Short-term potentiation of miniature excitatory synaptic currents causes excitation of supraoptic neurons

Magnocellular neurons (MCNs) of the hypothalamic supraoptic nucleus (SON) secrete vasopressin and oxytocin. With the use of whole-cell and nystatin-perforated patch recordings of MCNs in current- and voltage-clamp modes, we show that high-frequency stimulation (HFS, 10-200 Hz) of excitatory afferents induces increases in the frequency and amplitude of 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX)-sensitive miniature excitatory postsynaptic currents (mEPSCs) lasting up to 20 min. This synaptic enhancement, referred to as short-term potentiation (STP), could be induced repeatedly; required tetrodotoxin (TTX)-dependent action potentials to initiate, but not to maintain; and was independent of postsynaptic membrane potential, N-methyl-D-aspartate (NMDA) receptors, or retrograde neurohypophyseal neuropeptide release. STP was not accompanied by changes in the conductance of the MCNs or in the responsiveness of the postsynaptic non-NMDA receptors, as revealed by brief application of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate. mEPSCs showed similar rise times before and after HFS and analysis of amplitude distributions of mEPSCs revealed one or more peaks pre-HFS and the appearance of additional peaks post-HFS, which were equidistant from the first peak. STP of mEPSCs was not associated with enhanced evoked responses, but was associated with an NBQX-sensitive increase in spontaneous activity of MCNs. Thus we have identified a particularly long-lasting potentiation of excitatory synapses in the SON, which has a presynaptic locus, is dissociated from changes in evoked release, and which regulates postsynaptic cell excitability.     

Evidence for excitatory actions of histamine on supraoptic neurons in vitro: mediation by an H1-type receptor

The effects of histamine on the firing of supraoptic neurosecretory neurons in the rat were examined in vitro using acutely prepared, hypothalamo-neurohypophysial explants perifused with an artificial cerebrospinal fluid. Extracellular action potentials meeting the criteria of antidromic invasion from neurohypophysial stalk stimulation were recorded from 135 neurons in the tuberal portion of the supraoptic nucleus, which lies superficially along the tuber cinereum and consists of mostly vasopressin-containing neurons. Units could be classified as slow/silent (76.3%), phasic (21.5%) or continuous (2.2%) on the basis of their spontaneous activity. Histamine applied briefly to the perifusate excited approximately one-third of the slow silent neurons and approximately two-thirds of the phasic neurons, with a wide range (10(-3)-10(-9)) in the effective concentration across neurons. The H1-receptor agonists 2-pyridylethylamine and 2-thiazolylethylamine mimicked these excitations in 10 of 12 and 3 of 6 neurons tested, respectively. The H2-receptor agonists dimaprit (4 neurons) and impromidine (5 neurons) failed to excite any of the tested neurons previously excited by histamine. The H1-receptor antagonist promethazine antagonized histamine's excitatory effect in 8 of 9 cells, while the H2-receptor antagonist cimetidine had little effect on the 9 cells tested. Histamine also modified bursts of activity induced in some slow/silent neurons by antidromic stimulation without having an observable effect in the absence of an antidromic burst. In 10 of 18 neurons histamine produced an elongation of burst duration and a modest increase in intraburst firing rate when applied during an antidromically evoked burst. In an additional 5 of 17 neurons, which had neither previously responded to histamine nor shown an antidromically-evoked burst, the pairing of histamine application and antidromic shocks resulted in an antidromically evoked burst. The effects of histamine on evoked bursts also appeared to be mediated by an H1-receptor. Histamine's excitation of supraoptic neurons is thus dependent on the electrical activity expressed by the neuron at the time of testing. Conductances activated by depolarization of the neuron may be modified by histamine or this compound may alter the threshold for burst generation. Considered with data showing H1-receptor localization and histamine-immunoreactive fibers within the supraoptic nucleus, the present results, as well as those showing the potency of centrally applied histamine in releasing vasopressin, suggest histamine may act physiologically by altering the electrical activity of vasopressin-secreting neurons.     

