Plateau potentials in developing antennal-lobe neurons of the moth, Manduca sexta.

Using whole cell recordings from antennal-lobe (AL) neurons in vitro and in situ, in semi-intact brain preparations, we examined membrane properties that contribute to electrical activity exhibited by developing neurons in primary olfactory centers of the brain of the sphinx moth, Manduca sexta. This activity is characterized by prolonged periods of membrane depolarization that resemble plateau potentials. The presence of plateau potential-generating mechanisms was confirmed using a series of tests established earlier. Brief depolarizing current pulses could be used to trigger a plateau state. Once triggered, plateau potentials could be terminated by brief pulses of hyperpolarizing current. Both triggering and terminating of firing states were threshold phenomena, and both conditions resulted in all-or-none responses. Rebound excitation from prolonged hyperpolarizing pulses could also be used to generate plateau potentials in some cells. These neurons were found to express a hyperpolarization-activated inward current. Neither the generation nor the maintenance of plateau potentials was affected by removal of Na+ ions from the extracellular medium or by blockade of Na+ currents with TTX. However, blocking of Ca2+ currents with Cd2+ (5 x 10(-4) M) inhibited the generation of plateau potentials, indicating that, in Manduca AL neurons, plateau potentials depend on Ca2+. Examining Ca2+ currents in isolation revealed that activation of these currents occurs in the absence of experimentally applied depolarizing stimuli. Our results suggest that this activity underlies the generation of plateau potentials and characteristic bursts of electrical activity in developing AL neurons of M. sexta.

The influence of endogenous dopamine levels on the density of [3H]SCH23390-binding sites in the brain of the honey bee, Apis mellifera L.

This paper examines the relationship between endogenous dopamine (DA) levels and the density of [3H]SCH23390-binding sites in the brain of the adult worker honey bee. DA levels were reduced pharmacologically using a single 10 microl injection of either alpha-methyl-DL-p-tyrosine (AMT; 250 microg or 500 microg) or alpha-methyl-DL-tryptophan (AMTP; 250 or 500 microg) into the haemolymph of the bee. In all cases, maximum depletion of DA was observed 3 h after treatment, but in bees treated with AMTP (250 or 500 microg) or with 250 microg AMT, DA levels returned to normal within 24 h of treatment. Neither AMT nor AMTP was selective for DA: both drugs also reduced serotonin (5-hydroxytryptamine, 5HT) levels in the brain. However, AMTP was more effective than AMT at depleting 5HT, whereas for DA, the reverse was true. Depletion of DA levels, using 250 microg AMT, led to a dramatic decline in the levels of specific binding of [3H]SCH23390, defined in this study as binding in the presence of 5x10(-6) M cis-(Z)-flupentixol (see Ref. [28] ). In contrast, naturally occurring diel fluctuations in DA levels, identified in the optic lobes of the brain, and changes in brain DA levels resulting from queenlessness, had no significant effect on the density of [3H]SCH23390-binding sites in the brain of the bee. Overall, these results indicate that under normal physiological conditions, there is no direct link in honey bees between changes in endogenous brain DA levels and the density of D(1)-like receptors labelled by [3H]SCH23390.

Serotonin enhances central olfactory neuron responses to female sex pheromone in the male sphinx moth manduca sexta.

In the brain of the sphinx moth Manduca sexta, sex-pheromonal information is processed in a prominent male-specific area of the antennal lobe called the macroglomerular complex (MGC). Whole-cell patch-clamp recordings from identified projection (output) neurons in the MGC have shown that serotonin [5-hydroxytryptamine (5-HT)] increases both the excitability of MGC projection neurons and their responses to stimulation with pheromone. At least two types of voltage-activated potassium currents in these cells are modulated by 5-HT. 5-HT decreases the maximal conductance of a transient potassium current (I(A)) and shifts its voltage for half-maximal inactivation to more negative potentials without affecting the half-maximal voltage for activation. This reduces the "window current" between the voltage activation and inactivation curves, decreasing the tonically active I(A) near the resting potential and causing the cell to depolarize. 5-HT's effect in this case is to decrease both the transient and resting K(+) conductance by modulating the same channel (I(A)). 5-HT also decreases the maximal conductance of a sustained potassium current [I(K(V))] without affecting its voltage dependence. Using HPLC, we show also that levels of 5-HT in the antennal lobes fluctuate significantly over a 24 hr period. Interestingly, 5-HT levels are highest at times when the moths are most active. We suggest that by controlling the responsiveness of antennal-lobe projection neurons to olfactory stimuli, 5-HT will have significant impact on the performance of odor-dependent behaviors.

