bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain-stem relays mediating stimulation-produced antinociception from the lateral hypothalamus in the rat
Authors: Aimone LD , Bauer CA , Gebhart GF
Published: March 2018

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to examine systematically the organization in the brain stem of pathways mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of lidocaine into the midbrain, dorsolateral pons, or medial medulla resulted in significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (89.1, 67.4, and 73.6%, respectively). Selective lesions of cell bodies in the midbrain or medulla by the neurotoxin ibotenic acid also produced significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (31.6 and 131.6%, respectively), thus revealing relays in the periaqueductal gray and the nucleus raphe magnus located between the LH and the lumbar spinal cord. The failure of ibotenic acid to affect LH- produced descending inhibition when microinjected into the dorsolateral pons, and the significant effect produced by lidocaine microinjected into the same area, implicates fibers of passage in the dorsolateral pons in descending inhibition of the TF reflex produced by focal electrical stimulation in the LH. The fluorescent dye Fast blue and HRP conjugated to wheat germ agglutinin were used to confirm that the area stimulated in the LH has reciprocal connections with the periaqueductal gray and nucleus raphe magnus.
Interactions between the lateral hypothalamus and the periaqueductal gray
Authors: Behbehani MM , Park MR , Clement ME
Published: March 2018

Anatomical and physiological experiments were conducted to characterize the interactions between the lateral hypothalamus (LH) and the periaqueductal gray (PAG) and to determine the role of neurotensin in their interaction. Anatomical studies using injection of Phaseolus vulgaris leucoagglutinin into the LH showed an extensive projection to the ventromedial and the ventrolateral PAG and a less dense projection to the medial and dorsal parts of this region. Physiological experiments were performed on both deeply and lightly anesthetized animals. Electrical stimulation of the LH caused excitation of PAG cells with an onset latency of approximately 14 msec. There was a strong correlation between the response of PAG cells to electrical stimulation and injection of glutamic acid into the LH. Electrical or chemical stimulation of the LH produced an increase in tail flick latency in the lightly anesthetized animals that outlasted the stimulation period. There was a strong correlation between the response of PAG cells to electrical and chemical stimulation of the LH and their response to pressure-injected neurotensin. It is concluded that an excitatory projection from the LH to PAG exists which may involve neurotensin. This pathway may be involved in the analgesia produced by LH stimulation.
Catecholamine toxicity in cerebral cortex in dissociated cell culture
Authors: Rosenberg PA
Published: March 2018

Identification of endogenous toxins and characterization of the mechanisms by which toxins produce cell injury and death may help understand both normal modeling of cell populations and connections in the CNS as well as abnormal cell loss. The toxicity of catecholamines intrinsic to the CNS was investigated using the model system of rat cerebral cortex in dissociated cell culture. All catecholamines tested, including norepinephrine (NE), dopamine, and epinephrine, were toxic to neurons as well as glia at a concentration of 25 microM when added to cultures 24 hr after plating. Toxicity was evident after 48 hr exposure to NE, as monitored by loss of cells from the cultures. Toxicity did not seem to be mediated by adrenergic receptors because, although the beta-adrenergic agonist isoproterenol (but not the alpha-adrenergic agonist phenylephrine) was similar in its toxic effect to NE, the beta- adrenergic antagonist atenolol did not block the toxic effect of NE. Toxicity could be mimicked by hydrogen peroxide, a product of the oxidative degradation of catecholamines. Toxicity of NE was blocked by catalase. The neurotoxin 6-hydroxydopamine (6-OHDA), supposedly selective for catecholaminergic neurons, was found to be toxic over the same concentration range as NE. These results suggest that endogenous catecholamines may play a role in normal and abnormal cell death, and suggest that caution be used in relying on the specificity of 6-OHDA and other supposedly selective neurotoxins.
The interaction of agonists and noncompetitive antagonists at the excitatory amino acid receptors in rat retinal ganglion cells in vitro
Authors: Karschin A , Aizenman E , Lipton SA
Published: March 2018

The pharmacological properties of the interaction between the excitatory amino acid (EAA) analogs kainate and N-methyl-D-aspartate (NMDA) have been examined on the isolated rat retinal ganglion cell preparation. In addition, we have studied the effects on this interaction of 2 noncompetitive NMDA antagonists, the dissociative anesthetic phencyclidine (PCP) and the anticonvulsant MK-801. Electrophysiological measurements were performed with the whole-cell patch-clamp technique on cultured ganglion cells that had been back- labeled with a fluorescent dye. Whereas only 69% of the cells showed responses to NMDA (in the absence of extracellular Mg2+), every ganglion cell responded to kainate under the same conditions. When a given cell was voltage-clamped at -60 mV, the large inward currents elicited by 125 microM kainate generally exceeded the responses evoked by 200 microM NMDA, when present, by 1 or 2 orders of magnitude. There was a poor correlation between the magnitudes of the currents produced by both agonists for the population of cells tested. Furthermore, NMDA proved to be an antagonist for the kainate receptor binding site. Without influencing the kainate-activated currents, PCP (75 microM) and MK-801 (20 microM) completely and reversibly blocked the responses evoked by NMDA (200 microM), independent of the membrane holding potential. The degree of block produced by a submaximal concentration of either antagonist was accentuated by increasing the concentration of NMDA. The independence of NMDA and kainate currents was examined. In the presence of NMDA and PCP (or MK-801), kainate-induced responses were comparable in amplitude to those generated by the application of kainate and NMDA together. Thus, kainate continued to produce an increase in membrane conductance at a time when NMDA-activated currents were blocked by either antagonist. The NMDA antagonism of kainate- induced currents was shown to be constant and independent of PCP or MK- 801. Our results suggest that the 2 EAA analogs might not share a common ionophore, but rather activate separate receptor-ion channel complexes in rat retinal ganglion cell membranes.
The adhesion molecule on glia (AMOG) incorporated into lipid vesicles binds to subpopulations of neurons
Authors: Antonicek H , Schachner M
Published: March 2018

