Acetylcholine levels increase in rat hippocampus following acute septal lesions: Evidence for interactions between cholinergic and noncholinergic neurons

Acute septal lesions in rat brain resulted in elevation of the amount of particle-bound acetylcholine in the hippocampus irrespective of the extent of damage to the cholinergic septohippocampal projection. Changes in the high affinity choline uptake in the hippocampus were, however, proportional to the degree of destruction of this projection. The results are discussed in terms of possible interactions between the cholinergic and noncholinergic pathways in the system investigated.     

Evidence for interactions between target selection and visual fixation for saccade generation in humans

We examined the processes controlling selective orientation, specifically the processes required for generating saccadic eye movements in humans. Before a saccadic eye movement can be initiated, active visual fixation must be disengaged from the current point of fixation and a new target selected. We investigated whether these neural processes occur independently or interactively by devising a simple, multimodal choice reaction task in which subjects were asked to direct their gaze away from a central visual fixation target to an eccentric visual target while ignoring a simultaneous auditory distractor. Subjects had more difficulty suppressing incorrect movements toward the distractor when the fixation target was extinguished prior to onset of the eccentric target than when the fixation target remained illuminated during eccentric target presentation. Subjects with the shortest saccadic reaction times produced the most incorrect movements. These results support a recent hypothesis suggesting that the processes of disengaging active visual fixation and selecting a new saccade target are interrelated and arise, at least in part, from a change of activity within the superior colliculus.     

Reciprocal synapses between cholinergic axons and small granule-containing cells in the rat cardiac ganglion

Electron microscopy of the rat cardiac ganglion shows occurence of small granule-containing cells that form reciprocal synaptic junctions with cholinergic terminals. At the synaptic junctions which are from axon to granule-containing cell, the intraaxonal vesicles are clustered against the junctional axolemma, but dense-cored vesicles in the postynaptic cell do not cluster towards the membrane densities in these synapses. By contrast, the synaptic zone polarized in the opposite direction shows an absence of axonal vesicles in close proximity to the postsynaptic axolemma, but there is a marked aggregation of dense-cored vesicles towards the presynaptic specializations of granule-containing cells. The synaptic zones are multifocal rather than bifocal, and the minimal distance separating each synaptic zone is about 0.3 μ. These findings may indicate that cholinergic excitation of some or all granule-containing cells causes a reciprocal inhibition of one or more cholinergic terminals.     

Influence of age on nuclear bodies and nuclear volume in pituicytes of the rat neurohypophysis

This study has analyzed age-related changes in the nuclear organization of pituicytes of the rat. The cytological study of the cell nucleus and the quantitative analysis of nuclear bodies (NBs) were performed on ultrathin sections. Nuclear diameter, perimeter, and area were measured on semithin sections, and nuclear volume was estimated from these data. The nucleolus was mainly composed of a few large fibrillar centers with their associated dense fibrillar component, whereas the granular component tended to form large masses at the nucleolar periphery. The most frequent configuration of NBs was a globular inclusion composed of a fibrillar capsule with a core that contained a few electron-dense granules. Intranuclear glycogen was detected on rare occasions and only in old rats. The proportion of nuclear sections containing NBs increased significantly from 1.5% in 3-month-old rats to 8.6% in 18-month-old rats. A significant increase in the nuclear volume was detected in older rats with respect to the younger ones (157 +/- 69 vs. 98 +/- 43 microns 3, mean +/- S.D.). Our results suggest an age-related activation of nuclear metabolism in pituicytes resulting in a nuclear expansion and an increase in the frequency of appearance of NBs. This activation might be a reactive cellular event induced by the degenerative changes in neurosecretory nerve endings naturally occurring in older animals.     

Evidence for a dissociation between cardiovascular and behavioral reactivity in the spontaneously hypertensive rat

Spontaneously hypertensive rats of the Okamoto strain (SHR) were compared with normotensive rats of the Wistar-Kyoto strain (WKY) on the acoustic startle response in rats prepared for simultaneous blood pressure recordings. Blood pressure was continuously recorded by means of an indwelling cannula in the caudal tail artery. The presentation of the startle stimulus caused a blood pressure response in both strains consisting of an initial increase in blood pressure followed by a decrease and after that a longer lasting, but less pronounced second increase in pressure. The startle-elicited increase in blood pressure was significantly elevated in SHRs and at the same time the acoustic startle response was depressed as compared to WKY rats. These data indicate a dissociation between cardiovascular and behavioral reactivity in the SHR.     