Development of dopamine-immunoreactive neurons associated with the antennal lobes of the honey bee, Apis mellifera.

This report examines the development of the dopaminergic system in the primary antennosensory centres (antennal lobes) of the brain of the honey bee, Apis mellifera, and the effects of dopamine on neurite outgrowth of antennal-lobe neurons in vitro. Antibodies raised against dopamine were used to follow the development of a small population of dopamine-immunoreactive neurons that invade the antennal lobes during metamorphic adult development. Immunopositive somata associated with the antennal lobes were first detected at stage 2 of the nine stages of metamorphic adult development, but processes of these neurons within the antennal-lobe neuropil did not exhibit immunostaining until pupal stage 3. Severe depletion of primary sensory input to the right antennal lobe early in metamorphic adult development or removal of the right antenna from newly emerged bees did not alter the expression of dopamine immunoreactivity in the antennal-lobe neuropil. The presence of dopamine in developing antennal lobes was confirmed by using high performance liquid chromatography with electrochemical detection. Levels of dopamine were significantly higher at pupal stage 4 than at all other stages examined. This surge in dopamine levels coincided with rapid growth and compartmentalisation of the antennal-lobe neuropil. Exogenously applied dopamine (50 microM) enhanced the growth of antennal-lobe neurons in vitro, but only in cells derived from pupae at stage 5 of metamorphic adult development. The early appearance of dopamine-immunoreactive neurons and the effects of dopamine on stage 5 antennal-lobe neurons in vitro support the view that dopamine plays a role in the developing brain of the honey bee.

Distribution of dopamine receptors and dopamine receptor homologs in the brain of the honey bee, Apis mellifera L.

In the brain of the honey bee, Apis mellifera, the radioligands [3H]-SCH23390 and [3H]-spiperone recognise D1- and D2-like receptors, respectively. In addition to being pharmacologically distinct and exhibiting significantly different expression profiles during the lifetime of the bee, [3H]-SCH23390- and [3H]-spiperone-binding sites differ markedly in their distribution within the brain. Estimates of [3H]-SCH23390-binding site density are highest in the somatal rind, whereas [3H]-spiperone-binding sites are most concentrated in the beta lobe neuropil of the mushroom bodies. Molecular cloning techniques have been used to identify two honey bee genes encoding dopamine receptor homologs. The first is the honey bee counterpart of a Drosophila D1-like dopamine receptor and is expressed in the mushroom bodies of both workers and drones. The second is related to D2-like dopamine receptors from vertebrates and is expressed in the brain of the bee, but the precise distribution of expression is not yet known.

Voltage-activated currents from adult honeybee (Apis mellifera) antennal motor neurons recorded in vitro and in situ.

Voltage-activated currents from adult honey bee antennal motor neurons were characterized with in vitro studies in parallel with recordings taken from cells in situ. Two methods were used to ensure unequivocal identification of cells as antennal motor neurons: 1) selective backfilling of the neurons with fluorescent markers before dissociation for cell culture or before recording from cells in intact brains, semiintact brains, or in brain slices or 2) staining with a fluorescent marker via the patch pipette during recordings and identifying antennal motor neurons in situ on the basis of their characteristic morphology. Four voltage-activated currents were isolated in these antennal motor neurons with pharmacological, voltage, and ion substitution protocols. The neurons expressed at least two distinct K+ currents, a transient current (IA) that was blocked by 4-aminopyridine (4-5 x 10(-3) M), and a sustained current (IK(V)) that was partially blocked by tetraethylammonium (2-3 x 10(-2) M) and quinidine (5 x 10(-5) M). IA activated above -40 to -30 mV and the half-maximal voltages for steady-state activation and inactivation were -8.8 and -43.2 mV, respectively. IK(V) activated above -50 to -40 mV and the midpoint of the steady-state activation curve was +11.2 mV. IK(V) did not show steady-state inactivation. Additionally, two inward currents were isolated: a tetrodotoxin (10(-7) M)-sensitive, transient Na+ current (INa) that activated above -35 mV, with a maximum around -5 mV and a half-maximal voltage for inactivation of -72.6 mV, and a CdCl2 (5 x 10(-5) M)-sensitive Ca2+ current that activated above -45 to -40 mV, with a maximum around -15 mV. This study represents the first step in our effort to analyze the cellular and ionic mechanisms underlying the intrinsic properties and plasticity of antennal motor neurons.

The effects of queenlessness on the maturation of the honey bee olfactory system.