The role of the actin cytoskeleton in oxytocin and vasopressin release from rat supraoptic nucleus neurons

Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K⁺, 50 m m ) or TG (200 n m ) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 μ m ) or depolymerizing agents (latrunculin B, 5 μ m ) to treat SON and neural lobe (NL) explants in vitro and measure high K⁺-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K⁺-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K⁺-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.     

Mechanisms underlying type I mGluR-induced activation of lobster gastric mill neurons

In addition to ionotropic effects, glutamate and acetylcholine have metabotropic modulatory effects on many neurons. Here we show that in the stomatogastric ganglion of the lobster, glutamate, one of the main ionotropic neurotransmitters, modulates the excitability of gastric mill neurons. The neurons in this well-studied system produce rhythmic output to a subset of lobster foregut muscles. Recently, metabotropic glutamate receptor (mGluR) agonists were suggested as modulators of the rhythmic output, in addition to the previously described muscarinic modulation by acetylcholine. However, the cellular mechanisms responsible for these effects on the pattern are not known. Using intracellular recording methods and calcium imaging, we show that glutamate has an excitatory effect on specific neurons in the stomatogastric ganglion, which is mediated by mGluRs. Responses to the application of mGluR type I agonists are transient oscillations in the system, probably arising from network interactions. We show that the excitatory effect is sensitive to phospholipase-C and IP(3) and is G-protein dependent. The G-protein dependency was demonstrated by GDPbetaS and GTPgammaS injection into identified neurons. The depolarizations and oscillations were accompanied by an increase of intracellular Ca(2+) levels and correlated Ca(2+) oscillations. By using cyclopiazonic acid, an endoreticular Ca(2+) uptake inhibitor, we show that some internal calcium release may augment the response, but is not crucial for its production. Interestingly, although Ca(2+) concentration increase is typically associated with the phosphoinositide pathway, in the lobster, the Ca(2+) concentration increase-either voltage dependent or independent-cannot account for the observed depolarization.     

Mechanisms of long-interval selectivity in midbrain auditory neurons: roles of excitation, inhibition, and plasticity

Stereotyped intervals between successive sound pulses characterize the acoustic signals of anurans and other organisms and provide critical information to receivers. One class of midbrain neuron responds selectively when pulses are repeated at slow rates (long intervals). To examine the mechanisms that underlie long-interval selectivity, we made whole cell recordings, in vivo, from neurons in the anuran inferior colliculus (anuran IC). In most cases, long-pass interval selectivity appeared to arise from interplay between excitation and inhibition; in approximately 25% of these cases, the delayed inhibition to a pulse overlapped with the excitation to the following pulse at fast pulse repetition rates (PRRs), resulting in a phasic "onset" response. In the remaining cases, inhibition appeared to precede excitation. These neurons did not respond to fast PRRs apparently because delayed excitation to a pulse overlapped with the inhibition to the following pulse. These results suggest that the relative timing of inhibition and excitation govern differences in the response properties of these two cell types. Loading cells with cesium increased their responses to fast AM rates, supporting a role for inhibition in long-interval selectivity. Three cells showed little or no evidence of inhibition and exhibited strong depression of excitation. These findings are discussed in the context of current models for long-pass interval selectivity.     

Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord

We used anatomical and physiological approaches to characterize nicotinic receptors (AChRs) on Renshaw cells of the neonatal rat spinal cord. Confocal imaging of Renshaw cells, identified by their characteristic pattern of gephyrin immunoreactivity, revealed that these neurons are immuno-positive for the alpha4 and beta2 AChR subunits but not for the alpha7 subunit. We used whole cell recording in spinal cord slices to characterize synaptic transmission from alpha-motor neurons to Renshaw cells, which could be identified pharmacologically by the sensitivity of transmission to d-tubocurarine. alpha-Motor neuron-to-Renshaw cell synapses were blocked by 10 microM dihydro-beta-erythroidine (dHbetaE), but not 50 nM methyllycaconitine (MLA), a selective alpha7 antagonist. These findings support a role for alpha4beta2-like AChRs, but not alpha7 AChRs, in rapid excitatory transmission between alpha-motor neurons and Renshaw cells in rat spinal cord.     

Effect of gonadal steroids on the oxytocin-induced excitation of neurons in the bed nuclei of the stria terminalis at parturition in the rat.