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC₅₀ of ∼2 n m , VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V_1a receptor antagonist. This pharmacological profile matches the one for VP- and OT-evoked calcium signals in pituicytes, consistent with the fact that VP-induced taurine efflux is blocked by BAPTA-AM. However, BAPTA-AM also blocks the taurine efflux induced by a 270 mosmol l⁻¹ challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium-mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) suggests the same pathway for VP-induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l⁻¹). Moreover, it is likely that the osmosensitivity of the taurine channel is up-regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.     

Inhibitory interactions between spiny projection neurons in the rat striatum

The spiny projection neurons are by far the most numerous type of striatal neuron. In addition to being the principal projection neurons of the striatum, the spiny projection neurons also have an extensive network of local axon collaterals by which they make synaptic connections with other striatal projection neurons. However, up to now there has been no direct physiological evidence for functional inhibitory interactions between spiny projection neurons. Here we present new evidence that striatal projection neurons are interconnected by functional inhibitory synapses. To examine the physiological properties of unitary inhibitory postsynaptic potentials (IPSPs), dual intracellular recordings were made from pairs of spiny projection neurons in brain slices of adult rat striatum. Synaptic interactions were found in 9 of 45 pairs of neurons using averages of 200 traces that were triggered by a single presynaptic action potential. In all cases, synaptic interactions were unidirectional, and no bidirectional interactions were detected. Unitary IPSPs evoked by a single presynaptic action potential had a peak amplitude ranging from 157 to 319 microV in different connections (mean: 277 +/- 46 microV, n = 9). The percentage of failures of single action potentials to evoke a unitary IPSP was estimated and ranged from 9 to 63% (mean: 38 +/- 14%, n = 9). Unitary IPSPs were reversibly blocked by bicuculline (n = 4) and had a reversal potential of -62.4 +/- 0.7 mV (n = 5), consistent with GABA-mediated inhibition. The findings of the present study correlate very well with anatomical evidence for local synaptic connectivity between spiny projection neurons and suggest that lateral inhibition plays a significant role in the information processing operations of the striatum.     

Relationship between scaffold channel diameter and number of regenerating axons in the transected rat spinal cord

Regeneration of endogenous axons through a Schwann cell (SC)-seeded scaffold implant has been demonstrated in the transected rat spinal cord. The formation of a cellular lining in the scaffold channel may limit the degree of axonal regeneration. Spinal cords of adult rats were transected and implanted with the SC-loaded polylactic co-glycollic acid (PLGA) scaffold implants containing seven parallel-aligned channels, either 450 μm (n = 19) or 660 μm in diameter (n = 14). Animals were sacrificed after 1, 2 and 3 months. Immunohistochemistry for neurofilament expression was performed. The cross-sectional area of fibrous tissue and regenerative core was calculated. We found that the 450 μm scaffolds had significantly greater axon fibers per channel at the 1 month (186 ± 37) and 3 month (78 ± 11) endpoints than the 660 μm scaffolds (90 ± 19 and 40 ± 6, respectively) (p = 0.0164 and 0.0149, respectively). The difference in the area of fibrous rim between the 450 and 660 μm channels was most pronounced at the 1 month endpoint, at 28,046 ± 6551 and 58,633 ± 7063 μm², respectively (p = 0.0105). Our study suggests that fabricating scaffolds with smaller diameter channels promotes greater regeneration over larger diameter channels. Axonal regeneration was reduced in the larger channels due to the generation of a large fibrous rim. Optimization of this scaffold environment establishes a platform for future studies of the effects of cell types, trophic factors or pharmacological agents on the regenerative capacity of the injured spinal cord.     

Regeneration of long spinal axons in the rat

Summary To investigate regeneration of long spinal axons, the right lateral column of the rat spinal cord was cut at high cervical, low cervical, midthoracic or lumbar level, and one end of an autologous sciatic nerve segment was grafted to the spinal cord at the site of incision. Three to six months after operation, the origin of axons in the grafts was traced retrogradely with horseradish peroxidase injected into the grafts and, in some cases, anterogradely with radioautography of tritiated amino acids injected into the brainstem. Axons from each of the major lateral spinal tracts arising in the brainstem as well as axons ascending from the lower spinal cord succeeded in growing into low cervical grafts. However, long descending axons rarely regenerated after midthoracic or lumbar injury; axons ascending from lumbar segments of the spinal cord usually failed to enter high cervical grafts. Differences in axonal regrowth at the four segmental levels were not simply attributable to dwindling of axonal number in fibre tracts. Axonal regeneration from Clarke's column or the red nucleus was observed only with lesions causing atrophy of many neurons.There was no obvious example of a fibre tract in the lateral spinal columns from which axons failed to regenerate nor from which axons regenerated exceptionally well. Under the conditions of these experiments, the distance from cell body to injury appeared to be an important determinant of axonal regeneration.     