During the first week of adult life the olfactory system of the honey bee undergoes a critical period of maturation [Masson and Arnold, Organisation and plasticity of the olfactory system of the honeybee, Apis mellifera, in: Menzel and Mercer (Eds.), Neurobiology and Behaviour of Honeybees. Springer-Verlag, Berlin, 1987, pp. 280 295]. This is accompanied by dramatic increases in the volume of the antennal lobes [Winnington et al., Structural plasticity of identified glomeruli in the antennal lobes of the adult worker honey bee. J. Comp. Neurol., 365 (1996) 479-490], centres of the brain that receive direct input from primary olfactory receptor neurons housed in the antennae of the bee. Here, we show that during the first 4-6 days of adult life there is a significant increase in the percentage of bees that respond to a conditioned olfactory stimulus after a single conditioning trial and, furthermore, that the ontogeny of this olfactory learning behaviour is altered significantly if the queen is removed from the colony. The absence of a queen during early adult life also has site-specific effects on the maturation of the antennal lobes of the brain. These results show for the first time that the queen's presence in a colony has a significant impact not only on the behaviour of the adult worker honey bee, but also on the structure of the brain.

Activity-dependent changes to the brain and behavior of the honey bee, Apis mellifera (L.).

To explore the origins and possible behavioral consequences of structural plasticity in an insect brain, we have taken advantage of the following: (1) the highly compartmentalized nature of the primary antenno-sensory centers (antennal lobes) of the brain, (2) the ease with which individual compartments, or glomeruli, within the antennal-lobe neuropil can be identified, and (3) the predictability of changes to readily identifiable glomeruli in the antennal lobes of the adult worker honey bee. Treatment with the juvenile hormone analog methoprene and hive manipulation techniques are used to induce precocious foraging behavior in young worker honey bees. The impact of these treatments on the ontogeny of olfactory learning performance and on the volumes of readily identifiable glomeruli in the antennal lobes of the bee brain are examined in parallel. The study reveals that (1) significant changes in glomerular volume are activity dependent and (2) associative learning of floral odors improves with experience. Improvements in associative learning performance coincide temporally with increases in glomerular volume. This raises an important question: are changes in glomerular volume that result from shifts in behavior simply a consequence of changes in the use of peripheral sensory pathways, or are they associated with events that underlie learning and the formation of long-term memories?

Antennal lobe neurons of the honey bee, Apis mellifera, express a D2-like dopamine receptor in vitro.

We have used the D2-specific dopamine receptor ligand spiperone [N-(p-aminophenethyl) spiperone; NAPS] coupled to the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) to visualize dopamine receptors expressed in vitro by neurons of the primary antennosensory centers (antennal lobes) of the brain of the honey bee, Apis mellifera. Changes in the percentage of antennal lobe neurons exhibiting spiperone binding sites over time in culture and at different stages of metamorphic adult development have been investigated. Neurons obtained from animals at all stages of development exhibited spiperone binding sites, but only after 2 days or more in vitro. The percentage of antennal lobe neurons in vitro expressing spiperone binding sites increased significantly with the development of the antennal lobe neuropil. Fluorescently labelled spiperone (120 nM) could be displaced effectively by 1 mM dopamine but not by the same concentration of tyramine, octopamine, or serotonin. In addition, the D2 antagonist spiperone and the D2/D1 antagonist fluphenazine were more effective at displacing the fluorescent ligand than the D1-specific antagonist SCH23390. Our results indicate that Apis antennal lobe neurons in culture express a dopamine receptor and that this receptor is more likely to be D2-like than D1-like in nature. The receptor is expressed early in the metamorphic adult development of the antennal lobe neuropil of the brain.

Structural plasticity of identified glomeruli in the antennal lobes of the adult worker honey bee.

Adult worker honey bees alter their behaviour with age but retain a strong reliance on sensory information from the antennae. The antennae house a diverse array of receptors, including mechanoreceptors, hygroreceptors, olfactory receptors, and contact chemoreceptors, which relay information to the brain. Antennal sensory neurons that project to the antennal lobes of the brain converge onto second-order interneurones to form discrete spheres of neuropil, called glomeruli. The spatial organisation of glomeruli in the antennal lobes of the honey bee is constant, but the central distribution of information from receptors tuned to different sensory modalities is unknown. Here we show that the glomerular neuropil of the antennal lobes undergoes constant modification during the lifetime of the adult worker bee. Changes in morphology are site specific and highly predictable. The total volume of the glomerular neuropil of the antennal lobe increased significantly during the first 4 days of adult life. Each of the five readily identifiable glomeruli examined in this study exhibited a unique pattern of growth. The growth of two of the five glomeruli changed dramatically with the shift to foraging duties. Furthermore, significant differences were identified between the antennal lobes of bees performing nectar- and pollen-foraging tasks. The highly compartmentalized nature of the antennal lobes, the ease with which specific glomeruli can be identified, and the predictability of changes to the antennal lobe neuropil make this an ideal system for examining the mechanisms and behavioural consequences of structural plasticity in primary sensory centres of the brain.

