Synaptic actions of identified peptidergic neuron R15 in Aplysia. I. Activation of respiratory pumping

The purpose of the study described in this and the following two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 increases the frequency of bursting in the R25/L25 network that triggers respiratory pumping. This excitatory modulatory effect appears to be mediated by R15 alpha 1 peptide. R15 activates both strong and weak modes of respiratory pumping. In contrast, the two R20 cells, which are thought to use the neuropeptides SCPA and SCPB as transmitters, elicit only strong episodes of respiratory pumping. The synaptic actions of R15 also differ from those of the R20 cells in being longer lasting and in exhibiting profound desensitization. Chronic recording of R15 activity in vivo indicates that it does not burst spontaneously in the intact animal, so the synaptic actions of R15 are not chronically desensitized. The neuroendocrine bag cells, which initiate egg laying, had been shown by others to excite R15 and the R25/L25 network that triggers respiratory pumping. Our data indicate that the excitatory effects of the bag cells on the R25/L25 cells are mediated in part by R15.     

Respiratory pumping: Neuronal control of a centrally commanded behavior in aplysia

Respiratory pumping in Aplysia californica is a relatively stereotyped behavioral pattern with three components: (1) withdrawal of gill, siphon and mantle shelf; (2) closing of parapodia; (3) heart inhibition accompanied by a decrease in vasomotor tone. This phasic behavior is triggered by a central burst-generating network of interneurons in the abdominal ganglion. During respiratory pumping, motor neurons innervating the several effector organs receive a burst of either excitatory or inhibitory synaptic input which has previously been attributed to an unidentified central command cell called Interneuron II. Several of these motor cells are also concomitantly release from tonic synaptic input, which is opposite in sign to that which they receive from Interneuron II. This tonic input has been attributed to an unidentified cell called Interneuron XI. In this paper we identify and describe some of the neurons which contribute to the burst generating network; specifically, we focus on the neurons that produce the synaptic action attributed to Interneurons II and XI. The synaptic actions attributed to Interneuron XI are produced by a single, spontaneously active neuron, cell L24. This cell is a multi-action interneuron: it produces inhibitory synaptic potentials in some follower motor neurons, excitatory synaptic potentials in other follower cells, and a conjoint excitatory-inhibitory synaptic action onto gill motor neuron L7. At low frequency, L24 is excitatory to L7. With high frequency firing of L24, the synaptic potential produced in L7 converts from excitatory to inhibitory. In contrast to Interneuron XI, which is a single cell, the synaptic potentials previously attributed to Interneuron II are actually produced by a cluster of at least 3 respiratory command cells which we call L25, L26 and L27. Each of these cells accounts for only a limited portion of the synaptic input that drives the motor neurons during respiratory pumping. For most motor neurons innervated by both the respiratory command cells and Interneuron XI, the two synaptic inputs are opposite in sign. Mutually inhibitory connections between Interneuron XI and some of the central respiratory command cells ensure that the synaptic potentials from these two sources are constrained to occur at different times. Thus, centrally commanded synaptic inhibition or excitation of these motor neurons is made more effective by simultaneous disexcitation or disinhibition of Interneuron XI input. In addition to their role in generating respiratory pumping, L24 and L26 also contribute to the mediation of the defensive gill and siphon withdrawal reflex.     

Synaptic actions of identified peptidergic neuron R15 in Aplysia. III. Activation of the large hermaphroditic duct

The purpose of the study described in this and the preceding two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 activates anterograde peristaltic movements in the segment of the large hermaphroditic duct through which eggs move during egg-laying behavior. This action is mimicked by R15 alpha 1 peptide, a putative transmitter of R15. The neuroendocrine bag cells, which initiate egg laying when they fire in a population burst, have been shown by others to excite R15. Our data suggest that R15 mediates excitatory effects of the bag cells on the large hermaphroditic duct. Taken with the results of the two companion papers, these data support the hypothesis that R15 integrates various aspects of egg-laying behavior. The desensitization of R15's postsynaptic actions may complement the long-lasting refractoriness of the bag cells described by others, with both effects contributing to the episodic nature of egg laying.     

Nonsynaptic characteristics of neurotransmission mediated by egg-laying hormone in the abdominal ganglion of Aplysia

The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.     

