In competition experiments, applications of saturating concentrations of AMPA and either kainate or quisqualate produce responses intermediate between the response to either agonist alone, indicating commonality in the mechanism of these agents. Applications of AMPA with the NMDA-selective agonist aspartate give an additive response. Analysis of current fluctuations indicates that AMPA, quisqualate, and kainate gate a channel with a primary conductance near 20 pS. Differences in maximal macroscopic current evoked by saturating concentrations of AMPA, kainate, and quisqualate cannot be explained by differences in mean channel open time as the most efficacious agonist, kainate, has the shortest channel open time (AMPA = 5.9 +/- 0.4 msec, kainate = 2.7 +/- 0.1 msec, quisqualate = 5.0 +/- 0.5 msec). Rather, kainate induces a greater frequency of channel opening. This finding contrasts with results obtained at the nicotinic ACh receptor, where the most efficacious agonists have the longest mean channel open time. Our results suggest that AMPA acts at the same receptor-channel complex as kainate and quisqualate on chick motoneurons and support the hypothesis that only 2 classes of excitatory amino acid receptor complexes exist in this preparation.
Inward rectification of resting and opiate-activated potassium currents in rat locus coeruleus neurons
Authors: Williams JT , North RA , Tokimasa T
Published: March 2018

Intracellular recordings were made from rat locus coeruleus neurons in vitro, and membrane currents were measured at potentials from -50 to - 130 mV. In the absence of any applied agonists, the slope conductance of the cells increased 3-fold when the cell was hyperpolarized from -60 to -120 mV. This conductance increase was complete within 5 msec of the onset of a hyperpolarizing command and was subsequently independent of time for several seconds. The conductance increase was blocked by cesium chloride (1–2 mM), rubidium chloride (1–2 mM), or barium chloride (1–100 microM). The membrane potential range over which the conductance increased was centered at the potassium equilibrium potential (EK; extracellular potassium concentration, 2.5–10.5 mM): the current/voltage (I/V) relation of the cell could be well described by supposing that there were 2 potassium conductances, one voltage independent (G1) and the other (inward rectifier, Gir) activated according to the expression Gir = Gir,max/(1 + exp[(V - EK)/k]), where k ranged from 15 mV in 2.5 mM potassium to 6 mV in 10.5 mM potassium. The additional membrane potassium conductance that developed when agonists at mu-opioid and alpha 2-adrenoceptors were applied also became larger with membrane hyperpolarization, and this voltage dependence was also reduced or blocked by rubidium, cesium, and barium; in the presence of these agonists the current also reached its final value within 5 msec. However, the conductance increased by the agonists (Gag) was not well expressed by simply increasing the values of G1 and Gir,max. It was best described by a potassium conductance that increased according to Gag,max/(1 + exp[(V - Vm)/k]), where Vm (the potential at which the conductance was half-maximum) was close to the resting potential of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)
Motor neuronal receptive fields delimit patterns of motor activity during locomotion of the locust
Authors: Laurent G , Hustert R
Published: March 2018

During walking, the muscles of a leg undergo a typical sequence of activity, which is partly shaped by phasic sensory feedback. To assess the role played by such feedback, we characterized intracellularly the receptive fields of tarsal motor neurons in the locust Schistocerca gregaria and considered these receptive fields within the context of a step cycle. The depressor motor neurons, active during the stance phase, are excited by ventral tarsal contact or an imposed levation and are inhibited by dorsal contact or an imposed depression. Partial deafferentation of the anterior tarsus reduces this stance phase depressor activity. The levator motor neuron, active during the swing phase, has the opposite receptive field. The retractor unguis motor neurons, synergistic to the depressors, are, like them, excited by ventral contact but, like the levator, are inhibited by afferents which can signal the end of the stance phase. The inhibition of the retractors could constitute a preparation for the swing phase, by reducing the grip on the substrate. The motor neuronal receptive fields thus appear to support the patterns of muscular activity recorded during walking. Excitation of the motor neurons by extero- and proprioceptors is usually direct: hair, canal, campaniform, and chordotonal afferents all evoke 1:1 EPSPs in motor neurons after a central latency of 1–1.5 msec. Inhibition is indirect, as IPSPs occur at least 2 msec later than the EPSPs. The motor neurons of one pool have overlapping but not necessarily identical, receptive fields. Parallel, supplementary excitatory and inhibitory pathways involving nonspiking local interneurons also exist, which can allow gain control of a specific reflex. The weight given to a reflex response will thus depend, first, on the number of motor neurons in a pool affected by the stimulus and, second, on the existence and state of intercalated interneurons.
Context-US learning in Aplysia californica
Authors: Colwill RM , Absher RA , Roberts ML
Published: March 2018

