Fast synaptic transmission in the goldfish CNS mediated by multiple nicotinic receptors.

Usually nicotinic receptors in the central nervous system only influence the strength of a signal between neurons. At a few critical connections, for instance some of those involved in the flight response, nicotinic receptors not only modulate the signal, they actually determine whether a signal is conveyed or not. We show at one of the few such connections accessible for study, up to three different nicotinic receptor subtypes mediate the signal. The subtypes appear to be clustered in separate locations. Depending on the number and combination of the subtypes present the signal can range from short to long duration and from low to high amplitude. This provides a critical connection with a built-in plasticity and may enable it to adapt to a changing environment.

Transient electrical coupling regulates formation of neuronal networks.

Electrical synapses are abundant before and during developmental windows of intense chemical synapse formation, and might therefore contribute to the establishment of neuronal networks. Transient electrical coupling develops and is then eliminated between regenerating Helisoma motoneurons 110 and 19 during a period of 48-72 h in vivo and in vitro following nerve injury. An inverse relationship exists between electrical coupling and chemical synaptic transmission at these synapses, such that the decline in electrical coupling is coincident with the emergence of cholinergic synaptic transmission. In this study, we have generated two- and three-cell neuronal networks to test whether predicted synaptogenic capabilities were affected by previous synaptic interactions. Electrophysiological analyses demonstrated that synapses formed in three-cell neuronal networks were not those predicted based on synaptogenic outcomes in two-cell networks. Thus, new electrical and chemical synapse formation within a neuronal network is dependent on existing connectivity of that network. In addition, new contacts formed with established networks have little impact on these existing connections. These results suggest that network-dependent mechanisms, particularly those mediated by gap junctional coupling, regulate synapse formation within simple neural networks.

Transient electrical coupling delays the onset of chemical neurotransmission at developing synapses.

The formation and subsequent elimination of electrical coupling between neurons has been demonstrated in many developing vertebrate and invertebrate nervous systems. The relationship between the disappearance of electrical synaptic connectivity and the appearance of chemical neurotransmission is not well understood. We report here that identified motoneurons from the snail Helisoma formed transient electrical and chemical connections during regeneration both in vivo and in vitro. Electrical connections that formed in vivo were strongest by day 2 and no longer detectable by day 7. During elimination of this electrical connection, an inhibitory chemical connection from 110 onto 19 formed. This sequence of synaptic development was recapitulated in cell culture with a similar time course. The relationship between the appearance of transient electrical coupling and its possible effects on the subsequent chemical synaptogenesis were examined by reducing transient intercellular coupling. Trophic factor-deprived medium resulted in a 66% reduction in coupling coefficient. In these conditions, the unidirectional chemical connection formed readily; in contrast, chemical synaptogenesis was delayed in cell pairs exposed to trophic factors where transient electrical coupling was strong. Dye coupling and synaptic vesicle cycling studies supported electrophysiological results. Exposure to cholinergic antagonists, curare and hexamethonium bromide, which block chemical neurotransmission in these synapses, resulted in prolonged maintenance of the electrical connection. These studies demonstrated an inverse relationship between chemical and electrical connectivity at early stages of synaptic development and suggest a dynamic interaction between these forms of neuronal communication as adult neural networks are constructed or regenerated.

Distribution and physiological effects of B-type allatostatins (myoinhibitory peptides, MIPs) in the stomatogastric nervous system of the crab Cancer borealis.

The crustacean stomatogastric ganglion (STG) is modulated by a large number of amines and neuropeptides that are found in descending pathways from anterior ganglia or reach the STG via the hemolymph. Among these are the allatostatin (AST) B types, also known as myoinhibitory peptides (MIPs). We used mass spectrometry to determine the sequences of nine members of the AST-B family of peptides that were found in the stomatogastric nervous system of the crab Cancer borealis. We raised an antibody against Cancer borealis allatostatin-B1 (CbAST-B1; VPNDWAHFRGSWa) and used it to map the distribution of CbAST-B1-like immunoreactivity (-LI) in the stomatogastric nervous system. CbAST-B1-LI was found in neurons and neuropil in the commissural ganglia (CoGs), in somata in the esophageal ganglion (OG), in fibers in the stomatogastric nerve (stn), and in neuropilar processes in the STG. CbAST-B1-LI was blocked by preincubation with 10(-6) M CbAST-B1 and was partially blocked by lower concentrations. Electrophysiological recordings of the effects of CbAST-B1, CbAST-B2, and CbAST-B3 on the pyloric rhythm of the STG showed that all three peptides inhibited the pyloric rhythm in a state-dependent manner. Specifically, all three peptides at 10(-8) M significantly decreased the frequency of the pyloric rhythm when the initial frequency of the pyloric rhythm was below 0.6 Hz. These data suggest important neuromodulatory roles for the CbAST-B family in the stomatogastric nervous system.

Serotonin regulates electrical coupling via modulation of extrajunctional conductance: H-current.

