Multiple excitatory receptor types on individual olfactory neurons: implications for coding of mixtures in the spiny lobster

The aim of our paper was to investigate whether single olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus functionally express more than one type of receptor, examine the consequences of this on coding of mixtures, and compare principles of odorant mixture coding by spiny lobsters with that by the channel catfish, which has been studied extensively using the same experimental and analytical procedures (Caprio et al. 1989; Kang and Caprio 1991). We examined responses of individual taurine-sensitive ORNs to binary mixtures of excitatory compounds, either competitive agonists (taurine, β-alanine, hypotaurine) or non-competitive agonists (taurine, l-glutamate, ammonium chloride, adenosine-5′-monophosphate). Responses to mixtures were compared to two indices: mixture discrimination index (MDI) and independent component index (ICI). Binary mixtures of competitive agonists had MDI values close to 1.0, as expected for competitors. Mixtures of non-competitive agonists had ICI values averaging 0.83, indicating the effects of the components are not independent. We conclude that individual olfactory cells of spiny lobsters can express more than one type of receptor mediating excitation, one of which typically has a much higher density or affinity, and that spiny lobster and catfish olfactory cells encode mixtures of two excitatory agonists using similar rules. Accepted: 20 December 1996     

Complex binding interactions between multicomponent mixtures and odorant receptors in the olfactory organ of the Caribbean spiny lobster Panulirus argus

Our study was designed to examine how components of complex mixtures can inhibit the binding of other components to receptor sites in the olfactory system of the spiny lobster Panulirus argus. Biochemical binding assays were used to study how two- to six-component mixtures inhibit binding of the radiolabeled odorants taurine, L-glutamate and adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane of olfactory receptor neurons. Our results indicate that binding inhibition by mixtures can be large and is dependent on the nature of the odorant ligand and on the concentration and composition of the mixture. The binding inhibition by mixtures of structurally related components was generally predicted using a competitive binding model and binding inhibition data for the individual components. This was not the case for binding inhibition by most mixtures of structurally unrelated odorants. The binding inhibition for these mixtures was generally smaller than that for one or more of their components, indicating that complex binding interactions between components can reduce their ability to inhibit binding. The magnitude of binding inhibition was influenced more by the mixture's precise composition than by the number of components in it, since mixtures with few components were sometimes more inhibitory than mixtures with more components. These findings raise the possibility that complex binding interactions between components of a mixture and their receptors may shape the output of olfactory receptor neurons to complex mixtures.      

Perception of odor mixtures by the spiny lobster Panulirus argus

We used spiny lobsters (Panulirus argus) in a discriminationlearning procedure with aversive conditioning to examine theirbehavioral discrimination of adenosine-5'-monophosphate (AMP),betaine, L-cysteine and their binary mixtures. Our results showthat spiny lobsters can clearly discriminate among binary mixturesand their components. Lobsters aversively conditioned to avoidresponding to a binary mixture continued to respond to thatmixture's components, and lobsters that were aversively conditionedto avoid responding to a compound tended to continue to respondto binary mixtures containing that compound. Thus, responsesof conditioned lobsters to binary mixtures were not usuallyintermediate between the responses to the mixtures' components,which might be expected for response-matched compounds. Thisresult might arise from any of several factors. First, it mightresult from mixture interactions in the peripheral olfactorysystem, if the responses of olfactory receptor neurons to onecomponent of a binary mixture were suppressed by the other component,making the response to the mixture more similar to the suppressingcomponent. Electrophysiological data from a population of 50singly-recorded olfactory receptor neurons (Daniel and Derby,1994) do not consistently support this idea. A second possiblereason for the behavioral response to a binary mixture not beingintermediate between the responses to its components involveshigher order processing, such as mixture interactions generatedin olfactory interneurons in the CNS (which is known to occur:Derby et al., 1985; Ache, 1989), configural learning or associativeprocessing.     