To investigate the functional role of the novel adhesion molecule on glia (AMOG) in cell surface interactions, immunoaffinity-purified AMOG was incorporated into liposomes and measured for its ability to bind to cells in monolayer cultures. AMOG could be incorporated into liposomes in functionally active form after solubilization from membranes in 1% cholate buffer containing soybean lecithin, elution from the AMOG monoclonal antibody column with 4 M MgCl2, containing 1% octylglucoside, and removal of detergent for liposome incorporation by gel filtration. AMOG-containing liposomes bound to neurons, but not to oligodendrocytes, astrocytes, or fibroblasts in early postnatal cerebellar cultures. AMOG-containing liposomes also bound to the pheochromocytoma cell line PC12, but not to neurons in cultures of spinal cord and dorsal root ganglia after various times in vitro. Fab fragments of monoclonal AMOG antibodies, but not of L3 monoclonal antibodies directed against a carbohydrate structure on AMOG, inhibited binding of liposomes. Liposome binding was not reduced by preincubation of cerebellar cells with antibodies to AMOG, to the neuron adhesion molecule L1, the neural cell adhesion molecule N-CAM, or the L3 carbohydrate structure, nor with 2 monoclonal antibodies reacting with neuronal cell surface glycoproteins related to the L2/HNK-1 family. These results show that AMOG is indeed a ligand in adhesion and binds to particular subpopulations of neurons in L1- and N-CAM-independent mechanisms.
Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes
Authors: Hartikka J , Hefti F
Published: March 2018

To characterize the role of NGF in the development of forebrain cholinergic neurons, we established primary cell culture systems to grow these cells under controlled in vitro conditions. Cultures of dissociated cells were prepared from the septal area of fetal (E17) rats, which contained part of the group of basal forebrain cholinergic neurons. Cultures were treated either with NGF (100 ng/ml) or with an antiserum against NGF (1:500 dilution). To assess the influence of non- neuronal cells, 2 types of high-density cultures were prepared: mixed neuronal-glial cultures and pure neuronal cultures. Cholinergic neurons were identified using choline acetyltransferase (ChAT) immunocytochemistry and AChE cytochemistry. Receptors for NGF (NGF-R) were located immunocytochemically using monoclonal antibodies against rat NGF-R. We report that, first, NGF-R are exclusively localized on cholinergic neurons in septal cultures. All neurons labeled with antibodies against NGF-R also contained AChE. Twenty-one percent of all AChE-positive neurons were not stained in NGF-R immunocytochemistry (AChE has earlier been shown to be colocalized with ChAT in septal cultures). Second, NGF treatment increases and anti-NGF treatment reduces the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF promotes survival of septal cholinergic neurons in these cultures. In cultures of high plating density, NGF increased the number of NGF-R and ChAT-positive neurons without affecting the number of AChE-positive neurons in these cultures. These results suggest that exogenous NGF is not required for survival of cholinergic neurons in high-density cultures but stimulates the expression of ChAT and NGF-R. Third, NGF stimulates fiber growth of septal cholinergic neurons, as assessed by computerized image analysis of AChE-positive neurons. Fourth, NGF specifically increases ChAT and AChE activities in septal cultures. These NGF-mediated increases in enzyme activities are more pronounced when neurons are grown together with glial cells. In pure neuronal cultures, NGF increased ChAT and AChE activities by 101 and 16%, and in mixed neuronal-glial cultures by 318 and 87%, respectively. Anti-NGF blocked the effects of NGF but failed to reduce ChAT and AChE activities below control levels in cultures of high plating density. Fifth, astrocytes attenuate the expression of ChAT and AChE by septal neurons in the absence of NGF.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential expression of pseudoconditioning and sensitization by siphon responses in Aplysia: novel response selection after training
Authors: Erickson MT , Walters ET
Published: March 2018