Two experiments explored learning about the relation between an unconditioned stimulus (US) and the context in which that US was delivered in Aplysia californica. Subjects were exposed to 2 different contexts. They received a series of moderate electric shock USs in one context but not in the other. Experiment 1 revealed that the context- shock pairings were sufficient to establish an association between that context and the shock. That association was demonstrated by the ability of the reinforced context to enhance a defensive reflex and to reduce learning about a conditioned stimulus (CS) that was subsequently paired with the shock US. Experiment 2 provided evidence that the context-US association could be extinguished by simple nonreinforced exposure to the reinforced context. These results show that context conditioning is not unique to vertebrate learning. We argue that this learning is difficult to accommodate within current contiguity-based neuronal models of learning.
Dendritic spines of rat cerebellar Purkinje cells: serial electron microscopy with reference to their biophysical characteristics
Authors: Harris KM , Stevens JK
Published: March 2018

We have used serial electron microscopy and 3-dimensional reconstructions of dendritic spines from Purkinje spiny branchlets of normal adult rats to evaluate 2 questions about the relationship of spine geometry to synaptic efficacy. First, do relationships between spine geometry and other anatomical indicators of synaptic activity suggest that spine size and shape might be associated with synaptic efficacy? Reconstructed spines were graphically edited into head and neck compartments; the area of the postsynaptic density (PSD) was measured; the volume of spine smooth endoplasmic reticulum (SER) was computed; and all of the vesicles in the axonal varicosities were counted. Spine head volume and the volume of SER contained in the head are well correlated with the area of the PSD and the number of vesicles in the presynaptic axonal varicosity. Spine neck diameter does not fluctuate with PSD area, head volume, or the vesicle number. These results suggest that the dimensions of the spine head, but not of the spine neck, are likely to reflect differences in synaptic efficacy. Second, does the geometry of cerebellar spine necks reduce the transfer of synaptic charge to the recipient dendrite from the theoretical maximum that could be transferred if the synapse were on a dendritic shaft? Comparison of volume to surface area showed that the spine heads are approximately spherical and the necks are approximately cylindrical. Application of results from a biophysical model that assumed these geometrical shapes for spines (Wilson, 1984) showed that the cerebellar spine necks are unlikely to reduce transfer of synaptic charge by more than 5–20% even if their SER were to completely block passage of current through the portion of the neck that it occupies. We suggest that the constricted spine neck diameter might serve to isolate metabolic events in the vicinity of activated synapses by reducing diffusion to neighboring synapses, without significantly influencing the transfer of synaptic charge to the postsynaptic dendrite.
A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina
Authors: Lukasiewicz P , Werblin F
Published: March 2018