Coding of blend ratios of binary mixtures by olfactory neurons in the Florida spiny lobster, Panulirus argus

The aim of this study was to investigate quality coding of blend ratios of binary mixtures by olfactory receptor cells in the spiny lobster. Three odorants (adenosine-5′-monophosphate, l-glutamate, and taurine) at 0.1–100 μmol · l⁻¹ and seven blend ratios of each of their binary mixtures at a total concentration of 100 μmol · l⁻¹ were used. The olfactory cells recorded (n = 48) evoked across-neuron patterns for single odorants that were well separated from each other. Across-neuron patterns varied with stimulus concentration but less than with stimulus type. Blend ratios of the three mixtures evoked across-neuron patterns that were orderly placed within a continuum between those elicited by the components. Mixture interactions, defined as a lack of independent effects by a mixture's components, occurred in 25, 24 and 37% of responses to blend ratios of glutamate/taurine, adenosine-5′-monophosphate/taurine, and glutamate/adenosine-5′-monophosphate, respectively. These mixture interactions did not have a large enough effect on the across-neuron patterns for the mixtures such they would be novel relative to those of the single components. These results suggest that despite mixture interactions the quality of individual compounds is not lost when mixed. This corroborates behavioral studies showing that spiny lobsters have the ability to elementally process odor mixtures. Accepted: 23 August 1996     

Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus)

Summary Intracellular calcium was measured in single olfactory neurons from the channel catfish (Icatalurus punctatus) using the fluorescent Ca²⁺ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (l-alanine,l-arginine,l-norleucine andl-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding ofl-alanine andl-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction.     

Morphology and physiology of multiglomerular olfactory projection neurons in the spiny lobster

Whole-cell patch-clamp recording was used to characterize olfactory projection neurons in an isolated brain preparation of the spiny lobster, Panulirus argus. Responses to electrical stimulation of the olfactory afferents were recorded from projection neuron somata using biocytin-filled electrodes. All projection neurons were multiglomerular, innervating up to 80% of all olfactory lobe glomeruli, but the innervation was heterogeneous. Most neurons densely innervated only 3–4 glomeruli; the remaining glomeruli in their dendritic arbor were sparsely innervated, thereby creating two distinct patterns of intraglomerular branching. Projection neurons responded to orthodromic stimulation with an initial depolarization and spiking followed by a 1–3 s hyperpolarization. The inhibitory phase of the response was lower in threshold and longer in latency than the excitatory phase, a response pattern also reported in olfactory projection neurons of insects and vertebrates. The somata of the projection neurons supported voltage-activated currents and TTX-sensitive action potentials, suggesting that the soma, although spatially separated from the axon and dendrites, may play a significant functional role in these cells. Dye coupling between some projection neurons correlated with the presence of multiple amplitude action potentials, suggesting that at least some projection neurons may be coupled via gap junctions.     

Responses of olfactory receptor neurons in the spiny lobster to binary mixtures are predictable using a noncompetitive model that incorporates excitatory and inhibitory transduction pathways

Coding of binary mixtures by a population of olfactory receptor neurons in the spiny lobster (Panulirus argus) was examined. Extracellular single-unit responses of 50 neurons to seven compounds and their binary mixtures were recorded. The ability of a noncompetitive model with correction for binding inhibition to predict responses to mixtures based on responses to their components was compared with the predictive abilities of other models. This model assumes that different compounds activate different transduction processes in the same neuron leading to excitation or inhibition, and it includes a term quantifying the degree to which binding of an odorant to its receptor sites is inhibited by other compounds. The model accurately predicted the absolute response magnitude of the population of neurons for 13 of 15 mixtures assessed, which is superior to the predictive power of any of the other models. The model also accurately predicted the across neuron patterns generated by the binary mixtures, as evaluated by multidimensional scaling analysis. The results suggest that there is no emergence of unique qualities for binary mixtures relative to components of these mixtures.Abbreviations AMP or A adenosine-5-monophosphate - ANP across neuron pattern - ARM absolute response magnitude - ASW artificial sea water - Bet or B betaine - Cys or C L-cysteine - Glu or G L-glutamate - MDS multidimensional scaling - MID mixture interaction distance - NC model noncompetitive model - NCBI model noncompetitive model with correction for binding inhibition - C model competitive model - CBI model competitive model with correction for binding inhibition - MEC more effective component - NH ₄ or N ammonium chloride - ORN olfactory receptor neuron - Suc or S DL-succinate - Tau or T taurine     

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons).Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.Abbreviations AL antennal lobe - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - IPSP inhibitory postsynaptic potential - LN local interneuron - MGC macroglomerular complex - OB olfactory bulb - PN projection neuron - TES N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid     