Nonassociative training with a noxious unconditioned stimulus (US) applied to the head or tail of freely moving Aplysia caused a qualitative change in siphon responses to midbody test stimulation, so that the midbody test responses came to resemble the unconditioned siphon response (UR) to the US when tested 1 d after exposure to the US. Such a nonassociative, US-induced transformation of test responses into responses resembling the UR has traditionally been termed “pseudoconditioning.” Short-term pseudoconditioning was compared to sensitization and to habituation in a reduced preparation that used a photocell to distinguish “head-type” siphon responses from qualitatively different “tail-type” responses. Transformation of test responses (pseudoconditioning) was observed only when the type of preexisting alpha response to the midbody test stimulus was different from the UR. Sensitization, defined as a US-induced enhancement of the alpha response to the test stimulus, was observed when the initial alpha response and the UR were of the same type. General sensory facilitation was excluded as a critical mechanism for pseudoconditioning by the observation that the same midbody test response could be transformed to either a head-type or tail-type response, depending on the site of the US, and by the observation that simply increasing the intensity of the midbody test stimulus in the absence of a head or tail US did not produce similar response transformations. These studies demonstrate pseudoconditioning in a preparation amenable to analysis at the level of identified neurons, and draw attention to a distinctive and widespread form of behavioral modifiability that has been neglected by investigators of learning.
Mechanisms of action of peripherally administered cholecystokinin octapeptide on brain stem neurons in the rat
Authors: Raybould HE , Gayton RJ , Dockray GJ
Published: March 2018

We have investigated the pathway and the mechanism by which cholecystokinin octapeptide (CCK-8), given systemically, may influence the discharge of brain stem neurons that have an input from the stomach. Extracellular recordings were made from neurons in the nucleus of the solitary tract (NTS), where vagal afferents terminate, and from neighboring regions of the dorsal medial medulla. Gastric distension and CCK-8 injected intra-aortically close to the stomach evoked either excitatory or inhibitory responses that were abolished by cervical vagal section. In animals from which the celiac/superior mesenteric ganglia were removed, or the gastric antrum resected 2 weeks earlier, responses to gastric distension and CCK-8 were maintained. The effects of CCK-8 are unlikely to be secondary to changes in smooth muscle tone because CCK-8 decreased pressure in the body of the stomach, while distension increased it. Moreover, intravenous noradrenaline and vasoactive intestinal peptide had effects similar to CCK-8 on intragastric pressure, but evoked different patterns of responses from brain stem neurons. The results are consistent with the idea that CCK-8 acts directly on vagal mechanoreceptive endings in the gastric corpus wall. It is well known that peripheral administration of CCK-8 influences short-term regulation of food intake. The effects described here may reflect the pathway by which peripheral CCK influences CNS function.
Regenerating afferent fibers stimulate the recovery of mauthner cell dendritic branching in the axolotl
Authors: Goodman LA , Covell DA , Model PG
Published: March 2018

In the medulla of the axolotl (Ambystoma mexicanum), Mauthner cells (M- cells) occur as a pair of large identifiable neurons at the level of entry of the vestibular nerve (nVIII). Each receives synapses from the ipsilateral nVIII; the terminals can be identified as club endings and are restricted to a specific set of M-cell dendritic branches. We have examined these branches for morphologic changes following long-term deafferentation in the presence and absence of nerve regeneration. Deafferentation was brought about in a group of young larvae by unilaterally severing nVIII. The nerve was allowed to regenerate in half of the larvae. In those remaining, the nVIII ganglion was damaged to preclude or limit nerve regeneration. The contralateral side served as control. After 3 months survival, the larvae were killed and the medullae prepared for microscopy. To estimate the extent of nerve regeneration, axons in the experimental nVIII tract were counted and compared with the number in the control. The mean number of axons in the nVIII tract ipsilateral to intact ganglia indicated that 69% of the fibers had regenerated. In contrast, only 31% regenerated in larvae with damaged ganglia. Electron microscopic analysis of selected sections revealed that the mean number of nVIII terminals per section through M-cells ipsilateral to destroyed ganglia was significantly less than the mean number in analogous sections through either control cells or cells ipsilateral to intact ganglia. Control and experimental M- cells were reconstructed from serial sections. Deprived M-cells had significantly reduced dendritic branching patterns in the region that normally receives nVIII input. On the other hand, the extent of branching on cells receiving regenerated afferents from intact ganglia was like that of their contralateral controls. The distribution of dendritic branches on many reinnervated M-cells, however, was broader than that on control cells. Electron microscopic examination of the displaced dendritic branches (those extending into adjacent tracts) revealed that they received vestibular synapses. Thus, in some animals, regenerated vestibular fibers were not restricted to the nVIII tract. Deafferentation of the M-cells results in a reduction of dendritic branches in the region deprived of vestibular contacts.(ABSTRACT TRUNCATED AT 400 WORDS)
An atlas of a rare neuronal surface antigen in the rat central nervous system
Authors: Stephenson DT , Kushner PD
Published: March 2018