Voltage-gated ganglion cell membrane currents were studied under whole- cell patch clamp in isolation and in retinal slices. The cells were identified by (1) backfilling their axons with rhodamine and later identifying them by their fluorescence in the slice or the mix of isolated cells or (2) by filling them with Lucifer yellow during recording in retinal slices. Both methods yielded cells with similar currents. In some cases, isolated cells lacked processes yet showed currents similar to other cells, suggesting that voltage-gated currents in all cells were located primarily at the soma. Both a conventional inactivating sodium current and a sustained calcium current were found. We describe 3 inactivating outward currents, ordered in their rate of inactivation. The fastest current resembled IA reported by Connor and Stevens (1971a, b). A slower current labeled IB inactivated with a time constant of 339 msec at 0 mV. The current with slowest inactivation is labeled IC here, inactivating with a time constant of 4.03 sec at 0 mV. An additional outward current was sustained and calcium dependent labeled IK(Ca). IB was the largest of these currents. It was slower than IA, was not blocked by 4AP, and inactivated over a much more positive potential range. IB appears to play an important role in spike generation, different from that of IA: Its inactivation leads to a slow depolarizing shift of the membrane during a current step, truncating spike activity after about 300–700 msec as the membrane potential enters the region of sodium inactivation. We analyze how the inactivating outward current acts to ensure a graded spiking response and to truncate the spiking output in the presence of large excitatory inputs.
Stereoscopic mechanisms in monkey visual cortex: binocular correlation and disparity selectivity
Authors: Poggio GF , Gonzalez F , Krause F
Published: March 2018

The neural signals in visual cortex associated with positional disparity and contrast texture correlation of binocular images are the subject of this study. We have analyzed the effects of stereoscopically presented luminous bars and of dynamic random-dot patterns on the activity of single neurons in cortical visual areas V1, V2, and V3-V3A of the alert, visually trained rhesus macaque. The interpretation of the results and considerations of possible neural mechanisms led us to recognize 2 functional sets of stereoscopic neurons. (1) A set of neurons, tuned excitatory (T0) or tuned inhibitory (TI), which respond sharply to images of zero or near-zero disparity. Objects at or about the horopter drive the T0 neurons and suppress the TI, while objects nearer and farther have the opposite effects on each type, inhibition of the T0 and excitation of the TI. The activity of these neurons may provide, in a reciprocal way, the definition of the plane of fixation, and the basic reference for binocular single vision and depth discrimination. (2) A second set of neurons includes tuned excitatory at larger crossed or uncrossed disparities (TN/TF) and neurons with reciprocal excitatory and inhibitory disparity sensitivity with cross- over at the horopter (NE/FA). Binocularly uncorrelated image contrast drives these neurons to a maintained level of activity, which shifts, in response to correlated images, toward facilitation or suppression as a function of positional disparity. These neurons may operate in the neural processing leading to stereopsis, both coarse and fine, and also provide signals for the system controlling binocular vergence. These results indicate that cortical visual neurons are binocularly linked to respond to the relative position and contrast of the images over their receptive fields, and also that both these aspects of binocular stimulation may be utilized by the brain as a source of stereoscopic information.
Parabrachial innervation of the cat's dorsal lateral geniculate nucleus: an electron microscopic study using the tracer Phaseolus vulgaris leucoagglutinin (PHA-L)
Authors: Cucchiaro JB , Uhlrich DJ , Sherman SM
Published: March 2018

Ascending pathways from the brain stem play a key role, generally facilitatory, in controlling the transmission of retinal information through the lateral geniculate nucleus to the visual cortex (for reviews, see Singer, 1977; Burke and Cole, 1978; Sherman and Koch, 1986). In order to characterize the morphological basis of this brain- stem control, we used the electron microscope to study synaptic terminals labeled anterogradely from injections of the tracer Phaseolus vulgaris leucoagglutinin into the parabrachial region of the brain stem. The labeled axons, which are fine and unmyelinated in our material, form conventional synaptic contacts onto both relay cells and interneurons. These connections are surprisingly selective for certain postsynaptic elements such as the dendritic shafts and appendages of relay cells and the presynaptic dendritic terminals of interneurons. That is, the morphology of contacts made from parabrachial axons varies with the specific postsynaptic profile. Even a single axon can form symmetrical contacts onto F2 terminals, which are synaptic terminals deriving from dendrites of interneurons, and dendritic shafts of relay X cells, and form asymmetrical contacts onto dendritic appendages of the same relay X cells. Reconstructions of the dendritic segments postsynaptic to the labeled terminals show that the dendritic appendages receive retinal and parabrachial input in triadic relationships with F2 terminals: a retinal or parabrachial axon contacts the F2 terminal, and the F2 terminal plus the retinal or parabrachial axon contact the dendritic appendage. This positioning of the parabrachial innervation is well suited for control of retinal transmission. Finally, the dual morphology of the parabrachial synaptic contacts suggests that their actions may differ depending on the postsynaptic target.
Associative learning modifies two behaviors in the leech, Hirudo medicinalis
Authors: Sahley CL , Ready DF
Published: March 2018