Morphological plasticity and rearrangement of cytoskeletons in pituicytes cultured from adult rat neurohypophysis.

The adult rat neurohypophysis reveals drastic morphological plasticity of neuron-glial organization during chronic physiological stimulation. Pituicytes are modified astrocytes in the neurohypophysis, and shape conversion of them largely contributes to the morphological plasticity. The present study aimed to investigate the receptor-mediated mechanism for shape conversion of the pituicyte morphology, particularly in relation with changes of cytoskeletal organization. The cultured pituicytes from adult rat neurohypophysis were mostly flat amorphous shape in normal salt solution. Histochemical experiments showed that thick bundle of microfilament (stress fibers) and fine fibers of microtubule distributed evenly within the pituicyte. When pituicytes were treated with adenosine (more than 1 microM), isoproterenol (IPR); beta-agonist, more than 10 nM), and dibutyryl cyclic AMP (dBcAMP, 1 mM), the pituicyte morphology changed from flat to stellate shape. Upon treatment with dBcAMP, stress fibers within pituicyte cytoplasm disappeared, and microtubule assembled in the cellular processes and cytoplasm surrounding the nucleus. Pretreatment with colchicine (microtubule-disrupting agent, 25 microM) and orthovanadate (tyrosine phosphatase inhibitor, 1 mM) prevented dBcAMP-induced stellation of the pituicyte morphology. Treatment with sphingosine (protein kinase C inhibitor, 10 microM), W-7 (calmodulin dependent protein kinase inhibitor, 40 microM), ML-9 (myosin light chain kinase inhibitor, 20 microM), and cytochalasinB (CytB; microfilament disrupting agent, 5 microM), induced stellation of the pituicyte morphology. Treatment of endothelin-1 (more than 0.1 nM) and endotheline-3 (more than 0.1 nM) reverted dBcAMP-induced stellation of the pituicyte morphology to original flat one and also reverted arrangement of cytoskeletons of stress fiber and microtubules as seen in control one. The present results reveal that pituicyte shape conversion is mediated via beta-adrenergic, adenosine and endotheline and depend on rearrangement of stress fibers and microtubules. In addition, the mechanism of shape conversion of pituicytes cultured from adult neurohypophysis is quite similar to that of astrocytes cultured from neonatal brains and possibly is useful for understanding morphological plasticity of adult brains.     

Bursts and hyperexcitability in non-myelinated axons of the rat hippocampus

Strict control over the initiation of action potentials is the primary task of a neuron. One way to lose proper spike control is to create several spikes, a burst, when only one should be initiated. We describe a new site for burst initiation in rat hippocampal CA3 neurons: the Schaffer collateral axons. These axons lack myelin, are long, extremely thin, and form synapses along their entire paths, features typical for many, if not most cortical axons in the mammalian brain. We used hippocampal slices and recorded from individual Schaffer collateral axons. We found that single action potentials were converted into bursts of two to six action potentials after blocking 4-aminopyridine (4-AP) sensitive K⁺ channels. The CA3 somata and initial part of their axons were surgically removed in these experiments, leading to the conclusion that the bursts were initiated far out in the axons. This conclusion was supported by two additional kinds of experiments. First, local application of 4-AP to one out of two stimulated axonal branches of the same neuron showed bursting only at the 4-AP exposed branch. Second, intracellular recordings from CA3 somata showed that some spontaneously occurring bursts were resistant to somatic hyperpolarization. We then investigated a hyperexcitable period that follows individual spikes in the Schaffer collaterals. With extracellular excitability testing, we showed that the time course of this hyperexcitability was compatible with that of the bursts, so this hyperexcitability could be the underlying cause of the bursts. Furthermore, the hyperexcitability was enhanced by low doses of 4-AP (20 μM), α-dendrotoxin (α-DTX) or margatoxin (MgTX). Kv1.2 containing channels may therefore dampen the hyperexcitability, but because bursting was observed only at high 4-AP concentration (1 mM), other channels may be needed to prevent axonal bursting.     

Kinesin cross-bridges between neurosecretory granules and microtubules in the mouse neurohypophysis

Neurosecretory granules are conveyed along microtubules in the neurohypophysial axon. By both quick-freeze deep-etch and thin section electron microscopies, we found cross-bridges between the granules and microtubules. The length of the cross-bridges (mean 26.6 +/- 15.2 nm) was not uniform, and its histogram was multi-phasic showing the highest peak around 20 nm and several lower peaks in the range of 40-100 nm. This implies that cross-bridges are complexes of several kinds of constituents. Immunofluorescence microscopy showed the expression of a motor protein kinesin in the mouse neurohypophysis. Immunoelectron microscopy detected kinesin at the contact sites of neurosecretory granules to microtubules. These results suggest that kinesin is a major component of the cross-bridges, and involved in the neurosecretory granule transportation.     