The mature nervous system contains functional synaptic networks composed of neuronal sets and subsets whose identity and maintenance may rely on external surface molecules specific for these neuronal subdivisions. Such molecules may reside permanently on specific neurons, serving to identify those neurons within a complex population. From a collection of monoclonal antibodies made to the Torpedo cholinergic synaptosome preparation, we have identified several antibodies that bind the surface of some, but not all, neurons of the mature rat brain (Kushner and Stephenson, 1983; Kushner, 1984). In the present study we catalog which neurons of the entire rat brain and spinal cord are immunopositive for one of these antibodies, Tor 23. The atlas we have compiled can be used (1) on a practical level to guide affinity purifications and neuronal cell sorting, and (2) more esoterically to address whether surface antigenic sets of neurons share or define a common functional property. In the forebrain, Tor 23- positive neurons predominate as a rare cell type of the inner cortical laminae. In the midbrain, few cells stain. In the hindbrain, labeled neurons are dispersed among several nuclei. The exceptions to these observations, areas that almost exclusively contain Tor 23- immunopositive neurons, are the spinal cord ventral horn, the deep cerebellar nuclei, some cerebellar-related nuclei, selected auditory nuclei, the supraoptic and paraventricular nuclei of the hypothalamus, and the CA2 pyramidal neurons of the hippocampus. The neurons that bind Tor 23 are related biochemically by virtue of that shared epitope alone. Whether they are related in a functional manner is a separate issue. Tor 23 demarks many motor-related structures, specifically, the ventral horn motor lamina, the orofacial motor nuclei 5 and 7, the ambiguus nucleus, the deep cerebellar nuclei, the pontine reticulotegmental nucleus, the lateral reticular nucleus, the gigantocellular reticular nucleus, the red nucleus, and neurons within the motor and somatosensory cortices. The overlap of Tor 23 with motor structures suggests that Tor 23 is a motor system marker.
Immunocytochemical localization by electron microscopy of 2'3'-cyclic nucleotide 3'-phosphodiesterase in developing oligodendrocytes of normal and mutant brain
Authors: Braun PE , Sandillon F , Edwards A , Matthieu JM , Privat A
Published: March 2018

Oligodendrocytes or their putative progenitors were the only cells found to be immunoreactive to polyclonal antisera against the enzyme 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in developing and mature brains of rats and mice, as visualized by light and electron microscopy. Prior to myelination (day 6), oligodendrocytes of the corpus callosum have reticular networks of CNP-containing filopodia, in addition to abundant CNP throughout the cytoplasm. Some glioblast-like cells of the subventricular zone are also immunoreactive to anti-CNP, suggesting that, as progenitors of oligodendroglia, they express this myelination-related protein as one of the earliest events in myelinogenesis. Following the commencement of myelination (day 15), many oligodendrocytes lose much of their lacelike network of fine projections, possessing, instead, larger CNP-filled processes that extend to myelin-bearing fibers. CNP was always found only in the cytoplasm-containing compartments of the cells and myelin sheaths; neither lamellae nor cellular membranes were immunostained. These data support our contention that CNP is not an intrinsic membrane protein, despite its strong interaction with membrane components when cells are disrupted. In mutant (mld) mice (day 25), the many distended and uncompacted oligodendroglial processes that invest axons with only a few turns of membrane contained cytoplasmic CNP, accounting for the elevated levels of CNP activity previously noted in tissue fractions.
Proprioceptive inputs to nonspiking local interneurons contribute to local reflexes of a locust hindleg
Authors: Burrows M , Laurent GJ , Field LH
Published: March 2018

Local reflexes of a leg of the locust Schistocerca gregaria (Forskal) can be elicited by selective stimulation of a proprioceptor (the femoral chordotonal organ) at the femorotibial joint. Motor neurons are either excited or inhibited, so that a coordinated reflex response of a leg results. At the same time, some nonspiking local interneurons are either excited or inhibited by the inputs from these proprioceptive afferents. Altering the membrane potential of an individual, nonspiking interneuron can either increase or decrease the response of the participating motor neurons to the proprioceptive stimulus and thereby alter the gain of the reflex. To determine the pathways, and to understand the role of the nonspiking interneurons in mediating these reflex effects, recordings were made simultaneously from these interneurons and afferent neurons. The excitation of a particular nonspiking local interneuron is produced monosynaptically by the afferent neurons. Chemically mediated EPSPs consistently follow sensory spikes with a latency that is the same as that for the known parallel, direct connections made by these sensory neurons with motor neurons (Burrows, 1987a). The chordotonal afferents and the branches of the local interneurons project to the same regions of neuropil. In contrast, the simplest inhibitory pathway is disynaptic, involving spiking local interneurons. The afferents make direct excitatory connections with some of these spiking interneurons, which then make direct inhibitory connections with a nonspiking interneuron. Interactions between the local interneurons add to the complexity of the pathways.(ABSTRACT TRUNCATED AT 250 WORDS)
The extending astroglial process: development of glial cell shape, the growing tip, and interactions with neurons
Authors: Mason CA , Edmondson JC , Hatten ME
Published: March 2018