We report that 2 behaviors, stepping and shortening, are modified by associative learning in the leech, Hirudo medicinalis. Experiment 1 explored conditioning of the “stepping” response. Paired presentations of touch to the medial dorsal surface of the leech and shock to the tail of the leech resulted in the development of stepping to the touch. Leeches in control groups experiencing the CS alone, US alone, or explicitly unpaired presentations of the CS and US did not. In experiments 2–4, classical conditioning explored conditioning of the touch-elicited shortening reflex. We found that the reflex was enhanced following paired CS-US presentations but not following CS alone, US alone, or explicitly unpaired presentations of the stimuli. Moreover, the learning was extinguished following 15 unreinforced presentations of the CS but was retained for at least 24 hr without extinction training. Moreover, the associative effect was not evident when the CS and US were presented in a backward relationship. That is, no learning was observed when the US preceded the CS. Lastly, the hand-held stimuli were replaced with implanted electrodes. Using a 3 V pulse that mimicked the touch stimulus (CS), we found that paired CS-US presentations produced a significant enhancement in the shortening reflex. Again, no enhancement was observed following unpaired CS, US presentations.
The effects of hyperstriatal lesions on one-trial passive-avoidance learning in the chick
Authors: Davies DC , Taylor DA , Johnson MH
Published: March 2018

The roles of different forebrain structures in memory formation in the chick were investigated using restricted radio-frequency lesions. Young chicks will spontaneously peck at a small bright bead. If the bead has been coated with a distasteful substance, the chicks learn not to peck a similar bead on subsequent presentation. Thus, in a single trial chicks learn not to peck at an aversive stimulus. Bilateral lesions of the intermediate part of the medial hyperstriatum ventrale (IMHV) prevented the acquisition of this 1-trial passive-avoidance learning task. However, neither bilateral lesions of the lateral cerebral area (LCA) nor sham operation affected learning. IMHV has previously been shown to be critically involved in the learning process of imprinting in the chick. The present experiment demonstrates that the role of IMHV in learning is not restricted to imprinting. The function of IMHV in imprinting has been suggested to be concerned with the recognition of objects. In the present study sham-operated and LCA-lesioned chicks recognized the visual characteristics of the training bead and did not avoid a bead of a different color in the test. We propose that IMHV- lesioned chicks fail to learn the task because they are unable to recognize the visual characteristics of the training bead and consequently cannot make the association between the bead and the unpleasant taste.
Characterization of antinociception produced by glutamate microinjection in the nucleus tractus solitarius and the nucleus reticularis ventralis
Authors: Randich A , Roose MG , Gebhart GF
Published: March 2018

