Mechanical tension modulates local and global vesicle dynamics in neurons.

Growing experimental evidence suggests that mechanical tension plays a significant role in determining the growth, guidance, and function of neurons. Mechanical tension in axons contributes to neurotransmitter clustering at the Drosophila neuromuscular junction (NMJ) and is actively regulated by neurons both in vitro and in vivo. In this work, we applied mechanical strain on in vivo Drosophila neurons and in vitro Aplysia neurons and studied their vesicle dynamics by live-imaging. Our experiments show that mechanical stretch modulates the dynamics of vesicles in two different model systems: (1) The global accumulation of synaptic vesicles (SV) at the Drosophila NMJ and (2) the local motion of individual large dense core vesicles (LDCV) in Aplysia neurites. Specifically, a sustained stretch results in enhanced SV accumulation in the Drosophila NMJ. This increased SV accumulation occurs in the absence of extracellular Ca(2+), plateaus after approximately 50 min, and persists for at least 30 min after stretch is reduced. On the other hand, mechanical compression in Aplysia neurites immediately disrupts LDCV motion, leading to decreased range and processivity. This impairment of LDCV motion persists for at least 15 min after tension is restored. These results show that mechanical stretch modulates both local and global vesicle dynamics and strengthens the notion that tension serves a role in regulating neuronal function.

Mass spectrometric imaging of the nervous system.

Mass spectrometric imaging (MSI) integrates multiple fields of analytical and biomedical research with the goal of generating chemical maps that present the identity and location of the elements, molecules, and molecular complexes that comprise biological structures. Rapid advances in the development of MSI, which include a broad range of sampling and mass spectrometry strategies, allow the increasingly information-rich creation of chemical images of structurally complex tissues, individual cells, and even single chromosomes. Here we describe a variety of MSI techniques available to investigate the nervous system, with particular focus on the capability of MSI to examine both normal and diseased brain function. An important investigative tool, MSI offers tremendous potential in fundamental studies of brain chemistry, localization of pharmaceutical compounds, and the discovery of biomarkers for different neuropathologies.

A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons.

A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250-310 nm, 310-400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L-1 for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics.

Identification and characterization of the feeding circuit-activating peptides, a novel neuropeptide family of aplysia.

We use a multidisciplinary approach to identify, map, and characterize the bioactivity of modulatory neuropeptides in the circuitry that generates feeding behavior in Aplysia. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the cerebral-buccal connective (CBC), a nerve containing axons of many interneurons that control feeding behavior of Aplysia, was used to identify neuropeptides that may participate in generation and shaping of feeding motor programs. Using this functionally oriented search, we identified a novel family of peptides that we call the feeding circuit-activating peptides (FCAPs). Two peptides with masses identical to those observed in the CBCs (molecular weight 1387 and 1433) were purified from buccal ganglia and partially sequenced using mass spectrometry. The amino acid sequence was then used to clone the FCAP precursor, which encodes multiple copies of eight different FCAPs. The two FCAPs present in highest copy number correspond to those observed in the CBC. The distribution of FCAP expression was mapped using Northern analysis, whole-mount in situ hybridization, and immunocytochemistry. Consistent with our initial findings, FCAP-immunopositive axons were observed in the CBC. Furthermore, we found that FCAP was present in some cerebral-buccal and buccal-cerebral interneurons. As their name suggests, FCAPs are capable of initiating rhythmic feeding motor programs and are the first neuropeptides with such activity in this circuit. The actions of FCAPs suggest that these peptides may contribute to the induction and maintenance of food-induced arousal. FCAPs were also localized to several other neuronal systems, suggesting that FCAPs may play a role in the regulation of multiple behaviors.

Direct assay of Aplysia tissues and cells with laser desorption/ionization mass spectrometry on porous silicon.

Desorption/ionization on porous silicon (DIOS) is a form of laser desorption mass spectrometry that allows for the direct mass analysis of a variety of analytes without the addition of organic matrix. Protocols are described for the direct analysis of exocrine tissue and single neurons using DIOS-MS. The atrial gland of Aplysia californica was blotted on to porous silicon and analyzed with DIOS-MS in the range m/z 1000-4000. The ability to culture invertebrate neurons directly on porous silicon is also presented. Isolated bag cells regenerated neuronal processes in culture on porous silicon. DIOS-MS allowed the direct detection of the peptides contained in individual cultured neurons indicating that with appropriate protocols, DIOS can be used with biological samples with considerable thickness.