Inhibitors of calcium-dependent enzymes prevent the onset of afterdischarge in the peptidergic bag cell neurons of Aplysia

Experiments with isolated bag cell neurons of Aplysia have produced evidence that changes in neuronal excitability may be brought about by the activation of calcium dependent enzymes such as calcium-dependent protein kinases. We have now examined the effects of agents which have been shown to inhibit several calcium-dependent enzymes on the properties of bag cell neurons in situ. In response to brief electrical stimulation the bag cell neurons of Aplysia generate an afterdischarge during which they release neuroactive peptides. We have found that the ability of stimulation to trigger an afterdischarge in the bag cell neurons is inhibited by trifluoperazine (TFP, 50-100 microM), N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide (W7) (25-50 microM) and calmidazolium (40-100 microM), each of which has previously been shown to inhibit calmodulin-dependent enzymes and the calcium-phospholipid-dependent protein kinase in the bag cell neurons. Further analysis of the effects of TFP showed that this inhibition occurs at concentrations which do not inhibit synaptic transmission or the endogenous bursting of another neurosecretory neuron, R15. Secretion of neuroactive peptides from the bag cell neurons was measured both electrophysiologically and biochemically. No attenuation of secretion could be observed at concentrations of TFP below those which inhibited the initiation of afterdischarge. Our results indicate that these agents inhibit secretion from these neurons primarily by inhibiting the onset of the afterdischarge and are consistent with the hypothesis that a calcium-dependent enzyme plays a role in triggering the stimulus-induced transformation in the electrical properties of these neurons.     

Peptidergic modulation of neuronal circuitry controlling feeding in Aplysia

We examined the effects of 3 neuropeptides and the bioactive amine 5-HT on identified motoneurons (B15 and B16) and interneurons (B4, B5) involved in the control of feeding behavior in Aplysia californica. The application of egg-laying hormone (ELH), small cardioactive peptide b (SCPb), and 5-HT elicits distinct patterns of synaptically induced bursting in the neurons, while PheMetArgPheamide (FMRFamide) inhibits firing due to synaptic activity. Repetitive IPSPs recorded in B15 and B16 are induced by 5-HT and SCPb and inhibited by FMRFamide. The substances also may act directly: In solutions that block synaptic transmission SCPb excites B15, ELH excites B16, 5-HT excites B15, B16, and B4, and FMRFamide both inhibits B15 and B16 and excites B4. We suggest that the output of a buccal ganglion central pattern generator may be modulated to produce distinct patterns of motoneuron activity by these candidate transmitters. We also noted differences in the intrinsic properties of the 2 motoneurons. B15 contains SCPb immunoreactivity while B16 does not. This finding suggests that B15 may be the source for the SCPb immunoreactivity previously found at the ARC muscle and that SCPb may be acting in an autocrine mode. Also, B15 has a significantly lower resting potential than B16 and contains a large transient outward (Ia-like) current. The candidate transmitters act by exciting or inhibiting elements at every level within the hierarchically organized motor system that controls feeding. This expands the diversity of behavioral repertoires that may be elicited from a particular neural circuit.     

Metabolic regulations of the rhythmic activity in pacemaker neurons. II. Metabolically induced conversions of beating to bursting pacemaker activity in isolated Aplysia neurons.

In pacemaker neurons of the sea hare Aplysia californica, isolated from their synaptic, ephaptic and humoral inputs, conversion of the regular beating to a bursting discharge pattern can be induced by certain cell metabolites. Administration of the phosphofructokinase (PFK) activator fructose-6-phosphate (F-6-P), or its nonmetabolizable analogue 1-deoxy-F-6-P, induced bursting discharges in R3, R5, R6 and R11 neurons, with spike doublets and triplets appearing transiently in the time pattern. With another PFK activitor, adenosine-5-monophosphate, only double spikes have been noted in R7, R8 and R14 neurons. Burst activity was induced also in the presence of the fructose-1,6-diphosphatase activators, citrate and 3-phosphoglycerate, in R9, R10 and R12 neurons. Cyclic 3',5'-AMP, which also activates the PFK (beside other effects on cellular metabolism), induced bursting discharges in all R3-R14 neurons. In contrast, the inhibitors of the PFK, citrate and ATP, decreased the spike activity of the bursting L3 and L6 neurons, even changing L3 neurons to the regular beating type. Among a variety of cell metabolites tested only pyruvate was able to induce a burst-like tendency in R9 neurons. The characteristic bursting patterns which appeared in the presence of the described metabolic effectors could not be duplicated by low Ca2+ and/or high K+ media nor by artificial shifts in membrane potential triggered by depolarizing and hyperpolarizing currents.     