Synaptic strength can be highly variable from animal to animal within a species or over time within an individual. The process of synaptic plasticity induced by neuromodulatory agents might be unpredictable when the underlying circuits subject to modulation are themselves inherently variable. Serotonin (5-hydroxytryptomine; 5HT) and serotonergic signaling pathways are important regulators of animal behavior and are pharmacological targets in a wide range of neurological disorders. We have examined the effect of 5HT on electrical synapses possessing variable coupling strengths. While 5HT decreased electrical coupling at synapses with weak electrical connectivity, synapses with strong electrical coupling were less affected by 5HT treatment, as follows from the equations used for calculating coupling coefficients. The fact that the modulatory effect of 5HT on electrical connections was negatively correlated with the strength of electrical coupling suggests that the degree of electrical coupling within a neural network impacts subsequent neuromodulation of those synapses. Biophysical studies indicated that these effects were primarily due to 5HT-induced modulation of membrane currents that indirectly affect junctional coupling at synaptic contacts. In support of these experimental analyses, we created a simple model of coupled neurons to demonstrate that modulation of electrical coupling could be due solely to 5HT effects on H-channel conductance. Therefore, variability in the strength of electrical coupling in neural circuits can determine the pharmacological effect of this neuromodulatory agent.

Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins.

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10(-6)M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.

Behavioral and physiological characterization of sensorimotor gating in the goldfish startle response.

Prepulse inhibition (PPI) is typically associated with an attenuation of auditory startle behavior in mammals and is presumably mediated within the brainstem startle circuit. However, the inhibitory mechanisms underlying PPI are not yet clear. We addressed this question with complementary behavioral and in vivo electrophysiological experiments in the startle escape circuit of goldfish, the Mauthner cell (M-cell) system. In the behavioral experiments we observed a 77.5% attenuation (PPI) of startle escape probability following auditory prepulse-pulse stimulation. The PPI effect was observed for prepulse-pulse interstimulus intervals (ISIs) ranging from 20 to 600 ms and its magnitude depended linearly on prepulse intensity over a range of 14 dB. Electrophysiological recordings of synaptic responses to a sound pulse in the M-cell, which is the sensorimotor neuron initiating startle escapes, showed a 21% reduction in amplitude of the dendritic postsynaptic potential (PSP) and a 23% reduction of the somatic PSP following a prepulse. In addition, a prepulse evoked a long-lasting (500 ms) decrease in M-cell excitability indicated by 1) an increased threshold current, 2) an inhibitory shunt of the action potential (AP), and 3) by a linearized M-cell membrane, which effectively impedes M-cell AP generation. Comparing the magnitude and kinetics of inhibitory shunts evoked by a prepulse in the M-cell dendrite and soma revealed a disproportionately larger and longer-lasting inhibition in the dendrite. These results suggest that the observed PPI-type attenuation of startle behavior can be correlated to distinct postsynaptic mechanisms mediated primarily at the M-cell lateral dendrite.

Effects of temperature acclimation on a central neural circuit and its behavioral output.

In this study, we address the impact of temperature acclimation on neuronal properties in the Mauthner (M-) system, a brain stem network that initiates the startle-escape behavior in goldfish. The M-cell can be studied at cellular and behavioral levels, since it is uniquely identifiable physiologically within the intact vertebrate brain, and a single action potential in this neuron determines not only whether a startle response will occur but also the direction of the escape. Using animals acclimated to 15 degrees C as a control, 25 degrees C-acclimated fish showed a significant increase in escape probability and a decrease in the ability to discriminate escape directionality. Intracellular recordings demonstrated that M-cells in this population possessed decreased input resistance and reduced strength and duration of inhibitory inputs. In contrast, fish acclimated to 5 degrees C were behaviorally similar to 15 degrees C fish and had increased input resistance, increased strength of inhibitory transmission, and reduced excitatory transmission. We show here that alterations in the balance between excitatory and inhibitory synaptic transmission in the M-cell circuit underlie differences in behavioral responsiveness in acclimated populations. Specifically, during warm acclimation, synaptic inputs are weighted on the side of excitation and fish demonstrate hyperexcitability and reduced left-right discrimination during rapid escapes. In contrast, cold acclimation results in transmission weighted on the side of inhibition and these fish are less excitable and show improved directional discrimination.

Otolith endorgan input to the Mauthner neuron in the goldfish.

The Mauthner (M-) cell of the goldfish, Carassius auratus, triggers the rapid escape response of the fish in response to various stimuli, including visual and auditory. The large size and accessibility of the M-cell make it an ideal model system for the study of synaptic transmission, membrane properties, and sensory-motor gating. Although physiological recordings have suggested that afferents from all three of the inner ear endorgans (the saccule, lagena, and utricle) synapse directly on the ipsilateral M-cell, the specific contacts and anatomical distributions of these inputs along the M-cell lateral dendrite remain unknown. We traced specific branches of the auditory (VIIIth) nerve from the three otolith organs of the fish inner ear to the M-cell. The goldfish sacculus gives rise to the vast majority of inputs that contact a large portion of the M-cell lateral dendrite, and these inputs vary greatly in size. In contrast to the ubiquitous distribution of saccular inputs, those from the lagena are segregated to distal regions of the M-cell and synapse on the distal dorsal branch of the lateral dendrite. Similarly, inputs from the utricle are also segregated to distal regions, synapsing on the ventral branch of the lateral dendrite. These results demonstrate that nerves from all three endorgans contact the M-cell, with input-specific segregation of synapses along the M-cell lateral dendrite.