Carlos Collin

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.

Neurotrophins act at presynaptic terminals to activate synapses among cultured hippocampal neurons.

We have recently demonstrated that embryonic E16 hippocampal neurons grown in cultures are unable to form fast synaptic connections unless treated with BDNF or NT‐3. This experimental system offers an opportunity to define the roles of neurotrophins in processes leading to formation of functional synaptic connections. We have used ultrastructural and electrophysiological methods to explore the cellular locations underlying neurotrophin action on synaptic maturation. The rate of spontaneous miniature excitatory postsynaptic currents (mEPSCs) evoked by hyperosmotic stimulation was 7–16‐fold higher in neurotrophin‐treated cells than in controls. In addition, the potent neurotransmitter‐releasing drug α‐latrotoxin was virtually ineffective in the control cells while it stimulated synaptic events in neurotrophin‐treated cells. Likewise, the membrane‐bound dye FM1‐43 was taken up by terminals in neurotrophin‐treated cultures five‐fold more than in controls. Both the total number and the number of docked synaptic vesicles were increased by neurotrophin treatment. Activation of synaptic responses by neurotrophins occurred even when postsynaptic glutamate receptors and action potential discharges were pharmacologically blocked. These results are consistent with a presynaptic locus of action of neurotrophins to increase synaptic vesicle density which is critical for rapid synaptic transmission. They also suggest that neurotrophins can activate synapses in the absence of pre‐ and postsynaptic neuronal activity.

Intracellular calcium signals are enhanced for days after Pavlovian conditioning

Abstract: Abstract: Previous observations have implicated GABA as a neurotransmitter released by the vestibular sensory neurons („hair cells”) of the snail Hermissenda onto visual sensory neurons, the type B cells, whose cell bodies are the sites of biophysical and biochemical changes during and following Pavlovian conditioning. Still other observations demonstrated that light‐GABA pairings that simulate stimuli presented during Pavlovian conditioning cause prolonged elevatiori of intracellular Ca2+ and transformation of GABA‐induced synaptic inhibition into excitation. Intracellular Ca2+ signals in response to GABA perfused onto the postsynaptic: type B terminal branches are shown here to be prolonged on days after conditioning, but not after control paradigms. These and past results demonstrate two separate sites, i.e., the cell body and the terminal branches, for learning‐induced changes after Pavlovian conditioning.

GABA-mediated synaptic interaction between the visual and vestibular pathways of Hermissenda.

Abstract: The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual‐vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CMS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 μM), the cholinergic antagonist atropine (250 μM), and the serotonergic antagonist imipramine (50 μM). In contrast, the GABAA antagonist bicuculline (250 μM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 μM)M) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 μM) onto the terminal branches of the B cell resulted in a concentration‐dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABAB agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from −70 to −80 mV and were consistent with mixed Cl‐ and K+ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual‐vestibular interactions in this animal, and suggest a possible role of GABA in visual‐vestibular associative learning.

Effects of α2‐Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Abstract: Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presyn‐aptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (≥ 100 μM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cells resting potential as well as after a light step. Clonidine reduces voltage‐dependent outward K+ currents (IA, an early current, Ica2+‐K+, a late Ca2+‐dependent current) that control the type B cells excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the α2‐receptor antagonist, yohimbine (0.5 μM), but not the α1‐blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde‐induced fluorescence or Falck‐Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine‐like synaptic effect on type B cells, therefore, could amplify conditioning‐specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.

CONFOCAL MICROSCOPY OF CELLS IMPLANTED INTO TISSUE BLOCKS: CELL MIGRATION IN LONG-TERM HISTOCULTURES

SummaryIn three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, then implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.

The role of calcium in prolonged modification of a GABAergic synapse

Caudal hair cell impulses cause postsynaptic inhibition of ipsilateral type B photoreceptors in the snail Hermissenda. This inhibition is shown to be GABAergic according to a number of criteria. HPLC, mass spectrophotometric, and immunocytochemical techniques demonstrated the presence of GABA in the hair cells and their axons. GABA agonists and antagonists mimic and block the synaptic effect in a manner consistent with endogenous GABAergic transmission. Other properties, including I-V relations, conductance changes and reversal potentials, are comparable for exogenous GABA responses and endogenous effects of the hair cell impulses. This inhibitory synapse has been found to undergo a long-lasting transformation into an excitatory synapse if GABA release is paired with post-synaptic depolarization. GABA, via GABAA and GABAB receptors in the B cell, causes the opening of calcium sensitive chloride and potassium channels that leads to the post-synaptic hyperpolarization. GABA also induces a long-lasting intracellular calcium elevation at the terminal branches of the B cell that greatly outlasts the voltage responses. Synaptic transformation induced by pairings is caused by a decrease in both GABA induced chloride and potassium conductances in the post-synaptic B cell, as well as a significant prolongation of the intracellular calcium accumulation in the B cells terminal axonal branches.

Sequential changes of potassium currents in Hermissenda Type B photoreceptor during early stages of classical conditioning

Classical conditioning of the marine snail Hermissenda can be produced in a single session of 50 pairings of light and rotation stimuli. Voltage clamp measurements of two outward K+ currents, IA and ICa2(+)-K+ were obtained from medial Type B photoreceptors that were isolated from the nervous system 1 day after animals were exposed to paired light and rotation stimuli or control procedures (Unpaired, or no exposure to light and rotation), ICa2(+)-K+ was found to be unchanged 18-30 h after 50 training trials. This result is consistent with a previous study where ICa2(+)-K+ was found to be unchanged after 50 light and rotation trials, although significantly reduced by 100 trials. In the present study 50 pairings of light and rotation produced a significant reduction in IA, suggesting an important role for this current in the earliest stages of classical conditioning.

Dye-coupling in three-dimensional histoculture of rat lingual frenulum

SummaryA three-dimensional histoculture of wet stratified squamous epithelium of rat lingual frenulum was cultured on a liquid-air interface. The tissue retained its morphology for many days in culture. During this period the vast majority of the epithelial cells remained viable and exhibited dye (lucifer yellow) coupling in all living epithelial strata. Dye coupling was determined using two methods: the conventional intracellular injection method, and a new method—“cut-loading.” In the cut-loading method, an incision is made in the epithelium in the presence of dye, and intracellular diffusion of dye throughout the epithelium was measured using confocal microscopy. The basolateral surface of the lingual frenulum also acted as a substrate for neuroblastoma cells to grow without exogenously added trophic factors. These neuroblastoma cells grow neurites that establish contacts with epithelial cells. This preparation can serve as a model for investigating interactions among epithelial cells and between nerves and epithelial cells.