Analysis of cellular release using capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

In order to increase our understanding of the mechanisms of learning and memory in the central nervous system, it is necessary to know the neurotransmitters and neuromodulators used in the specific neuronal circuits under study. Methods have been developed to identify the peptides released from single neurons and neuronal clusters from the common neuronal model Aplysia californica. Specifically, solid-phase extraction (SPE), capillary electrophoresis (CE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) are combined for profiling neuropeptide releasates. A variety of combinations of SPE and CE were coupled off-line with MALDI-TOF-MS to reduce the high physiological salts, to concentrate the analytes, and to reduce the complexity of the mass spectra using separation. With these protocols, peptides and proteins up to 11000 Da were detected in releasates, offering a much wider mass range compared to direct MALDI analysis of the same releasates. A number of expected and unknown neuropeptides, including egg-laying hormone (ELH) and the partially processed delta/gamma-bag cell peptide were observed in the SPE-treated releasates from a single Aplysia-cultured bag cell neuron. However, by adding a CE separation after the SPE step preceding off-line MALDI-TOF-MS detection, the most complete neuropeptide profiles were obtained.

Cerebrin prohormone processing, distribution and action in Aplysia californica.

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction.

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.

Measuring the peptides in individual organelles with mass spectrometry.

New sampling protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allow the assay of single dense core vesicles. Understanding the packaging of vesicles is important as vesicles are the quanta of information for intercellular communication. Using vesicles from the exocrine atrial gland of Aplysia californica as the model, a wide range of bioactive peptides are detected within each vesicle. Although the expression of the egg-laying hormone gene family of type 1 atrial gland cells has been previously examined, chemical characterization of individual 1-2 microm diameter vesicles demonstrates that products from several genes are colocalized. The mass sensitivity of MALDI MS can be further improved to enable the analysis of even smaller subcellular organelles.

Independent optimization of capillary electrophoresis separation and native fluorescence detection conditions for indolamine and catecholamine measurements.

Separation conditions in capillary electrophoresis with native fluorescence detection often represent a compromise in terms of the separation and detection figures of merit. As both the separation and fluorescence properties greatly depend on pH, the ability to independently optimize pH in the separation capillary and the detection region can improve many complex separations. When using a sheath flow cell, the pH at the detection zone can be adjusted independently of the electrophoresis buffer pH. Using capillary electrophoresis with 257-nm excitation and native fluorescence detection, more than an order of magnitude improvement in the limits of detection for dopamine (from 1400 to 120 nM) and epinephrine (from 850 to 60 nM) is achieved by maintaining the basic separation conditions and an acidified sheath buffer. The detection of dopamine in an individual Aplysia californica cerebral ganglion neuron is demonstrated.

Insulin prohormone processing, distribution, and relation to metabolism in Aplysia californica.

The first Aplysia californica insulin gene is characterized and its proteolytic processing from prohormone to final peptides elucidated using a combination of biochemical and mass spectrometric methods. Aplysia insulin (AI) is one of the largest insulins found, with a molecular weight of 9146 Da, and an extended A chain compared with other invertebrate and vertebrate insulins. The AI prohormone produces a series of C peptides and also a unique N-terminally acetylated D peptide. AI-producing cells are restricted to the central region of the cerebral ganglia mostly within the F and C clusters, and AI is transported to neurohemal release sites located on the upper labial and anterior tentacular nerves. The expression of AI mRNA decreases when the animal is deprived of food, and injections of AI reduce hemolymph glucose levels, suggesting that the function of insulin-regulating metabolism has been conserved.

Characterization of the Aplysia californica cerebral ganglion F cluster.