Synaptic organization in the lamina of the superposition eye of a skipper butterfly, Parnara guttata.

The first optic neuropil of the compound eye, the lamina, of the skipper butterfly Parnara guttata, was examined by light microscopy after Golgi-impregnation and by electron microscopy (EM) to clarify the cellular and synaptic organization. In the lamina, five different types of lamina neurons (L neurons) were characterized by using Golgi-impregnation. By EM, each cartridge was found to contain all nine receptor axons from an ommatidium, five L neurons, and a few putative centrifugal elements. Axons from photoreceptors (retinula cells) R2, R3, R4, R6, R7, and R8 terminate as short visual fibers (svfs) in the lamina cartridge. Those from R1, R5, and R9 penetrate the lamina and terminate in the medulla as long visual fibers (lvfs). In the cartridges, the synaptic contacts were formed from svfs onto L neurons, from the lvfs of R1 and/or R5 to the lvf of R9 and L neurons, and from the lvf of R9 to L neurons. The putative centrifugal fibers also make synapses to svfs and L neurons. At the most distal level of the cartridge, one of the centrifugal fibers containing dense-core vesicles makes presynaptic contacts to the putative long collaterals of the L neuron. A novel characteristic feature of this lamina is that svfs of R3 and R7 and the lvfs of R1 or R5 have long collaterals extending into neighboring cartridges. Presynaptic contacts were confirmed in such long collaterals from the svf. These results imply that receptor axons provide direct intercartridge connections as well as providing indirect connections to neighboring cartridges by way of their input upon L neurons.     

Protein synthesis in phenotypically different, single neurons of Aplysia

Different molecular weight classes of proteins from single phenotypically different neuron somas and their subcellular fractions were analyzed in terms of the relative concentration and relative labeling using sodium dodecyl sulfate (SDS) polyacrylamide microgel electrophoresis. A 12,000 (12K) molecular weight class of protein found in low relative concentration but highly labeled was shown to be present in some endogenously firing neurons, L3, L6, and L11, but not in others such as L4. A specific 12K class of protein has been found to be common to all neurosecretory neurons, R15, R14 and R3–13, studied. A high relative concentration of 6–9K class of protein in R15 and bag cells and a highly labeled 6–9K protein shown to be in R14 and R3–13 have been correlated with neurosecretion. In subcellular fractionation studies a specific 12K class of proteins was found in the membrane fraction of neuron L11. Several proteins, which have molecular weights similar to filarin (77K), tubulin (58–55K) and actin (43K) have been shown to be common to all neurons studied. Hence this study has demonstrated protein specificity in single phenotypically different neurons. Suggestive correlations of specifically translated small molecular weight proteins with neurosecretion in neurons R3–15 were made.     

VIP enhances synaptic transmission to hippocampal CA1 pyramidal cells through activation of both VPAC1 and VPAC2 receptors

We previously described that vasoactive intestinal peptide (VIP) increases synaptic transmission to hippocampal CA1 pyramidal cells at concentrations known to activate VIP-selective receptors (VPAC1 and VPAC2) but not the PACAP-selective PAC1 receptor. We now investigated the involvement of VPAC1 and VPAC2 receptors in the effects elicited by VIP as well as the transduction pathways activated by VIP to cause enhancement of synaptic transmission. Blockade of either VPAC1 or VPAC2 receptors with PG 97-269 (100 nM) or PG 99-465 (100 nM) inhibited VIP-induced enhancement of synaptic transmission. Selective activation of VPAC1 receptors with [K15, R16, L27] VIP(1-7)/GRF(8-27) (10 nM) or of VPAC2 receptors with RO 25-1553 (10 nM) increased synaptic transmission to CA1 pyramidal cells, and this increase was larger when both agonists were applied together. Inhibition of either PKA with H-89 (1 microM) or PKC with GF109203X (1 microM) attenuated the effect of VIP (1 nM). GF109203X (1 microM) abolished the effect of the VPAC1 agonist [K15, R16, L27] VIP(1-7)/GRF(8-27) (10 nM) on hippocampal synaptic transmission but that effect was not changed by H-89 (1 microM). The effect of RO 25-1553 (100 nM) obtained in the presence of both the PAC1 and VPAC1 antagonists, M65 (30 nM) and PG 97-269 (100 nM), was strongly inhibited by H-89 (1 microM) but not GF109203X (1 microM). It is concluded that VIP enhances synaptic transmission to CA1 pyramidal cell dendrites through VPAC1 and VPAC2 receptor activation. VPAC1-mediated actions are dependent on PKC activity, and VPAC2-mediated actions are responsible for the PKA-dependent actions of VIP on CA1 hippocampal transmission.     

