Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.     

Diversity and modulation of ionic conductances in leech neurons

A complete understanding of animal behavior at the cellular level requires detailed information on the intrinsic biophysical properties of neurons, muscles, and the synaptic connections they make. In the past 10 to 15 years, electrophysiological studies of leech neurons have revealed a diverse array of voltage-gated ionic conductances distinguished by their pharmacological sensitivity to classic ion channel blockers. Voltage-clamp studies have provided new information about the kinetics and voltage-dependence of Na⁺ conductances, several K⁺ currents, including I_A, I_K and I_K(Ca.)' and high- and low-voltage-gated Ca²⁺ conductances. These studies showed that the action potentials of most leech neurons result from the usual sequence of permeability changes to Na⁺, K⁺, and Ca²⁺ ions. They also added insight as to the role played by particular combinations of conductances in providing individual neurons with electrical properties appropriate for the particular information they encode. Evidence is accumulating on the modulatory actions of endogenous neurotransmitters such as FMRFamide, serotonin, and octopamine on motor behaviors in the animal. Parallel studies suggest that changes in behavior can be explained, at least in part, by the alteration of firing patterns of selected neurons and muscles resulting form modulation of multiple ion conductances. This makes the leech exceptionally attractive for neuroethological studies because it is one of the simplest organisms in which the methods of psychology and neurobiology can be combined. Information gathered from this animal will therefore increase our understanding regarding general principles underlying the cellular basis of behavior. © 1995 John Wiley & Sons, Inc.     

Anatomical analysis of contacts between identified neurons that control heartbeat in the leech Hirudo medicinalis

Summary The rhythmic constriction of the heart tubes in the leech Hirudo medicinalis is controlled by an identified set of motor neurons (HE cells) and interneurons (HN cells) (reviewed by Calabrese and Peterson 1983). Electrophysiological recordings have indicated particular synaptic relationships among HE and HN cells. In the present study, the synaptic framework mediating the interactions among HE cells and HN cells was examined anatomically. Using light and electron microscopy of physiologically identified, HRP-injected cells, we have examined the zones of interaction and types of contacts between specific cells. HE cells, which have very fine, threadlike processes, interact with their contralateral homologues throughout most of the middle third of the ganglionic neuropil. When HE-cell neuntes come together, the apposed plasma membranes are rigidly parallel, separated by an intercellular gap of 6 nm, for up to 6 m. These specializations must form the structural basis for the strong electrical coupling observed (Peterson 1983) between HE-cell pairs. HE cells also emit from the main neurite a series of extremely fine processes that extend dorsally. These appear in the light microscope to contact processes of the ipsilateral HN cell of the same ganglion, and are also in a position to make contact with the axons of more anterior HN cells. The intraganglionic processes of HN cells, which are studded with large varicosities, ramify in part of the region of neuropil occupied by HE-cell processes, as well as more posteriorly. Contacts between HE and HN cells, which are known to be mostly inhibitory synaptic contacts, are seen in the electron microscope to be formed between medium-diameter HN processes, which are filled with clear round synaptic vesicles, and multiple fine tendrils of the HE cell that surround the HN process. Certain HN cells form reciprocal inhibitory synapses with their contralateral homologues. These contacts occur near the midline, sometimes in the major mass of neuropil and sometimes embedded in the extracellular material that ensheathes the neuropil. The contacts are between medium-and small-diameter profiles that are both filled with synaptic vesicles. Our findings indicate that various classes of physiological interactions among HE and HN cells are mediated by anatomically distinct types of contacts and, at least in some cases, are segregated from each other on the neuritic trees of the cells.     

Molecular cloning and characterization of LKv1, a novel voltage‐gated potassium channel in leech

We have cloned a novel voltage‐gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage‐gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I_K. Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage‐gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker‐related K channels in jellyfish. Thus, this analysis indicates that this group of voltage‐gated K channels contains several evolutionarily divergent lineages. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 287–299, 1999     

Steroids,histamine, and serotonin in the medicinal leech salivary gland secretion

Lipids represent 20% of the total weight of the dried pool of medicinal leech salivary gland secretion (SGS) obtained from about 50 individual animals. SGS lacks phospholipids, but contains steroids. Immunochemiluminescent analysis of SGS revealed the presence of free steroid hormones: cortisol, progesterone, testosterone, estradiol, and dehydroepiandrosterone. Micro-chromatographic-mass spectrometric analysis of SGS and its low molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standard preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione, and testosterone) and histamine and serotonine have been identified in SGS.     