The cerebral ganglia neurons of Aplysia californica are involved in the development and modulation of many behaviors. The medially located F cluster has been characterized using morphological, electrophysiological and biochemical techniques and contains at least three previously uncharacterized neuronal population. As the three subtypes are located in three distinct layers, they are designated as top, middle, and bottom layer F-cluster neurons (CFT, CFM, and CFB). The CFT cells are large (92 +/- 25 microm), white, nonuniformly shaped, and located partially in the sheath surrounding the ganglion. These neurons exhibit weak electrical coupling, the presence of synchronized spontaneous changes in membrane potential, and a generalized inhibitory input upon electrical stimulation of the anterior tentacular (AT) nerve. Similar to the CFT neurons, the CFM neurons (46 +/- 12 microm) are mainly silent but do not show electrical coupling or synchronized changes in membrane potential. Unlike the CFT neurons, the CFM neurons exhibit weak action potential broadening during constant current injection. Comparison of the peptide profiles of CFT, CFM, and CFB (10-30 microm) neurons using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry demonstrates distinct peptide molecular weights for each neuronal subtype with the masses of these peptides not matching any previously characterized peptides from A. californica. The mass spectra obtained from the AT nerve are similar to the CFT neuron mass spectra, while upper labial nerve contains many peptides observed in the CFM neurons located in nongranular neuron region.

Formation of N-pyroglutamyl peptides from N-Glu and N-Gln precursors in Aplysia neurons.

Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry is used to examine the formation of N-pyroglutamate (pGlu) in single, identified neurons from Aplysia. Six pGlu peptides are identified in the R3-14 and the R15 neurons that result from in vivo processing of peptides containing either Glu or Gln at their respective N-termini. Moreover, we show that Glu-derived pGlu is not a sample collection or measurement artifact. The pGlu peptides are detected in isolated cell bodies, regenerated neurites in culture, interganglionic connective nerves, cell homogenates, and collected releasates. We also demonstrate that R3-14 cells readily convert a synthetic N-Glu peptide to its pGlu analogue, indicating the presence of novel enzymatic activity.

Opposite effects of interleukin-2 and interleukin-4 on GABA-induced inward currents of dialysed Lymnaea neurons.

1. The effects of interleukin-2 (rhIL-2) and interleukin-4 (rhIL-4) were investigated on gamma-aminobutyric acid (GABA)-induced inward currents on isolated, identified neurons of Lymnaea stagnalis L. (Mollusca, Gastropoda) by using a concentration clamp technique. 2. It was shown that the interleukins modified the GABA-induced inward current in an opposite direction: rhIL-2 (2-100 U/ml) decreased the peak value of IGABA in a dose-dependent manner, whereas rhIL-4 (0.2-100 U/ml), on the contrary, potentiated it. Both types of modulation were partially or fully reversible. 3. The reversal potential of IGABA was not shifted by these cytokines. 4. The time-to-peak value and inactivation time constant of the gamma-aminobutyric acid (GABA)-induced current was decreased by rhIL-4. The modulatory effect of rhIL-4 was eliminated after conjugation of this cytokine with its antibody. 5. It appears that cytokines could play a role in regulating the neural excitability through GABA-erg mechanisms.

Met-enkephalin and morphiceptin modulate a GABA-induced inward current in the CNS of Lymnaea stagnalis L.

1. The interaction between GABA and opioid peptides (met-enkephalin and morphiceptin) was studied on the identified, isolated and internally perfused neurons of Lymnaea stagnalis L. (Gastropoda, Basommatophora). 2. GABA (10(-7)-10(-5)M) activated a Cl-dependent inward current with about -20 mV equilibrium potential. Slow and fast GABA-induced inward currents were recorded with different kinetic parameters in distinct identified neurons. 3. Both types of GABA-induced inward currents were reduced or blocked by met-enkephalin (10(-7)-10(-5)M) and morphiceptin (10(-7)-10(-5)M) in a dose-dependent manner. GABA-activated fast inward current was modulated in a biphasic way in some neurons. Opioid reduction of the GABA-activated slow inward current was reversible, whereas the fast current was not. 4. The reversible inhibition of the GABA-induced slow inward current produced by met-enkephalin or morphiceptin was naloxone (10(-5)-10(-4)M)-sensitive, whereas the irreversible block of the fast GABA response was not antagonised by naloxone. Some additive effects between GABA and the peptides were also noted. 5. The modulatory effect of the opioid peptides on the GABA response altered the peak current, the time-to-peak and inactivation time-course of the GABA-induced current. 6. Thus, the identified, isolated and internally perfused neurons of Lymnaea stagnalis L. provide a useful model for studying postsynaptic mechanisms of interaction between GABA and opioid peptides. This interaction is a phenomenon of evolutionary significance because of it is also found in mammals.