Localization and differential interaction of R7 RGS proteins with their membrane anchors R7BP and R9AP in neurons of vertebrate retina

G protein signaling in the retina is crucially regulated by the R7 family of regulators of G protein signaling (RGS) proteins, which act to stimulate the rate of G protein inactivation. Recent findings indicate that R7 RGS proteins form complexes with two newly identified membrane anchors: RGS9 Anchor Protein (R9AP) and R7 Binding Protein (R7BP), which play essential roles in modulating the expression and localization of R7 RGS proteins. Here we demonstrate that the four R7 RGS proteins: RGS6, RGS7, RGS9 and RGS11 differentially associate with two membrane anchors. R9AP was found to form complexes with RGS9 and RGS11 which were substantially enriched in the photoreceptors. In contrast, complexes of R7BP with R7 RGS proteins were predominantly localized to the synaptic projections of retina neurons, suggesting their involvement in regulation of synaptic transmission between retina neurons. Furthermore, studies of knockout mice revealed that R9AP is necessary for the expression of only RGS9 but not for RGS6, 7 or 11. Together these data suggest that R7 RGS proteins in the retina are present as macromolecular complexes with their membrane anchors that could differentially regulate their function in various retina neurons.     

SCP-containing R20 neurons modulate respiratory pumping in Aplysia

Respiratory pumping in Aplysia consists of transient, synchronous pumping actions of the gill, siphon, mantle shelf, and parapodia. This behavior has previously been shown to be driven by a network of coupled interneurons in the abdominal ganglion, the R25 and the L25 cells. We describe here a pair of electrically coupled cells, the R20 cells, which when active can initiate respiratory pumping or increase its spontaneous rate of occurrence. This action is mediated by a slow, long-lasting excitation of the endogenous burst mechanism of the cells in the R25/L25 network. The R20 cells, which are located in the abdominal ganglion, also make slow inhibitory connections to the RB cells and to the RG cells in that ganglion, and to the gill motoneurons in the branchial ganglion. The R20 cells are immunoreactive to SCPB, a molluscan neuropeptide. Biochemical purification studies demonstrate that each of the R20 cells synthesizes not only SCPB, but also SCPA, a closely related molecule known to be encoded by the same gene as SCPB. The R20 cells also synthesize in abundance several other low-molecular-weight, methionine-containing peptides. The excitatory actions of the R20 cells on the R25/L25 network are mimicked by SCPA and SCPB. However, the inhibitory actions of the R20 cells on the RB cells, the RG cells, and on the cells of the branchial ganglion are not mimicked by the SCPs. Thus, the data support the hypothesis that the R20 cells release SCPA and SCPB and at least one other unidentified transmitter.     

Alpha bag cell peptide directly modulates the excitability of the neurons that release it

Brief electrical or hormonal stimulation of the bag cell neurons of Aplysia triggers a long-lasting discharge during which alpha bag cell peptide (alpha-BCP) and other neuropeptides are released from the cells. We have carried out experiments, using both intact abdominal ganglia and isolated neurons, demonstrating that alpha-BCP acts directly on the bag cell neurons to influence cAMP levels and voltage-dependent potassium currents. Exposure of the bag cell neurons within intact ganglia to alpha-BCP, at concentrations greater than 1 nM, inhibited an ongoing discharge. alpha-BCP also significantly reduced both basal and forskolin-stimulated levels of cAMP in bag cell clusters. The inhibition of the discharge by alpha-BCP could be prevented and reversed by pharmacological elevation of intracellular cAMP levels. Immunohistochemical staining of neurons maintained in cell culture showed that all isolated bag cell neurons exhibit immunoreactivity with antisera against alpha-BCP. Application of the adenylate cyclase activator forskolin to such isolated cells, in the presence of a phosphodiesterase inhibitor, attenuates the amplitude of the delayed voltage-dependent outward currents measured in voltage-clamp experiments. Pretreatment of the cells with alpha-BCP significantly reduced the ability of forskolin to attenuate these currents, demonstrating that alpha-BCP acts directly at autoreceptors on bag cell neurons. Experiments with the isolated cells showed that a second autoreceptor-mediated effect of alpha-BCP was the enhancement of an inwardly rectifying potassium current that was activated at potentials more negative than -40 mV. The reversal potential and conductance of the current induced by alpha-BCP were dependent on the external K+ concentration. This response to alpha-BCP could be blocked by rubidium, cesium, and barium ions. Our data demonstrate that alpha-BCP can exert inhibitory biochemical and electrophysiological actions on the bag cell neurons that release it and suggest that autoreceptors for alpha-BCP play an important role in the termination of a discharge in the bag cell neurons.     