To analyze how astroglial cells attain the complex shapes that support neuronal migration and positioning in vitro (Hatten et al., 1984; Hatten 1985), early postnatal mouse cerebellar cells were plated in microcultures, and glial process outgrowth was monitored by high- resolution time-lapse video microscopy combined with immunocytochemical localization of antisera to glial filament protein (GFP), and by electron microscopy. The 2 principal astroglial forms seen in these cultures, stellate and Bergmann-like (Hatten et al., 1984), begin to develop their distinctive shapes by the outgrowth of processes in the first 8 hr after the cells are plated. Glial process extension is most vigorous in this period, resulting predominantly in stellate forms. A second population of glial cells, having fewer, longer processes reminiscent of Bergmann glia in vivo, first appears about 5 hr after plating. During the next 16–24 hr, while the stellate cells only slightly increase their process length, the bipolar cells double their length. The most striking feature of the elongating glial process is its highly motile tip, which rapidly extends microspikes and lamellopodia. Unlike the neuronal growth cone, which is the expanded terminal of a thin neurite shaft, the glial growing tip forms the end of a wide, paddle-like process that is filled with motile mitochondria and masses of glial filaments, and is bordered by an undulating lamella fringed by microspikes. Soon after the emergence of glial processes, cell-cell interactions between the growing glial process tip and granule neurons occur. Within minutes of an initial encounter between the glial process and the neuron, contact relationships that are stable during the observation period form between the cells. Subsequently, many neurons extend a small neurite onto the glial process, and astroglial process extension continues by the movement of the glial growing tip out beyond the neuron. Thus, cerebellar astroglia in vitro develop complex shapes in the same fashion as do neurons: the outgrowth of processes tipped by a motile ending. The growing tips of astroglial processes interact with neurons, resulting in the stable association of neurons and glia.
Changes in cytoskeletal proteins in the rat facial nucleus following axotomy
Authors: Tetzlaff W , Bisby MA , Kreutzberg GW
Published: March 2018

Changes in L-35S-methionine incorporation into cytoskeletal proteins of the facial nucleus of the rat were studied at various times after unilateral crush or resection of the facial nerve by using 2- dimensional gel electrophoresis and fluorography. We found an increase in labeling of actin and tubulin and a decrease in the 68 kDa and 150 kDa neurofilament polypeptides (200 kDa was not studied). The increase in actin and decrease in neurofilament polypeptide labeling was already significant by 24 hr after nerve resection. These changes were more pronounced after nerve resection than after nerve crush on day 7; actin labeling increased to 270%, and tubulin to 205% of contralateral normal nuclei after resection, whereas both proteins increased to only 165% after crush. Neurofilament labeling decreased to 28% of the contralateral side after resection and to 50% after crush. Immunocytochemistry with a monoclonal antibody to the 150 kDa neurofilament component revealed decreased immunoreactivity in the axotomized facial axons at the inner facial genu, 1 cm proximal to the crush. In contrast, neurofilament immunoreactivity was not decreased in the axotomized perikarya of the facial motoneurons. All changes returned to normal 3 weeks after crush. When axonal regeneration was impeded by nerve resection, incorporation into tubulin remained elevated and into neurofilament proteins remained depressed. Actin returned to normal after either nerve resection or crush. We conclude that the synthesis of tubulin and neurofilament proteins following axotomy is regulated by successful axonal regeneration and/or target contact. Actin synthesis seems to be regulated independently of target- derived factors.
Compartmentalization of anterogradely and retrogradely transported organelles in axons and growth cones from chick optic tectum
Authors: Cheng TP , Reese TS
Published: March 2018

Previous work suggests that organelles contacting microtubules in axons are in fast transport. Here, we examine the distribution of organelles contacting microtubules in growing axons and growth cones from chick optic tectum. Five axon segments, each 10 microns long, and 4 entire growth cones were reconstructed from serial electron micrographs of quick-frozen, freeze-substituted chick optic tectum. Organelles contacting microtubules in axons are evenly distributed along all microtubules. Smaller organelles, presumably in anterograde transport, are enclosed in fascicles of microtubules, while larger organelles in retrograde transport lie outside the fascicles. In contrast, organelles contacting microtubules are prevalent only in the most proximal parts of the growth cone, before the microtubule fascicles splay out more distally. The distance between noncontacting organelles and microtubules also becomes progressively greater, reaching a maximum in the mid- and more distal region of the growth cone. Contacts with microtubules of both the smaller, presumably anterogradely transported organelles, as well as the larger, presumably retrogradely transported organelles, abruptly become less frequent in the proximal midregion of the growth cone. It is therefore of possible significance in stopping and starting microtubule-based organelle transport that microtubules change from a straight to an undulating configuration in the midregion of the growth cone. The decrease in organelle binding to microtubules at the demarcations between the straight and undulating microtubule segments may depend on proteins or other local factors as well as the splaying out of the microtubule bundles.
Inward rectification in response to FMRFamide in Aplysia neuron L2: summation with transient K current
Authors: Thompson S , Ruben P
Published: March 2018