Behavioral changes induced by GABA-receptor agonists in Lymnaea stagnalis L.

1. GABA, the GABAA receptor agonist, muscimol, and the GABAB receptor agonist, baclofen, were tested to study the involvement of the GABA neurotransmitter system in control of behaviour in the freshwater pulmonate snail, Lymnaea stagnalis L. Single injections of GABA (1-10 micrograms/ gbw) into the haemocoel of intact snails elicits a sequence of behavioural changes subsequently affecting feeding, locomotion, escape reactions, male mating behaviour and respiration. 2. Both muscimol and baclofen mimic distinct aspects of GABA action implying that the GABA action is mediated by both types of receptors. 3. The modulatory actions of GABA, muscimol, and baclofen on feeding were manifested as opening of the mouth and triggering of specific radular movements (e.g., protraction, retraction, rasping). 4. Baclofen (1-10 micrograms/gbw) evoked the full erection of the penis for a time long enough for natural copulation, while GABA itself caused only partial eversion of the preputium. Muscimol was less effective than GABA. The latency to penis eversion varied in a dose-dependent manner. The data emphasize the participation of baclofen-sensitive receptors in control of reproduction. 5. GABA and baclofen induced withdrawal of the head and anterior part of the foot accompanied by arrest of locomotion and respiration could be considered to be an escape reaction. 6. Muscimol (1-5 micrograms/gbw) failed to elicit withdrawal reactions but caused the loss of normal orientation and longitudinal contractions of the foot. 7. All the agents tested inhibited locomotion in a dose-dependent manner, for a substantial period of time. A decrease in total locomotor activity lead to the failure of animals to attach to the underlying surface, to have foot contractions and also to diminish locomotor velocity. 8. The majority of animals maintained the stereotyped complex reactions of respiration, but with restricted clockwise turning of the shell after drug treatment. 9. All the substances were shown to shift the background behavioural state characterised by high BSSs to a state similar to "rest" which was awarded a low BSS. It is concluded that the GABA neurotransmitter system can play an important role in activation and coordination of neuronal ensembles underlying behaviour and behaviour selection in Lymnaea stagnalis L.

Characterization of the GABA response on identified dialysed Lymnaea neurons.

1. The effect of gamma-aminobutyric acid has been studied on identified, internally perfused dialysed neurons of Lymnaea stagnalis L. (Pulmonata, Basommatophora). It was shown that: On the majority of neurons GABA (10(-8)-10(-3) M) depolarized the membrane with a decrease in input resistance and activated a Cl- dependent inward current with -20 +/- 4 mV E(GABA). In some cells, the outward current with -67 +/- 8 mV E(GABA) was also recorded. 2. The GABA-induced inward current was fast (1.5 +/- 0.3 sec, n = 4) or slow (3.2 +/- 0.2 sec, n = 3) peaking in a voltage-independent manner. The inactivation phase could be fitted by one or two exponentials characterized with fast (tau = 0.7 sec) and slow (tau = 3.6 sec) time constants. The outward current component was slow and activated at more positive Vh(-30-20 mV). 3. The agonist effects (GABA and muscimol) indicated the involvement of GABA(A) receptors and Cl-permeability changes in activating inward current. Picrotoxin (10(-5)-10(-4) M) and Cd2+ completely inhibited the GABA-activated inward current also affecting E(GABA). Furosemide was without effect on the peak value of GABA-induced inward current, but slightly modified the slope of inactivation. 4. High concentrations of Ca-ions and their substitution with Ba-ions in extracellular saline failed to alter the GABA-induced inward current. However, omission of Cl-ions from extracellular media shifted E(GABA) to the right by 18 +/- 8 mV (n = 4). 5. Omission of Cl-ions from intracellular saline led to inhibition of the fast component of GABA-induced inward current. Full recovery followed readdition of Cl-ions. 6. The results are in agreement with the data obtained on cloned Lymnaea GABA receptors.