Relationship between two types of vesicular glutamate transporters and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies

Our previous study demonstrated GABAergic and glycinergic synapses onto neurokinin‐1 receptor (NK1R)‐immunoreactive (ir) neurons in the pre‐Bötzinger complex (pre‐BötC), the hypothesized kernel of normal respiratory rhythmogenesis. In the present study, we aimed to identify glutamatergic synapses onto NK1R‐ir pre‐BötC neurons, as excitatory synaptic transmission is a prerequisite to normal respiratory rhythmogenesis. Two types of vesicular glutamate transporters (VGLUT), VGLUT1 and VGLUT2, have been recently implicated in glutamate‐mediated transmission. The present study used immunofluorescence and immunogold‐silver staining to determine the relationship between the transporters and NK1R‐ir neurons in the pre‐BötC of adult rats. Under the confocal laser‐scanning microscope, VGLUT2‐ir boutons were found to be widely distributed in the pre‐BötC, some of which were in close apposition to NK1R‐ir somas and dendrites. VGLUT1‐ir boutons were relatively rare and only a few were found to be in close apposition to NK1R‐ir somas and dendrites. Electron microscopic observation revealed that approximately 41% of VGLUT2‐ir terminals were in close apposition to, or made asymmetric synapses with NK1R‐ir somas and dendrites in the pre‐BötC. On the other hand, 50.5% of NK1R‐ir dendrites were closely apposed to, or synapsed with VGLUT2‐ir terminals. Occasionally, VGLUT1‐ir terminals were found in close apposition to NK1R‐ir somas or dendrites, but we were unable to identify synapses between them. The present findings provide the morphological basis for excitatory synaptic inputs onto NK1R‐ir neurons in the pre‐BötC. VGLUT2 may be involved in a dominant excitatory synaptic pathway for normal respiratory rhythmogenesis.     

Giant Aplysia neuron R2 reliably forms strong chemical connections in vitro

The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.     

Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Convergent inputs to single neurons in two different subdivisions of somatosensory forepaw digit cortex of the raccoon.

The aim of this study was to compare the physiological properties of single neurons in the glabrous (G) and heterogeneous (H) subdivisions of primary somatosensory digit 3 cortex of adult raccoons. Extracellular recordings were obtained from 50 G neurons whose receptive fields (RFs) were confined to the glabrous skin of a digit, and 41 H neurons whose RFs were located on hairy skin, claws, or mixtures of skin types. Both electrical and mechanical stimulation of the digits were used to assess excitatory neuronal responsiveness. The two sets of neurons, which had nearly identical depth distributions, differed considerably in their input convergence: (i) the percentage of neurons (%N) responding to electrical or mechanical stimulation of each off-focus digit and (ii) the number of digits from which individual cells could be driven were significantly greater for H neurons. Those G and H cells which could be excited by off-focus inputs were examined for probability of response (P), number of spikes per response (S/R), and latency of response (L) to digit stimulation. Surprisingly, for input from any one digit, there were no significant differences in these response properties between the two sets of neurons. However, inputs from different (on-focus versus off-focus) digits varied significantly and revealed patterns of response properties that were qualitatively similar for both G and H neurons. Specifically, %N and P decreased while L increased symmetrically with distance of each off-focus digit from the central on-focus digit 3, reflecting corresponding variations in the synaptic accessibility and conduction time of off-focus excitatory inputs. In contrast, S/R values were very similar for all digits, suggesting that the synaptic strength of off-focus inputs is regulated independently of accessibility. Finally, preliminary findings indicated that denervation of the third digit caused a decrease in off-focus response latencies, while the normal latency profile across digits was retained. This suggests that the previously existing pattern of off-focus inputs to G and H neurons provides a template for denervation-induced cortical reorganization, whereby the synaptic efficacy of off-focus inputs is increased by disinhibition or facilitation.     