The response of Aplysia abdominal ganglion neuron L2 to the molluscan neuroactive peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) was studied in voltage-clamp experiments. In all of the experiments, focal application of the peptide to the soma activated an inward rectifier current and reduced the apparent amplitude of the transient K current, IA. In a few cells, Na and K currents were activated in addition to these effects. Voltage-jump experiments were performed to study the ionic dependence, kinetics, and voltage dependence of the inward rectifier. Inward rectification increased exponentially during hyperpolarizing pulses and recovered exponentially on return to the resting potential. The reversal potential was variable, but was near -40 mV at the beginning of experiments. Inward rectification was insensitive to changes in external Na, Ca, or K concentration, but lowering the external Cl concentration had complicated effects on current amplitude. When KCl microelectrodes were used, perfusion with low-Cl external saline increased the amplitude of the peptide-dependent inward rectifier and shifted its reversal potential to a more positive voltage. With KAc microelectrodes, perfusion with low-Cl saline reduced the amplitude of the current. Inward rectification increased when a KAc microelectrode was withdrawn and replaced with a low-resistance KCl electrode, even when there was no measurable change in reversal potential. These results suggest that the FMRFamide-dependent inward rectifier is a Cl current that, like the current described by Chesnoy-Marchais (1982, 1983), is modulated by intracellular Cl. FMRFamide reduced the apparent amplitude of IA without affecting the voltage dependence of IA activation or inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Organization of adult motor cortex representation patterns following neonatal forelimb nerve injury in rats
Authors: Donoghue JP , Sanes JN
Published: March 2018

Somatotopic representation patterns in the motor cortex (MI) of rats that had a unilateral forelimb amputation on the first postnatal day were examined after 2–4 months of survival. Intracortical electrical stimulation and recording techniques were used to map the somatic representation in MI and in the somatic sensory cortex (SI). In normal rats, vibrissa, forelimb, and hindlimb areas comprise the bulk of the MI representation. Stimulation within the forelimb area elicits elbow, wrist, or digit movements at the lowest current intensities. The proximal limb representation appears to be contained within the distal forelimb area, since shoulder movements are nearly always evoked by stimulating at higher current intensities at some distal forelimb sites. In agreement with previous studies, the distal forelimb representation overlapped the adjacent part of the granular SI cortex. Following removal of the forelimb at birth, 3 novel features of MI organization were observed. First, the areas from which stimulation evoked movements of the vibrissa or the shoulder musculature were larger than normal. Stimulation thresholds were lower than those required for comparable movements in normal rats throughout these areas, suggesting that nerve section had not simply unmasked a high- threshold representation. Second, vibrissa movements were more commonly paired with movements of the proximal forelimb muscles at the same site. Third, stimulation in the adjacent granular SI cortex failed to evoke shoulder or trunk movements, although receptive-field mapping in this region showed that cells were responsive to cutaneous stimulation of the trunk and shoulder region. These results indicate that several organizational features develop differently in MI following perinatal nerve injury: certain remaining muscle groups have enlarged cortical representations, there is a strengthening of some normally weak connections from MI to the proximal musculature, and muscles are grouped in unusual combinations. These data demonstrate that the formation of MI representation patterns is strongly influenced by nerve injury during the perinatal period.
A monoclonal antibody to the insect prothoracicotropic hormone
Authors: O'Brien MA , Katahira EJ , Flanagan TR , Arnold LW , Haughton G , Bollenbacher WE
Published: March 2018

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. “Big” PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high- pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole- mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole- mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.
Distribution of somatostatin immunoreactivity in the human dentate gyrus
Authors: Amaral DG , Insausti R , Campbell MJ
Published: March 2018

In previous immunohistochemical studies in the rat and monkey, a system of somatostatin-positive neurons and fibers was observed in the dentate gyrus of the hippocampal formation. In both species, somatostatin- immunoreactive cell bodies are located primarily in the deep or polymorphic layer of the dentate gyrus, and they give rise to a fiber system that terminates principally in the outer two-thirds of the molecular layer. In the present study, we employed the same antisera and staining procedures to determine whether the organization of the somatostatin system in the human dentate gyrus is similar to that seen in the rat and nonhuman primate. Sections of human postmortem brain material incubated with antisera directed against somatostatin 28 (S320) or somatostatin 28 (S309) demonstrated a heterogeneous population of immunoreactive cells in the hilar region of the human dentate gyrus. Fiber staining was observed both in the hilar region and throughout the molecular layer, but the densest fiber and terminal plexus were observed in the outer two-thirds of the molecular layer. In addition, there were forms of somatostatin-immunoreactive profiles in the human sections that were not previously observed in the rat or monkey. Immunoreactive, grapelike clusters of apparently large, axonal varicosities were commonly observed, for example, as were dendritic profiles containing typical dendritic spines. In general, however, staining for somatostatin immunoreactivity in the human dentate gyrus presented a picture qualitatively similar to that observed in the rat and monkey. Thus, immunohistochemical methods have allowed the analysis of a chemically defined neural system in the human brain that has been extensively studied in rat and monkey brains with both experimental and immunohistochemical methods. That the pattern of labeling in the human sections closely parallels that observed in the experimental animals provides support for the contention that immunohistochemical methods can reliably be employed to determine the normal neuroanatomical organization of the human brain. These methods may also be particularly applicable for the analysis of pathological brain conditions. In particular, alterations of the hippocampal somatostatin system have been associated with both Alzheimer's disease and temporal lobe epilepsy. It would be of interest, therefore, to apply immunohistochemical procedures to determine whether the anatomical organization of the human hippocampal somatostatin system is altered in these diseases.
Spontaneous and harmaline-stimulated Purkinje cell activity in rats with a genetic movement disorder
Authors: Stratton SE , Lorden JF , Mays LE , Oltmans GA
Published: March 2018