Pharmacological and kinetic characterization of two functional classes of serotonergic modulation in Aplysia sensory neurons.

1. Modulation of mechanoafferent sensory neurons (SNs) by the neutrotransmitter serotonin (5HT) plays a significant role in behavioral sensitization of several withdrawal reflexes in Aplysia. The modulatory effects of 5HT on these SNs include increased excitability, increased input resistance, action potential broadening, and increased synaptic transmission. Based on a previously described dissociation of some of these modulatory effects, revealed with the 5HT-receptor antagonist, cyproheptadine, we investigated whether a similar dissociation could be found by systematically varying the concentration of the endogenous agonist, 5HT. 2. We first applied a range of 5HT concentrations to isolated pleural/pedal ganglia (containing tail SNs and tail motor neurons, respectively), and measured the magnitude of 5HT-induced modulation of spike broadening and increased excitability. The resulting dose-response curve showed that both forms of modulation increase monotonically as a function of 5HT concentration, but that excitability has a lower threshold for modulation by 5HT than does spike duration. 3. We further characterized the modulatory effects of 5HT on Aplysia SNs by comparing the time course of onset of modulation by 5HT and the time course of recovery after washout. Independent of 5HT concentration, modulation of excitability increases rapidly in the presence of 5HT and recovers rapidly (< 3 min) after washout. Similarly, input resistance increases and recovers rapidly, mirroring the profile of increased excitability. However, modulation of spike duration exhibits two profiles, dependent on 5HT concentration. Low concentrations of 5HT (0.5 and 1 microM) induce a rapid-onset and transient-recovery form of spike broadening, which resembles the kinetics of increased excitability and increased input resistance. Higher concentrations of 5HT (2.5 and 5 microM) induce a more slowly developing and prolonged-recovery form of spike broadening (> 9 min). At these higher concentrations, the recovery profile for prolonged spike broadening is significantly different from those observed for both increased excitability and increased input resistance. 4. We next compared the relationship between spike broadening and short-term synaptic facilitation. We found that significant facilitation of synaptic transmission requires a high 5HT concentration, which is comparable with that required to induce prolonged spike broadening. Similarly, the recovery profiles for spike broadening and synaptic facilitation are strikingly similar, recovering in parallel. 5. Our experiments show that the modulatory effects of 5HT in the tail SNs can be dissociated both by their sensitivity to different concentrations of 5HT and by their kinetics of serotonergic modulation. Based on these results, together with extensive evidence from other laboratories, we propose that the short-term modulatory effects of 5HT fall into two distinct functional classes. The first class, which includes excitability, input resistance, and transient spike broadening, has a low threshold for 5HT modulation and recovers rapidly. The second class, which includes prolonged spike broadening and short-term synaptic facilitation, has a higher threshold for modulation and recovers more slowly. It now will be of interest to determine the functional contribution of each of these classes to different aspects of sensitization.

Serotonin-induced changes in the excitability of cultured antennal-lobe neurons of the sphinx moth Manduca sexta.

The modulatory actions of 5-hydroxy-tryptamine (5HT or serotonin) on a morphologically identifiable class of neurons dissociated from antennal lobes of Manduca sexta at stages 9-15 of the 18 stages of metamorphic adult development were examined in vitro with whole-cell patch-clamp recording techniques. Action potentials could be elicited from approximately 20% of the cells. These cells were used to examine effects of 5HT (5 x 10(-6) to 5 x 10(-4) M) on cell excitability and action-potential waveform. 5HT increased the number of spikes elicited by a constant depolarizing current pulse and reduced the latency of responses. 5HT also led to broadening of action potentials in these neurons and increased cell input resistance. Modulation of potassium channels by 5HT is likely to contribute to these responses. 5HT causes reversible reduction of at least 3 distinct potassium currents, one of which is described for the first time in this study. Because effects of 5HT on antennal-lobe neurons in culture mimic those observed in situ in the brain of the adult moth, in vitro analysis should contribute to elucidation of the cellular mechanisms that underlie the modulatory effects of 5HT on central olfactory neurons in the moth.

Enhancement by serotonin of the growth in vitro of antennal lobe neurons of the sphinx moth Manduca sexta.