FMRF-amide-like substances in the leech. II. Bioactivity on the heartbeat system

In the preceding paper (Kuhlman, J. R., C. Li, and R. L. Calabrese (1985) J. Neurosci. 5: 2301-2309) FMRF-amide-like immunoreactivity was localized to a specific set of neurons in the leech. Three types of these neurons are involved in controlling the animal's heartbeat: HE motor neurons and HA modulatory neurons which directly innervate the hearts, and the swim-initiating interneurons (cells 204) which can accelerate the heartbeat central pattern generator. Application of synthetic FMRF-amide had effects on the hearts and the heartbeat central pattern generator that mimicked the actions of the HA and cell 204 neurons. Bath application of FMRF-amide (10(-7) to 10(-6) M) to the hearts activated their myogenic rhythm and increased their beat tension, thus mimicking the effects of activity in HA cells. Bath application of lower concentrations of FMRF-amide (10(-9) to 10(-8) M) to the isolated central nervous system dramatically accelerated the central motor program for heartbeat, thus mimicking the effects of activity in cell 204. These observations suggest that an FMRF-amide-like substance may be used as a chemical signal by HA and cell 204 neurons. The role of the FMRF-amide-like substance contained in HE motor neurons remains unclear, but it may be released along with the HE cell's neuromuscular transmitter, acetylcholine.     

Activation of mu-opioid receptors excites a population of locus coeruleus-spinal neurons through presynaptic disinhibition

The nucleus locus coeruleus (LC) plays an important role in analgesia produced by opioids and by modulation of the descending noradrenergic pathway. The functional role of micro-opioid receptors (muOR) in regulation of the excitability of spinally projecting LC neurons has not been investigated. In the present study, we tested the hypothesis that activation of presynaptic mu-opioid receptors excites a population of spinally projecting LC neurons through attenuation of gamma-aminobutyric acid (GABA)-ergic synaptic inputs. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye injected into the spinal dorsal horn of rats. Whole-cell current- and voltage-clamp recordings were performed on labeled LC neurons in brain slices. All labeled LC noradrenergic neurons were demonstrated by dopamine-beta-hydroxylase (DbetaH) immunofluorescence. In 37 labeled LC neurons, (D-Ala(2),N-Me-Phe(4),Gly-ol(5))-enkephalin (DAMGO) significantly increased the discharge activity of 17 (45.9%) neurons, but significantly inhibited the firing activity of another 15 (40.5%) cells. The excitatory effect of DAMGO on seven labeled LC neurons was diminished in the presence of bicuculline. DAMGO significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) in all nine labeled LC neurons. However, DAMGO had no effect on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in 12 of 15 neurons. Furthermore, DAMGO significantly inhibited the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in all 11 labeled neurons, but had no significant effect on the evoked excitatory postsynaptic currents (eEPSCs) in 10 of these 11 neurons. Thus, data from this study suggest that activation of micro-opioid receptors excites a population of spinally projecting LC neurons by preferential inhibition of GABAergic synaptic inputs. These findings provide important new information about the descending noradrenergic modulation and analgesic mechanisms of opioids.     

Proteolytic processing of the Aplysia egg-laying hormone and R3-14 neuropeptide precursors

A number of animal behaviors are influenced by the actions of neuropeptides that arise from the processing of complex protein precursors. In this report we investigate the proteolytic processing of neuropeptide precursors expressed in the Aplysia californica bag cells, which govern egg-laying, and neurons R3-14, which mediate aspects of cardiac output. Peptides were purified by fractionation on 2 high-pressure liquid chromatography systems followed by determination of amino acid compositions. Most of these compositions are indicative of processing products derived from the egg-laying hormone (ELH) and R3-14 precursors by cleavage at basic residues. We characterized 9 peptides that arise from the ELH precursor by cleavage of the signal sequence, as well as 7 out of 8 dibasic residues and at least 1 single Arg residue. The peptides range in size from 5 to about 60 amino acids. The R3-14 neuropeptide precursor is cleaved at 2 internal dibasic residues in addition to the signal sequence, resulting in 3 peptides. Shortened forms of several peptides probably result from amino- and carboxy-terminal peptidase action. It is likely that the complex mixtures of neuropeptides arising from these single protein precursors are co-secreted.