Optimizing dynamic interactions between a cardiac patch and inflammatory host cells

Damaged heart muscle has only a minimal ability for regeneration following myocardial infarction in which cardiomyocytes are lost to ischemia. The most clinically promising approach to regeneration of cardiac muscle currently under investigation is that of injecting cardiogenic repair cells or implanting a preformed tissue-engineered patch. While major advances are being made in the derivation of functional human cardiomyocytes and the development of tissue-engineering modalities for cardiac repair, the host environment into which the repair cells are placed is largely overlooked. Within seconds of myocardial ischemia, hypoxia sets in in the myocardium and the inflammatory response starts, characterized by rapid deployment of circulating cells and the release of paracrine and autocrine signals. Therefore, the inflammatory conditions under which these interactions take place, the design of the scaffold material used, and the maturity of the implanted cells will determine the outcomes of any stem cell-based therapy. We discuss here the interactions between implanted and inflammatory cells of the host, which are critical for the design of effective heart repair therapies.     

Evidence for direct reciprocal interactions between the central rhythm generators for spinal “respiratory” and locomotor activities in the rabbit

Summary 1. Rhythm generators for locomotion and respiration have been previously identified in the high spinal rabbit treated with nialamide and DOPA. In curarized preparations, with no sensory feedback, simultaneous recordings of motor commands from the nerves to the diaphragm and to several hindlimb nerves have demonstrated that central (intraspinal) interactions exist between these respiratory and locomotor activities. 2. The purpose of the present study was to investigate the nature of these interactions. Two main possibilities existed: (i) direct interactions taking place between the rhythm generators; (ii) the activity of one of the rhythm generators modifying the other generator's activity at its output (at the interneuronal or motoneuronal level). 3. The present analysis of the timing (and resetting) of activities in the phrenic, hindlimb extensor (gastrocnemius medialis) and flexor (tibialis anterior) nerves suggests a strong direct interaction between the two sets of rhythm generators. Each new locomotor cycle thus only begins at the termination of a long-lasting phrenic burst and a respiratory burst can only occur at certain parts of a locomotor cycle.Supported by the C.N.R.S. (ERA 411 and RCP 08.0803), the D.G.R.S.T. (DN 80-7-0252) and the INSERM (81-60-32)     

Glutamate immunoreactivity in rat dorsal root axons

Approximately 8.5% of the unmyelinated and 2.5% of the myelinated primary afferent axons in lumbar dorsal roots of normal rats are immunostained for glutamate. Thus unmyelinated fibers are the predominantly immunostained population under the conditions of our experiments. The mean size of the unmyelinated fibers is greater at L6 than at L4 and L2. The meaning of this is not clear, but it may imply that pelvic visceral afferents are slightly larger than afferents from other areas. We emphasize that the immunostained axons can be demonstrated in otherwise normal animals, so changes in the percentages of labeled axons in response to various stimuli will be of interest.     

Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons

Intracellular recordings of rat supraoptic nucleus neurons were obtained from perfused hypothalamic explants. Individual action potentials were followed by hyperpolarizing afterpotentials (HAPs) having a mean amplitude of -7.4 +/- 0.8 mV (SD). The decay of the HAP was approximated by a single exponential function having a mean time constant of 17.5 +/- 6.1 ms. This considerably exceeded the cell time constant of the same neurons (9.5 +/- 0.8 ms), thus indicating that the ionic conductance underlying the HAP persisted briefly after each spike. The HAP had a reversal potential of -85 mV and was unaffected by intracellular Cl- ionophoresis of during exposure to elevated extracellular concentrations of Mg2+. In contrast, the peak amplitude of the HAP was proportional to the extracellular Ca2+ concentration and could be reversibly eliminated by replacing Ca2+ with Co2+, Mn2+, or EGTA in the perfusion fluid. During depolarizing current pulses, evoked action potential trains demonstrated a progressive increase in interspike intervals associated with a potentiation of successive HAPs. This spike frequency adaptation was reversibly abolished by replacing Ca2+ with Co2+, Mn2+, or EGTA. Bursts of action potentials were followed by a more prolonged afterhyperpolarization (AHP) whose magnitude was proportional to the number of impulses elicited (greater than 20 Hz) during a burst. Current injection revealed that the AHP was associated with a 20-60% decrease in input resistance and showed little voltage dependence in the range of -70 to -120 mV. The reversal potential of the AHP shifted with the extracellular concentration of K+ [( K+]o) with a mean slope of -50 mV/log[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)