Four experiments examined the role of the nucleus tractus solitarius (NTS) and subjacent nucleus reticularis ventralis (NRV) in the production of antinociception. Experiment 1 showed that microinjection of glutamate (50 nmol) into the caudal NTS resulted in inhibition of the tail-flick reflex, hypotension, and mild bradycardia, whereas microinjection of glutamate into the rostral NTS resulted only in hypotension and mild bradycardia. Microinjections of glutamate into the NRV resulted in inhibition of the tail-flick reflex, hypertension, and mild bradycardia. Experiment 2 demonstrated that the magnitude of the antinociceptive and cardiovascular responses resulting from glutamate microinjections into both the depressor and pressor regions were dose dependent. Experiment 3 showed that the antinociceptive effects resulting from microinjections of glutamate into either the depressor or pressor regions could not be antagonized by phentolamine (30 micrograms), methysergide (30 micrograms), or naloxone (30 micrograms) alone, but the combined intrathecal administration of phentolamine and methysergide (7.5, 15, or 30 micrograms of each) attenuated the antinociception resulting from microinjection of glutamate into either depressor or pressor regions in a dose-dependent fashion. Experiment 4 showed that systemic administration of hexamethonium blocked the pressor response produced by microinjection of glutamate into the NRV but did not reduce the antinociceptive effect of the microinjection. These findings are consistent with a role for the NTS and NRV in the production of antinociception.
Colony-stimulating factors as promoters of ameboid microglia
Authors: Giulian D , Ingeman JE
Published: March 2018

Immunomodulators were tested for their ability to stimulate proliferation and biologic activity of ameboid microglia. Only the colony-stimulating factors (CSFs), multipotential-CSF (multi-CSF) and granulocyte/macrophage-CSF (GM-CSF), were potent mitogens for microglia. Other immunomodulators, including interleukin-1, interleukin- 2, interferon gamma, tumor necrosis factor, or granulocyte-CSF (G-CSF), had no effect upon microglial growth in vitro. Multi-CSF or GM-CSF were also observed to induce more rapid phagocytosis of polystyrene microspheres by cultured ameboid cells. In order to determine which immunomodulators alter brain inflammatory responses in vivo, we infused recombinant forms of GM-CSF, multi-CSF, macrophage-CSF, or G-CSF into the cerebral cortex of rats. Within 48 hr after infusion multi-CSF or GM-CSF stimulated the appearance of large numbers of mononuclear phagocytes at the site of injection. These same factors also accelerated the clearance of polystyrene microspheres from the brain. Our observations indicate that certain classes of immunomodulators which are mitogens and activators of ameboid microglia in vitro amplify the inflammatory response of the CNS in vivo by action upon intrinsic brain mononuclear phagocytes.
Do N-methyl-D-aspartate receptors mediate synaptic responses in the mudpuppy retina?
Authors: Coleman PA , Miller RF
Published: March 2018

Whole-cell recordings of amacrine and ganglion cells in the superfused retina-eyecup preparation of the mudpuppy were obtained in order to determine which excitatory amino acid receptor (EAAR) subtype mediates the synaptic responses of these neurons. All third-order retinal neurons tested were depolarized by kainic acid (KA, N-methyl-D- aspartate (NMDA), and quisqualate (QQ). The responses evoked by NMDA were blocked by the addition of D-2-amino-5-phosphonovaleric acid (D- AP5) and D-2-amino-7 phosphonoheptonoic acid (D-AP7) to the perfusate. When the actions of exogenously applied NMDA were completely blocked by D-AP5 and D-AP7, the light-evoked responses of inner retinal neurons persisted without any apparent reduction or, alternatively, a slight enhancement of the response was observed. Light-evoked responses of bipolar, amacrine, and ganglion cells associated with the On pathway were attenuated by L-AP5 in a manner similar to its lower-order homolog L-2-amino-4-phosphonobutyrate (AP4); nevertheless, L-AP5 was not an effective NMDA antagonist. Although synaptic transmission between retinal second- and third-order neurons appears to be mediated by EAARs, the NMDA receptor does not appear to play a prominent role under our experimental conditions. Nevertheless, our results suggest that the racemic mixture of AP5 should not be used as an NMDA antagonist in retinal research, due to the AP4-like actions of its L-enantiomer.
Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions
Authors: Chin GJ , Shapiro E , Vogel SS , Schwartz JH
Published: March 2018

We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3- fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid- dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.
Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum
Authors: Wolf ME , Kapatos G
Published: March 2018