Localization,physiology, and modulation of a molluskan dopaminergic synapse

We investigated the location, physiology, and modulation of an identified synapse from the central nervous system (CNS) of the mollusk Lymnaea stagnalis. Specifically, the excitatory synapse from interneuron right pedal dorsal one (RPeD1) to neurons visceral dorsal two and three (VD2/3) was examined. The gross and fine morphology of these neurons was determined by staining with Lucifer yellow or sulforhodamine. In preparations where RPeD1 was stained with Lucifer yellow and VD2/3 with sulfo-rhodamine, the axon collaterals occupied similar regions, suggesting that these neurons make physical contact in the CNS. Digital confocal microscopy of these preparations revealed that presynaptic varicosities made apparent contact (synapses) with smooth postsynaptic axon collaterals. The number of putative synapses per preparation was about five to 10. Regarding physiology, the synaptic latency was moderately rapid at 24.1 ± 5.2 ms. Previous work indicated that RPeD1 uses dopamine as a neurotransmitter. The RPeD1 → VD2/3 excitatory postsynaptic potential (EPSP) and the VD2/3 bath-applied dopamine (100-μM) response displayed a similar decrease in input resistance and a similar predicted reversal potential (−31 vs. −26 mV), indicating that the synapse and exogenous dopamine activate the same conductance. Finally, bath-applied serotonin (10 μM) rapidly and reversibly depressed the RPeD1 → VD2/3 synapse but did not affect the VD2/3 bath-applied dopamine (100-μM) response, suggesting a presynaptic locus of action for serotonin. The effect of serotonin was not associated with any changes to the pre- or postsynaptic membrane potential and input resistance, or the presynaptic action potential half-width. The RPeD1 → b3 VD2/3 synapse provides an opportunity to examine the anatomy and physiology of transmission, and is amenable to the study of neuromodulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 247–264, 1997     

Steps in the development of chemical and electrical synapses by pairs of identified leech neurons in culture

Experiments have been made to follow the development of chemical and electrical transmission between pairs of leech neurons in culture. 1. The cell bodies of identified neurons were isolated from the CNS by suction after mild enzyme treatment, together with a length of the initial segment (or 'stump'). The neurons tested were Retzius cells (R), annulus erector motoneurons (AE), Anterior pagoda cells (AP) and pressure sensory cells (P). Pairs of cells were placed together in various configurations, with different sites on their surfaces making contact. 2. When pairs of Retzius cells were apposed with their stumps touching, serotonergic, chemically mediated synaptic transmission became apparent before electrical transmission. By 2.5 h impulses in either of the two Retzius cells produced hyperpolarizing inhibitory potentials in the other. These potentials were reversed by raised intracellular Cl and showed clear facilitation. The strength of chemical transmission between Retzius cells increased over the next 72 h. 3. After chemical transmission had been established, weak non-rectifying electrical transmission became apparent between Retzius cells at about 24-72 h. By 4 days coupling became stronger and tended to obscure chemically evoked synaptic potentials. 4. When pairs of Retzius cells were aligned in culture with the tip of one cell stump touching the soma of the other, chemical transmission also developed rapidly. Transmission was, however, in one direction, from stump to soma. At later stages non-rectifying electrical coupling developed as with stump-stump configuration. With the cell bodies of two Retzius cells apposed, electrical coupling developed after several days, before chemical transmission could be observed. 5. When Retzius and P cells were cultured with their stumps in contact, inhibitory chemical synaptic transmission developed within 24 h. Transmission was always in one direction, from Retzius to P cell. Electrical coupling of Retzius and P cells never occurred whatever the spatial relations of the cells to one another. 6. Annulus erector motoneurons, which contain ACh and a peptide resembling FMRFamide, first developed electrical coupling when the two stumps were in contact and then, later, bi-directional chemical transmission. Anterior Pagoda pairs placed stump-to-stump showed electrical connections. 7. Electronmicrographs revealed the presence of synaptic structures within 24 h after Retzius-Retzius, Retzius-P or AE-AE stumps were apposed. 8. The specificity of connections between cultured cells was similar to that observed in earlier experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons

The lipid diacylglycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) was used to verify the existence of DAG‐sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca²⁺ ([Ca²⁺]i) in nearly 35% of the KCl‐responsive cells. These Ca²⁺ responses disappeared in a Ca²⁺‐free medium supplemented with EGTA. Mn²⁺ quench experiments showed that OAG activated Ca²⁺‐conducting channels that were also permeant to Ba²⁺. The OAG‐induced Ca²⁺ responses were unaffected by nifedipine or omega‐conotoxin GVIA (Sigma‐Aldrich, Saint‐Quentin Fallavier, France) but blocked by 1‐[β‐(3‐(4‐Methoxyphenyl)propoxy)‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride (SKF)‐96365 and Gd³⁺. Replacing Na⁺ ions with N‐methyl‐d ‐glucamine diminished the amplitude of the OAG‐induced Ca²⁺ responses showing that the Ca²⁺ entry was mediated via Na⁺‐dependent and Na⁺‐independent mechanisms. Experiments carried out with the fluorescent Na⁺ indicator CoroNa Green showed that OAG elevated [Na⁺]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca²⁺]i but not the protein kinase C activator phorbol 12‐myristate 13‐acetate. Moreover, the OAG‐induced Ca²⁺ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C‐type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca²⁺]i. Whole‐cell patch‐clamp recordings showed that hyperforin activated non‐selective cation channels. They were blocked by SKF‐96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin‐ and OAG‐sensitive Ca²⁺‐permeable channels displaying TRPC6‐like properties. This is the first report revealing the existence of second messenger‐operated channels in cortical neurons.     

Large-conductance cationic channels in the nuclear envelope of Purkinje neurons from the rat cerebellum

Using a patch-clamp technique, we studied the biophysical properties of large-conductance channels in the nuclear envelope of rat cerebellar Purkinje neurons. Our experiments showed that channels with identical conductance, selectivity, and kinetics are expressed in the external and internal nuclear membranes of these cells. These channels connect the perinuclear space with the cyto-and nucleoplasm; they are not channels of the complex of the nuclear pores for passive diffusion of ions and small molecules, as was believed earlier [17]. We hypothesize that large-conductance cationic channels in the membranes of the nuclear envelope are identical to ion channels of the endoplasmic reticulum and are necessary for functioning of the intermembrane space of the envelope as a calcium store. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 113–118, March–April, 2007.     

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.     

Characteristics of store-operated calcium channels activated by depletion of the endoplasmic reticulum ryanodine-sensitive calcium stores in rat dorsal root ganglion neurons

In neurons of the rat dorsal root ganglia (DRG), using a patch-clamp technique in the whole-cell configuration, we studied the characteristics of calcium channels activated by depletion of the ryanodine-sensitive calcium stores of the endoplasmic reticulum. Current-voltage (I-V) relationships of these store-operated calcium channels were obtained by subtraction of the integral I-V characteristics after application of caffeine from the integral I-V characteristics of calcium channels in the control. Currents through store-operated calcium channels could be induced by application of a series of hyperpolarization current pulses to the cell under conditions of replacement of a calcium-free solution containing caffeine by a caffeine-free solution containing 2 mM Ca²⁺. In this case, the following two main conditions were abserved: Voltage-operated calcium channels were inactivated, while a gradient of the electrochemical potential for calcium ions was increased, which made easier passing of these currents through store-operated calcium channels. Therefore, we found that in DRG neurons, despite the presence of great numbers of both voltage-operated and receptor-dependent calcium channels, one more mechanism underlying the entry of calcium through store-operated channels does exist. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 195–200, May–June, 2007.     