The genetically dystonic rat (dt) displays a complex movement disorder in the absence of morphological defects in the nervous system. This mutant is also insensitive to the tremorogenic effects of harmaline. Because harmaline is known to act on the cells of the inferior olive to induce activity at the tremor frequency in the olivocerebellobulbar pathway, this pathway has been investigated as a possible site of a defect in the dt rat. Biochemical studies suggested the presence of abnormalities at the level of the Purkinje cell or its afferent input. Thus, the present study investigated the harmaline response of Purkinje cells in dt rats and unaffected littermate controls with extracellular single-unit recording techniques. The spontaneous, simple spike and complex spike firing rates of dt rats were significantly lower than those of normal littermate controls. In normal rats, 2 responses to systemic harmaline injection were seen. Simple spikes were either completely suppressed for periods of 30–180 min, or were intermittently suppressed, pausing repeatedly for periods of 1–18 sec. Cells that showed complete suppression of simple spike activity also showed increased frequency and rhythmicity of complex spikes. In dt rats, intermittent simple spike responses were seen in a proportion (41%) similar to that in normal rats (53%). However, the proportion of cells showing high-frequency, rhythmic, complex spikes and complete suppression of simple spikes was low in the dt rats in comparison with littermate controls (18 versus 47%). In addition, 41% of the cells from dt rats displayed no change, or an anomalous change, in firing patterns in response to harmaline. Since the rhythmic activation of olivary neurons that results in the rhythmic, complex spike discharge of Purkinje cells is assumed to be responsible for the appearance of harmaline tremor, the failure of the dt rat to display tremor is most likely due to a failure at the olivocerebellar level, rather than at a site efferent to the cerebellum.
Serotonergic and serotonin-accumulating neurons in the goldfish retina
Authors: Marc RE , Liu WL , Scholz K , Muller JF
Published: March 2018

Autoradiography of goldfish retinas incubated in micromolar levels of 3H-serotonin displayed 3 kinds of labeled somas in the inner nuclear layer: S1 amacrine cells with heavy labeling, large somas, and a sparse distribution (approximately 93/mm2); S2 amacrine cells with moderate labeling, smaller somas, and a denser distribution (approximately 500/mm2); and a subset of bipolar cells with light labeling, small somas, and a very dense distribution (approximately 4000/mm2). Serotonin-like immunoreactivity was observed only in S1 amacrine cells and their synaptic terminals. Radiolabeled terminals in the inner plexiform layer formed 4 strata that were differentially assigned to the 3 cell types. S1 amacrine cells arborized in sublayers 1 and 5, received inputs from type a1 bipolar cells and amacrine cells, and made synapses on other amacrine cells, type a1 bipolar cells and unidentified processes. Thus, S1 amacrine cells seem to receive significant input from “off-center” pathways. S2 amacrine cells arborized in sublayer 3 and made synapses onto amacrine cells. Labeled bipolar cell terminals were exclusively located in sublayer 2 and were identified as type a2 mixed rod-cone bipolar cells. We conclude that the S1 amacrine cell is truly serotonergic and that radiolabeling of S2 amacrine cells and type a2 bipolar cells is due to cross-specificity for another carrier or processes unrelated to their neurochemical identities. These observations partially reconcile many previous observations on the types, numbers, and synaptologies of teleost retinal neurons identified by different markers for indoleaminergic transmission.
Regulation of hypothalamic magnocellular neuropeptides and their mRNAs in the Brattleboro rat: coordinate responses to further osmotic challenge
Authors: Sherman TG , Day R , Civelli O , Douglass J , Herbert E , Akil H , Watson SJ
Published: March 2018