Experiments were undertaken to examine the role of ovarian steroids in peripartum programming of oxytocin sensitivity of limbic neurons implicated in oxytocin-induced facilitation of the milk-ejection reflex. In vivo recordings of neurons in the bed nuclei of the stria terminalis and ventrolateral septum of pre-parturient rats which had undergone prior ovariectomy and hysterectomy showed that oestradiol significantly increased the excitatory responses of bed nuclei/ventrolateral septum neurons to intracerebroventricular oxytocin, compared to oil-treated controls. Oestradiol also increased the excitation of bed nuclei neurons to the selective oxytocin agonist, [Thr4,Gly7]oxytocin in brain slices from steroid pre-treated ovariectomized hysterectomized rats, so that both the proportion of responsive neurons, and the magnitude of their responses were significantly increased. Parallel autoradiographic studies showed that oxytocin binding in the medial bed nuclei and ventrolateral septum was selectively increased following oestradiol treatment. Progesterone pre-treatment had no effect on either oxytocin sensitivity of bed nuclei/ventrolateral septum neurons recorded in vivo, or on oxytocin binding in the medial bed nuclei and ventrolateral septum, compared to oil-treated controls. Mean responses to [Thr4,Gly7]oxytocin in bed nuclei neurons recorded in slices from progesterone-treated rats were larger than controls, but this effect was highly variable. These results demonstrate that oestradiol greatly enhances oxytocin receptor expression and sensitivity of bed nuclei/ventrolateral septum neurons to oxytocin over the peripartum period, consistent with involvement of this steroid in enhancing oxytocin regulation of neuroendocrine and behavioural adaptations required for lactation.     

Ultrastructural evidence of sexual dimorphism in supraoptic neurons: a morphometric study

Summary We have recently shown that in spite of the absence of receptors for gonadal steroids in the supraoptic nucleus (SON) of the rat hypothalamus, the volume of the nucleus and the size of its neurons are larger in males than in females, and that these differences between male and female rats are correlated with body weight and dependent on the vasopressinergic neurons. As supraoptic neurons and their organelles enlarge when they are engaged in active peptidergic secretion we have carried out a morphometric ultrastructural analysis to determine if cell structures involved in the synthesis and storage of neurosecretory material also display weight-dependent sex dimorphism. Groups of six male and six female rats aged 30,60 and 180 days were used. Nucleoli, rough endoplasmic reticulum and neurosecretory granules were analysed and we estimated their volume or surface densities and the total volume of nucleoli and rough endoplasmic reticulum, and total surface area of rough endoplasmic reticulum. We found that, with the exception of neurosecretory granules, the densities of the organelles did not differ among the groups studied, but total values were higher in males. These differences were found to be weight-dependent. Since the organelles studied are regarded as reliable indicators of the neurosecretory activity of supraoptic neurons, our data fully support the view that the weight-dependent sexual dimorphism observed in this nucleus reflects greater synthetic activity of its vasopressinergic neurons associated with the need to maintain water balance in larger bodies.     

Mechanisms underlying the sensitivity of neurons in the lateral superior olive to interaural intensity differences.

The initial processing of interaural intensity differences (IIDs), the major cue to the azimuthal location of high-frequency sounds in mammals, is carried out by neurons in the lateral superior olivary nucleus (LSO) that receive excitatory input from the ipsilateral ear and inhibitory input from the contralateral ear (IE neurons). The "latency" hypothesis asserts that it is the effects of intensity differences on the latency, and hence the relative timing, of the synaptic inputs to these neurons that is the basis of their sensitivity to IIDs, while other models assign the major role to changes in the relative amplitude of the inputs. To test the latency hypothesis and to determine the contributions of changes in the relative timing and amplitude of synaptic inputs to the IID sensitivity of LSO neurons, a method was developed of generating sets of stimuli that produced either the same changes in the relative timing of inputs without any change in their amplitude (equivalent interaural time difference stimuli) or the same differences in amplitude without any difference in timing (delay-cancelled IID stimuli) as a given range of IIDs. Data were obtained from a sample of IE neurons in the LSO of anesthetized rats using these stimulus paradigms and click and tone-burst stimuli. For click stimuli, the IID sensitivity of a small proportion of neurons was explained entirely by sensitivity to differences in input timing, but the sensitivity of most neurons reflected either sensitivity to the relative amplitude of inputs or to the joint operation of both factors. In neurons whose sensitivity was tested at a number of different absolute sound pressure levels (SPLs), the relative contributions of the two factors tended to differ at different SPLs. The IID sensitivity of onset responses to tone stimuli could be classified into the same three categories but was explained for a larger proportion of neurons by sensitivity to differences in input timing. The IID sensitivity of the late response component of neurons with sustained responses to tones in all cases reflected sensitivity to the relative amplitude of the inputs. The results confirm the contribution of changes in latency produced by intensity changes to the IID sensitivity of the onset responses of many IE neurons in LSO but require rejection of the strong form of the latency hypothesis, which asserts that this factor alone accounts for such sensitivity.     