Cell culture experiments have been used to examine the effects of serotonin [5-hydroxytryptamine (5-HT)] on the morphological development of antennal lobe (AL) neurons in the brain of the sphinx moth, Manduca sexta. The majority of cells used in this study were from animals at stage 5 of the 18 stages of metamorphic adult development. 5-HT did not affect the survival of M. sexta AL neurons in culture, but did increase the numbers of cells displaying features characteristic of certain cell types. Three morphologically distinct cell types were examined in detail. The principal effect of 5-HT on these neurons was enhancement of cell growth. The magnitude of responses to this amine was cell-type specific. Site-specific responses to 5-HT were apparent also in one cell type. Our results suggest that the effects of 5-HT can change during the course of metamorphic development. These changes coincide temporally with the development of fast, sodium-based action potentials.

Characterisation of dopamine receptors in insect (Apis mellifera) brain.

In vitro binding experiments using the vertebrate D1 dopamine receptor ligand [3H]SCH23390 and the vertebrate D2 dopamine receptor ligand [3H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [3H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent Kd of 6.3 +/- 1.0 nM and Bmax of 1.9 +/- 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [3H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol > or = chlorpromazine > fluphenazine > S(+)-butaclamol > spiperone. R(+/-)-SKF38393 and dopamine were the most effective agonists tested. [3H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D1 dopamine receptor subtype. [3H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [3H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent Kd of 0.11 +/- 0.02 nM and Bmax of 9.2 +/- 0.5 fmol/mg protein. The calculated values for the low affinity site were a Kd of 19.9 nM and Bmax of 862 fmol/mg protein. Kinetic analyses also indicated that [3H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [3H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [3H]spiperone binding was: spiperone > fluphenazine > S(+)-butaclamol > domperidone > R(+)-SCH23390 > S(-)-sulpiride.

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli.

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons.

Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta.

The modulatory effects of 5-hydroxytryptamine (5-HT or serotonin) on voltage-gated currents in central olfactory neurones of the moth Manduca sexta have been examined in vitro using whole-cell patch-clamp recording techniques. Central olfactory neurones were dissociated from the antennal lobes of animals at stage 5 of the 18 stages of metamorphic adult development. The modulatory actions of 5-HT on voltage-activated ionic currents were examined in a subset of morphologically identifiable antennal lobe neurones maintained for 2 weeks in primary cell culture. 5-HT caused reversible reduction of both a rapidly activating A-type K+ current and a relatively slowly activating K+ current resembling a delayed rectifier-type conductance. 5-HT also reduced the magnitude of voltage-activated Ca2+ influx in these cells. The functional significance of 5-HT-modulation of central neurones is discussed.

Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee.

Changes in biogenic amine levels associated with the morphological and behavioural development of the worker honeybee are examined. A significant increase in amine levels in the head of the honeybee is associated with transition from the larval to pupal stage. Adult emergence is also accompanied by a significant increase in 5-HT levels in the brain, but no significant change in brain dopamine (DA) levels. NADA (N-acetyldopamine) levels increase during larval and pupal development, but in contrast to both DA and 5-HT, drop significantly during the transition from pupa to adult. Levels of DA in the brain of nectar and pollen forager bees, presumed to be among the oldest adults sampled, were found to be significantly higher than in nurses, undertakers or food storers. These results suggest that an age-dependent change in amine levels occurs in the brain of the worker bee. In the optic lobes, levels of DA and 5-HT were found to be significantly higher in pollen forager bees than in all other behavioural groups. Significant differences in amine levels in the optic lobes of nectar foragers and pollen foragers indicate that some differences in amine levels occur independent of worker age. The functional significance of differences in brain amine levels and whether or not biogenic amines play a direct role in the control of honeybee behaviour has yet to be established.

Pharmacological dissociation of modulatory effects of serotonin in Aplysia sensory neurons.

In the mollusk Aplysia the neurotransmitter serotonin (5HT) has a fundamental modulatory role in several forms of learning and memory that involve an increase in the efficacy of synaptic transmission between tail sensory neurons (SNs) and motor neurons. The classical 5HT antagonist cyproheptadine (CYP) permits dissociation of three forms of serotonergic modulation in these SNs: (i) CYP reversibly blocks spike-broadening induced either by exogenous application of 5HT or by sensitizing stimulation of a tail nerve. (ii) CYP does not block 5HT-induced or tail input-induced increases in SN somatic excitability. (iii) Concomitant with its block of spike-broadening, CYP reversibly blocks 5HT-induced facilitation of synaptic transmission from SNs. These results suggest that endogenously released 5HT may act at different receptor subtypes that are coupled to different forms of neuromodulation in tail SNs of Aplysia.