Interleukin-4 potentiates voltage-activated Ca-currents in Lymnaea neurons.

The effect of interleukin-4 (IL-4) was studied on voltage-gated Ca-current of the neuron RPeD1 of Lymnaea stagnalis L. (Mollusca, Basommatophora), using two-microelectrode voltage-clamp method. It was found that: 1. The neuron RPeD1 possessed a high voltage-activated Ca-current characterized by -40 mV activation threshold and reaching its maximum at +5+5(-)+10 mV. The activation time constant of the current was found to be a monnexponential function of the membrane potential. Currents were almost completely inactivated within 200-300 ms. 2. IL-4 (10-200 U/ml) uniformly and reversibly increased the peak value of Ca-current in a dose- and time-dependent manner (at concentrations 75, 150 and 200 U/ml the potentiation was 5.3 +/- 1.8% (n = 3), 5.5 +/- 1. (n = 3) and 14.6 +/- 7.4% (n = 4), respectively. 3. No changes in the membrane resistance and holding current were observed during IL-4 application. 4. The time constants of activation and inactivation were not affected by IL-4. The maximal conductance as the only kinetic parameter was increased under the influence of IL-4. 5. The affect in modulation of HVA Ca-current caused by IL-4 was additive between the doses of 10-15o U/ml but saturated at concentrations > 200 U/ml. 6. IL-4 antiserum was a potent inhibitor for enhancement of Ca-current by IL-4.

Inhibition of calcium spikes by gamma-amino-butyric acid in the neurons of Lymnaea stagnalis L.

The modulation of action potentials (APs) and high-threshold calcium currents by gamma-aminobutyric acid (GABA) and GABAB receptor agonist baclofen was investigated in identified internally perfused neurons of Lymnaea stagnalis using current-clamp and whole-cell voltage-clamp techniques. GABA (5 x 10(-5) M) and baclofen (5 x 10(-5) M) reversibly reduced overshoot and afterhyperpolarization phase of the action potentials. Application of GABA or baclofen inhibited inward and outward voltage-gated ionic currents. GABA (10(-8)-10(-4) M) was found to suppress high-threshold calcium current in voltage- and dose-dependent manner. These effects were mimicked by baclofen (5 x 10(-5) M) but not by muscimol (5 x 10(-5) M). Inhibition of Ca(2+)-currents could not be prevented by pentylenetetrazole (5 x 10(-5) M) or with pertussis toxin (1 microgram/ml). These results provide evidences for the presence of GABAB receptors in the identified central neurons of Lymnaea stagnalis L.

HgCl2 potentiates GABA activated currents in Lymnaea stagnalis L. neurones.

Using the concentration clamp technique the effects of inorganic mercury (HgCl2) on gamma-aminobutyric acid (GABA) activated Cl- currents were studied on Lymnaea neurons. Hg2+ at concentrations of 0.01-1 microM caused a significant increase in the GABA induced chloride permeability of the cell membrane. With simultaneous application of GABA and 0.1 microM Hg2+ the amplitudes of the GABA activated inward Cl- currents were increased to a mean of 168%. The threshold concentration for potentiation was 0.01 microM Hg2+, while the GABA induced Cl- current was suppressed at concentrations higher than 100 microM. HgCl2 was also effective in decreasing the time-to-peak value and the time of inactivation of the GABA evoked Cl- current. In the presence of 0.1 microM mercury chloride the peak reached the maximum 1.6 times faster than without mercury. The effect was independent of the membrane potential, and mercury did not change the reversal potential of the GABA activated Cl- current. Application of mercury alone at concentrations effective to enhance GABA evoked Cl- permeability caused no or only a very small sustained inward current. However, higher mercury concentrations or repeated mercury application greatly increased the membrane permeability, and this effect could not be reversed by a 15 min wash. The results suggest, that low mercury concentrations act on GABA-ergic synapses at a postsynaptic site, and the potentiation of the ligand gated Cl- current may be an important factor in the mechanism of Hg2+ neurotoxicity.