Mechanisms underlying geroprotective effects of peptides

We review the role of peptides in aging and the mechanisms underlying the geroprotective effect of peptide preparations. Geroprotective properties of peptides are associated with their influence on systems maintaining homeostasis in the body and regulation of mechanisms underlying aging. Peptides normalize synthesis of tissue-specific proteins and regulate expression of genes responsible for proliferation and differentiation of cells. Thus, peptides maintain normal physiological functions and decelerate aging.     

Mechanisms underlying cytotoxicity of anti-DNA antibodies

We studied mechanisms underlying cytotoxicity of catalytic anti-DNA autoantibodies or DNA abzymes isolated from the blood of patients with systemic lupus erythematosus and autoimmune mice. Experiments were performed on L929, HL-60, Raji, and K562 cells. Treatment with DNA abzymes caused internucleosomal DNA fragmentation in target cells typical of apoptosis. Direct immunofluorescence assay demonstrated that DNA abzymes can penetrate into the nucleus of target cells. The dependence of antibody-mediated cytotoxicity on the duration of incubation indicated that cell death is realized via at least 2 mechanisms: penetration of DNA abzymes into the cell nucleus followed by degradation of nuclear DNA or induction of apoptosis.     

Mechanisms underlying the small intestinal fluid secretion caused by arachidonic acid, prostaglandin E1 and prostaglandin E2 in the rat in vivo

Prostanoids were given intraluminally (PGE2) or infused close intra-arterially (PGE1 and PGE2) or arachidonic acid was administered intraluminally to denervated jejunal segments of the rat in vivo. These experimental manoeuvres caused a net fluid secretion, although a 1,000-fold higher concentration of the prostanoids was needed from the luminal than from the vascular side. I.v. hexamethonium or serosally applied lidocaine diminished the induced fluid secretion suggesting that the prostanoids act mainly by eliciting local secretory reflexes in the enteric nervous system. This nerve-mediated secretion is not accompanied by any increase in tissue cAMP. However, at higher i.a. concentrations of PGE2 there seems to be a non-neurogenic effect on the enterocytes associated with an increase in tissue cAMP.     

Mechanisms underlying burst and regular spiking evoked by dendritic depolarization in layer 5 cortical pyramidal neurons

Apical dendrites of layer 5 pyramidal cells in a slice preparation of rat sensorimotor cortex were depolarized focally by long-lasting glutamate iontophoresis while recording intracellularly from their soma. In most cells the firing pattern evoked by the smallest dendritic depolarization that evoked spikes consisted of repetitive bursts of action potentials. During larger dendritic depolarizations initial burst firing was followed by regular spiking. As dendritic depolarization was increased further the duration (but not the firing rate) of the regular spiking increased, and the duration of burst firing decreased. Depolarization of the soma in most of the same cells evoked only regular spiking. When the dendrite was depolarized to a critical level below spike threshold, intrasomatic current pulses or excitatory postsynaptic potentials also triggered bursts instead of single spikes. The bursts were driven by a delayed depolarization (DD) that was triggered in an all-or-none manner along with the first Na+ spike of the burst. Somatic voltage-clamp experiments indicated that the action current underlying the DD was generated in the dendrite and was Ca2+ dependent. Thus the burst firing was caused by a Na+ spike-linked dendritic Ca2+ spike, a mechanism that was available only when the dendrite was adequately depolarized. Larger dendritic depolarization that evoked late, constant-frequency regular spiking also evoked a long-lasting, Ca2+-dependent action potential (a "plateau"). The duration of the plateau but not its amplitude was increased by stronger dendritic depolarization. Burst-generating dendritic Ca2+ spikes could not be elicited during this plateau. Thus plateau initiation was responsible for the termination of burst firing and the generation of the constant-frequency regular spiking. We conclude that somatic and dendritic depolarization can elicit quite different firing patterns in the same pyramidal neuron. The burst and regular spiking observed during dendritic depolarization are caused by two types of Ca2+-dependent dendritic action potentials. We discuss some functional implications of these observations.     