Dietary fat influences electric membrane properties of neurons in cell culture

1. SJL/J mice were maintained on semipurified diets which differed in the ratio of polyunsaturated/saturated fatty acid content (P/S). Exposure was from conception and was maintained for periods ranging from 6 to 34 weeks. 2. Neural cell cultures were prepared from dorsal root ganglia (DRG). After 6 and 20 days of culture, neuronal electric membrane properties were determined quantitatively by intracellular recording. 3. A number of significant differences were observed for the two dietary conditions. DRG from mice on the low-P/s diet had an increase in the rate of fall of both phases of repolarization which, in conjunction with the reduced action potential overshoot, led to a reduced action potential duration. This shift to shorter-duration action potentials was accompanied by a shift to more monophasic falling phases. The low-P/S neurons also exhibited a decreased afterhyperpolarization, decreased specific membrane resistance, and decreased membrane electrical time constant compared to high-P/S neurons. 4. It was concluded that the P/S ratio in the diet can have a significant effect on the electric properties of neurons. The high-P/S neurons tended to have action potentials with biphasic repolarizations and longer durations. In contrast, the low-P/S neurons tended to have action potentials with monophasic repolarizations and shorter durations. Moreover, the known ionic dependence of these two types of action potentials suggested that the low-P/S diet resulted in action potentials with a more exclusive Na dependence, while the high-P/S diet resulted in action potentials with both Na and Ca dependence.     

The effects of triethyl lead on the development of hippocampal neurons in culture

Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5mol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca²⁺]_in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mol/L, and overall production of neurites was reduced at 2 mol/L. The length of axons and the number of axons and dendrites were reduced at 1 mol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca²⁺]_in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca²⁺]_in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.Abbreviations BAPTA-AM 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester - [Ca²⁺]_in intracellular free calcium ion concentration - DMSO dimethyl sulfoxide - E18 embryonic day 18 - FBS fetal bovine serum - fura-2AM fura-2 acetoxymethyl ester - HBSS Hanks' Balanced Salt Solution - MEM Eagle's Minimum Essential Medium     

The ontogenetic origins of mirror neurons: evidence from 'tool-use' and 'audiovisual' mirror neurons

Since their discovery, mirror neurons-units in the macaque brain that discharge both during action observation and execution-have attracted considerable interest. Whether mirror neurons are an innate endowment or acquire their sensorimotor matching properties ontogenetically has been the subject of intense debate. It is widely believed that these units are an innate trait; that we are born with a set of mature mirror neurons because their matching properties conveyed upon our ancestors an evolutionary advantage. However, an alternative view is that mirror neurons acquire their matching properties during ontogeny, through correlated experience of observing and performing actions. The present article re-examines frequently overlooked neurophysiological reports of 'tool-use' and 'audiovisual' mirror neurons within the context of this debate. It is argued that these findings represent compelling evidence that mirror neurons are a product of sensorimotor experience, and not an innate endowment.     

Correlation of receptive field position of mechanosensory neurons and the strength of their connections to AP neurons in the CNS of the leech (Whitmania pigra)

An identified neuron of unknown function in the CNS of the leech, the anterior pagoda (AP) cell, receives multiple synaptic inputs from mechanosensory neurons that innervate the skin. Impulses in touch (T), pressure (P) and nociceptive (N) sensory cells on both sides of the ganglion produced electrical coupling potentials on both AP cells. Sensory cells with receptive fields contralateral to the cell body of the AP neuron always gave rise to larger synaptic potentials. In addition sensory cells supplying dorsal skin gave rise to larger synaptic potentials than those with lateral or ventral fields. It is suggested that integration by the AP cell can provide information about the position of mechanical stimuli impinging on the body wall of the animal.     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

A novel glutamate-mediated inhibitory mechanism linked with Ca2+/calmodulin-dependent protein kinase II in identified Euhadra neurons

The underlying mechanism(s) of the glutamate (Glu)-induced membrane hyperpolarizing response in identified Euhadra neurons was investigated using the voltage-clamp technique, pressure injection method, and pharmacologic agents. Under voltage-clamp conditions, bath-applied Glu elicits a slow outward potassium current (Glu current) accompanied by an increase in membrane conductance whose amplitude is dose dependent. Of the agonists tested, the Glu current was mimicked only by quisqualate (QA); its potency was approximately 10 times greater than that of Glu. Typical antagonists for the ionotropic type of Glu receptors and G protein inhibitors do not block this current. The Glu current is markedly enhanced by a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), KN-62 (1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) in a dose-dependent manner, while intracellularly injected CaM-KII suppresses the current. The potent protein kinase A inhibitors, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) and H-89 (N-[2-(p - bromocinnamylamino)ethyl] - 5 - isoquinolinesulfonamide) or the specific protein kinase C inhibitors staurosporine and K-252b had no effect on the Glu current. These results suggest the presence of a novel subtype of Glu receptor in Euhadra neurons, which may be coupled to the activation of potassium channels normally suppressed by CaM-KII. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 139–149, 1997.     

Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.