Fluorescence-activated cell sorting (FACS) was used to identify and isolate permeabilized dopaminergic nerve terminals from rat striatum based on immunofluorescent labeling of tyrosine hydroxylase (TH). Striatal synaptosomes were permeabilized by fixation with modified Zamboni fluid. A highly fluorescent subpopulation of particles was detected by FACS following sequential incubation with a monoclonal antibody to TH (LNC 1) and a fluorescein-conjugated secondary antibody. After correcting for nonsynaptosomal particles present in the synaptosomal fraction, analysis of these data suggested that approximately 12–15% of striatal synaptosomes were dopaminergic, consistent with previous estimates. Specific labelling by LNC 1 was decreased if synaptosomes were prepared from rats that had received intraventricular injections of 6-hydroxydopamine. The decrease in labeling was highly correlated with the extent of the lesion as determined by measurement of striatal dopamine levels, suggesting that LNC 1-labeled synaptosomes were derived from nigrostriatal dopamine terminals. In order to verify that LNC 1-labeled synaptosomes were enriched for TH, equal numbers of labeled and control synaptosomes were isolated by FACS and analyzed by SDS-PAGE. LNC 1-labeled synaptosomes were shown by Western blot techniques to be enriched 6-fold for TH compared with control synaptosomes, suggesting that the labeled population consisted almost entirely of dopaminergic synaptosomes.
Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis
Authors: Wallace TL , Johnson EM
Published: March 2018

Cytosine arabinoside (ARA C), a competitive inhibitor of the incorporation of 2′-deoxycytidine into DNA in other cell types, caused a concentration-dependent inhibition of KCl- and insulin-stimulated survival of postmitotic ciliary parasympathetic ganglion neurons, and the nerve growth factor (NGF)-stimulated survival of postmitotic dorsal root ganglion (DRG) sensory neurons in vitro. The IC50 for survival was 2 x 10(-8) M for both types of neurons after 4 d under the culture conditions used. The inhibition of DRG survival by ARA C in the presence of varying concentrations of NGF indicated that ARA C acted as an apparent noncompetitive antagonist of NGF. This cytotoxic effect of ARA C was blocked by 2′-deoxycytidine, but not by cytosine, 2′- deoxyadenosine, 2′-deoxyguanosine, or 2′-deoxythymine, indicating that ARA C was interfering with a deoxycytidine-specific survival process. Cytidine could block ARA C toxicity, but it was 40 times less potent than 2′-deoxycytidine. The blockade of the cytotoxic effect of ARA C by 2′-deoxycytidine indicated that 2′-deoxycytidine was an apparent competitive antagonist of ARA C toxicity. 2′-Deoxycytidine, by itself, was not survival-promoting. Other antimitotic agents, such as adenine arabinoside, thymine arabinoside, 5-fluorodeoxyuridine, 5- bromodeoxycytidine, 5-azadeoxycytidine, and aphidicolin had no effect on neuronal survival at a concentration 5000 times the EC50 of ARA C, indicating that inhibition of DNA synthesis or repair was probably not the mechanism by which ARA C inhibited neuronal survival and that other 2′-deoxynucleosides were not involved in the survival-promoting process. Nitrobenzylthioinosine, an inhibitor of 2′-deoxycytidine and ARA C membrane transport in other cell types, inhibited the cytotoxic effect of ARA C in neurons, suggesting that ARA C entered the neurons through a similar transport mechanism and that ARA C needed to gain access to the inside of the neuron to be effective. These results indicate that ARA C, in addition to being an antimitotic agent for dividing cells, is also cytotoxic for postmitotic neurons. This inhibition of neuronal survival by ARA C is hypothesized to be due to inhibition of a 2′-deoxycytidine-dependent process that is independent of DNA synthesis or repair. Thus, 2′-deoxycytidine may have an important and previously unrecognized role in cellular function that in the case of neurons is critical for survival.