A paradigm was developed for the chronic osmotic stimulation of homozygous diabetes insipidus rats of the Brattleboro strain, a strain that fails to synthesize vasopressin. This study examines the adaptation of 2 sets of coexisting peptide hormone magnocellular neurons in the hypothalamoneurohypophyseal system (HNS) of Long Evans (LE), Brattleboro heterozygote (HZ), and Brattleboro homozygote (DI) rats: (1) the arginine8-vasopressin (AVP)/dynorphin (DYN) neurons, and (2) the oxytocin (OT)/cholecystokinin (CCK8) neurons of the paraventricular and supraoptic nuclei, which project to the posterior pituitary. The regimen of chronic intermittent salt-loading (CISL) involved the replacement of 2% saline for normal drinking water for 18 hr/d. This protocol effectively increased plasma levels of AVP and OT in LE and HZ rats, oxytocin in DI rats, and maintained the posterior pituitary in a state depleted of AVP, OT, CCK, and peptides derived from pro-dynorphin: DYN A 1–17, DYN A 1–8, and DYN B 1–13. The ratio of pituitary DYN A 1–17 to DYN A 1–8 content in DI rats or in LE, HZ, and DI rats following 6 d of CISL suggests a preferential release of DYN A 1–17 during periods of chronic secretory activity. In response to chronic secretory activity, mRNAs for AVP, OT, DYN, and CCK increased 1.5–2-fold in all 3 AVP rat strains, with mRNAs for coexisting peptide hormones displaying parallel increases. Mutant AVP mRNA in the DI rat was expressed at very low levels and DYN mRNA in very high levels, with each of these mRNAs continuing to be regulated by CISL in a normal manner. These results suggest a regulatory relationship between AVP and OT neurons, in which vasopressin neurons are feedback-regulated by AVP, most likely via plasma osmolarity, and that oxytocin neurons are modulated by peptides derived from pro-dynorphin.
Differential expression of vasopressin alleles in the Brattleboro heterozygote
Authors: Sherman TG , Watson SJ
Published: March 2018

A solution hybridization/RNase protection assay for the molar quantitation of vasopressin and oxytocin mRNAs, using synthetic complementary RNA probes, is described. This assay was optimized to permit the identification of vasopressin (AVP) mRNAs containing the frame-shift point deletion causing inheritable diabetes insipidus in the Brattleboro strain of rat. Examination of RNA from hypothalamic magnocellular tissue punches found that of the 86.1 x 10(-18) mol [86.1 attomoles (amol)] of AVP mRNA detected in the Brattleboro heterozygote paraventricular (PVN) nucleus, 5.2% could be shown to be mutant AVP mRNA (AVPd RNA). The percentage of AVPd RNA increased dramatically to 18.1% after 6 d of chronic intermittent salt-loading. Similar percentages and percentage increases of AVPd RNA were detected in the heterozygote supraoptic nucleus (SON). These values were contrasted with those found in parallel studies in both Long Evans and Brattleboro homozygotes, and compared with values for oxytocin (OT) mRNA in all 3 AVP rat genotypes. The results of continued osmotic regulation of the mutant AVP gene, the low native levels of AVPd RNA found in both the Brattleboro heterozygote and homozygote, and the magnitudes of AVPd expression change with chronic osmotic challenge were interpreted as indicating that (1) in the diploid rat genome, both AVP alleles are transcribed, (2) the osmotic regulation of the mutant AVP gene is normal, and (3) the low levels of AVPd mRNA are consistent with a shorter-than-control effective mRNA half-life.
Neuropeptide Y action in the rat hippocampal slice: site and mechanism of presynaptic inhibition
Authors: Colmers WF , Lukowiak K , Pittman QJ
Published: March 2018

Neuropeptide Y (NPY), the most abundant peptide in mammalian CNS, has been shown to inhibit excitatory neurotransmission presynaptically at the stratum radiatum-CA1 synapse in the in vitro rat hippocampal slice. We examined the site and mechanism of this inhibition in a series of in vitro intra- and extracellular recordings in areas CA1 and CA3, the source of much of the excitatory synaptic input to the CA1 neurons. NPY's inhibitory action at the stratum radiatum-CA1 synapse was unaffected by high concentrations of the antagonists bicuculline, theophylline, or atropine, suggesting that it does not act by stimulating the release of the known presynaptic inhibitory transmitters GABA, adenosine, or ACh, respectively. Bath application of 10(-6) NPY, a concentration that strongly inhibited the stratum radiatum-CA1 synapse had no effect on CA3 neuron resting potential, input resistance or action potential amplitude, threshold, or duration. NPY also does not alter the amplitude or duration of the prolonged CA3 action potentials evoked in the presence of TTX, tetraethyl-ammonium, and elevated external Ca2+ or those evoked in the presence of TTX and Ba2+ ions. NPY therefore does not alter the passive or active properties of the somata of the presynaptic CA3 neurons. Neither the afferent fiber volley of the Schaffer collaterals in stratum radiatum of area CA1 nor the excitability of the CA3 terminals in CA1 was affected by NPY application. However, application of the transient K+ current blocker, 4-aminopyridine (4-AP) at concentrations of 10 and 50 microM, completely abolished the action of 10(-6) M NPY on the stratum radiatum-CA1 excitatory synaptic potentials. This action of 4-AP could be reversed by reducing extracellular Ca2+ concentrations from a control level of 1.5 to 0.7 mM (in 10 microM 4-AP) and to 0.5 mM (in 50 microM 4-AP). The evidence suggests that NPY inhibits excitatory synaptic transmission at the Schaffer collateral-CA1 synapse by acting directly at the terminal to reduce a Ca2+ influx.