Facilitation of actin polymerization by interfilamentous factors

Summary The polymerization of actin in low ionic strength buffer at 0° C in the presence of 0.25mm Mg²⁺ was studied by viscometry, turbidity and absorbance at 232 nm. Under these conditions, significant polymerization occurred only in the viscometer and not in isotropic mixtures. The polymerization rate with 0.25mm MgCl₂, as judged from shear viscosity, was equal to or greater than that observed with 0.1mm KCl and 0.25 mm MgCl₂ at 0° C, and was characterized by a longer nucleation period. Measurements of the turbidity at 350 nm (detecting filament formation and aggregation) and the absorbance at 232 nm (detecting conformational changes of the G-F transition) showed no evidence for polymerization or nucleation in a bulk solution at 0° C when Mg²⁺ was added to 0.25mm and, furthermore, F-actin nuclei were ineffective as seeds under these conditions. However, nucleation and polymerization by these criteria could be induced by raising the temperature to 20° C. These results demonstrate the existence of narrow conditions when elongation of F-actin is dissociated from nucleation of oligomeric acceptor nuclei, even if monitored on the sub-molecular level (absorbance at 232 nm). Under these conditions, elongation appears to require anisotropic F-Actin, I.e. that filaments are ordered by laminar flow.     

The role of different glutamate receptors in the mediation of glutamate-evoked excitation of red nucleus neurons after simulated microgravity in rat

The present study investigates changes in red nucleus (RN) neuronal activity and the role of glutamate receptors (GluRs) after simulated microgravity (tail-suspension) in the rat using single-unit recording and microinjection. The results showed that tail-suspension for 3, 7, and 14 days could induce a significant decrease in spontaneous firing rate of RN neurons in a time-dependent manner. Unilateral microinjection of glutamate into the RN significantly increased the firing rate of RN neurons, but the increased firing rate was significantly reduced following tail-suspension time. Microinjection of the NMDA receptor antagonist MK-801 or the non-NMDA receptor antagonist DNQX into the RN blocked this excitatory effect induced by glutamate. However, microinjection of the metabotropic glutamate receptor (mGluR) antagonist (±)-MCPG into the RN had no effect. These results suggest that simulated microgravity can reduce excitability of RN neurons following a functional impairment of glutamate receptors. NMDA and non-NMDA receptors, but not mGluRs, are involved in the mediation of glutamate-evoked excitation of RN neurons. The decrease in excitability of RN neurons may be involved in simulated microgravity-induced muscle atrophy.     

Capsaicin-induced excitation of locus coeruleus neurons

The noradrenergic pontine nucleus locus coeruleus (LC) seems to be involved in sensory processing. In the present study, the effect of capsaicin, a drug which specifically interferes with chemosensitive primary afferents, on LC firing rate was analysed, utilizing electrophysiological techniques. In control rats, low doses of capsaicin (1-8 micrograms kg-1 i.v.) caused a marked excitation of LC units. The effect was instantaneous in onset but short-lasting and no signs of tachyphylaxis were observed. The excitation was maintained in adult rats treated as neonates with high doses of capsaicin (50 mg kg-1 s.c.) but almost totally prevented by pretreatment of adult rats with high doses of capsaicin (300 mg kg-1 s.c.). According to our histological data, using selective silver impregnation techniques, the LC seems not to receive innervation by sensory primary afferents. It is proposed that the capsaicin-induced excitation of LC neurons is a centrally mediated effect and might, in part, be involved in the analgetic effect induced by the drug.