Mixture interactions of glutamate and betaine in single squid olfactory neurons

We used nystatin-patch techniques to characterize the responses of squid olfactory receptor neurons to the attractive odorant, L-glutamate, and to study mixture interactions between glutamate and the aversive odorant, betaine. We report that glutamate activates a cation-selective conductance that is permeable to Ca²⁺, K⁺, and Na⁺ and which would depolarize squid olfactory receptor neurons under physiological conditions. The responses to glutamate were concentration dependent. The EC₅₀ of individual cells ranged from 0.3 mmol · l⁻¹ to 85.0 mmol · l⁻¹. We found that individual cells were capable of responding to both glutamate and betaine, and that the relative magnitudes of these responses varied from cell to cell. Finally, we report that current responses to binary mixtures of glutamate and betaine are suppressed relative to the sum of the responses to the individual odors in single squid olfactory receptor neurons. Accepted: 20 October 1999     

Excitation, inhibition, and suppression by odors in isolated toad and rat olfactory receptor neurons

Vertebrate olfactoryreceptor neurons (ORNs) exhibit odor-induced increases in actionpotential firing rate due to an excitatory cAMP-dependent current. Fishand amphibian ORNs also give inhibitory odor responses, manifested asdecreases in firing rate, but the underlying mechanism is poorlyunderstood. In the toad, an odor-induced Ca²⁺-activatedK⁺ current is responsible for the hyperpolarizing receptorpotential that causes inhibition. In isolated ORNs, a third manner bywhich odors affect firing is suppression, a direct and nonspecificreduction of voltage-gated and transduction conductances. Here we showthat in whole cell voltage-clamped toad ORNs, excitatory or inhibitory currents were not strictly associated to a particular odorant mixture.Occasionally, both odor effects, in addition to suppression, wereconcurrently observed in a cell. We report that rat ORNs also exhibitodor-induced inhibitory currents, due to the activation of aK⁺ conductance closely resembling that in the toad,suggesting that this conductance is widely distributed amongvertebrates. We propose that ORNs operate as complex integrator unitsin the olfactory epithelium, where the first events in the process ofodor discrimination take place.

     

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-l-tryptophan (5-HTP) and l-3,4-dihydroxyphenylalanine (l-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5–1 μM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to l-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of l-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate-immunoreactive neurons in the rat enteric nervous system

l-Aspartate (l-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of l-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered l-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many l-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that l-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some l-Asp-immunoreactive neurons might function as interneurons. These results suggest that l-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric l-Asp-immunoreactive neurons provides strong support for the proposal that l-Asp is a neuromodulator in the rat ENS.     

Electro-olfactogram and multiunit olfactory receptor responses to binary and trinary mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that responses to binary and trinary mixtures of amino acids were predictable with knowledge obtained from previous cross-adaptation studies of the relative independence of the respective binding sites of the component stimuli. All component stimuli, from which equal aliquots were drawn to form the mixtures, were adjusted in concentration to provide for approximately equal response magnitudes. The magnitude of the response to a mixture whose component amino acids showed significant cross-reactivity was equivalent to the response to any single component used to form that mixture. A mixture whose component amino acids showed minimal cross-adaptation produced a significantly larger relative response than a mixture whose components exhibited considerable cross-reactivity. This larger response approached the sum of the responses to the individual component amino acids tested at the resulting concentrations in the mixture, even though olfactory receptor dose-response functions for amino acids in this species are characterized by extreme sensory compression (i.e., successive concentration increments produce progressively smaller physiological responses). Thus, the present study indicates that the response to sensory stimulation of olfactory receptor sites is more enhanced by the activation of different receptor site types than by stimulus interaction at a single site type.     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Identification of three classes of multiglomerular,broad-spectrum neurons in the crayfish olfactory midbrain by correlated patterns of electrical activity and dendritic arborization

We recorded electrical activity from three different classes of broad-spectrum, multiglomerular neurons in the crayfish (Procambarus clarkii, P. blandingi) olfactory midbrain. Responses were obtained to odorants and electrical stimuli applied to the antennules of isolated, perfused head preparations. All three neuronal types responded to a complex mixture of five amino acids as well as to solutions of a commercial fish food. At least two classes also responded to individual amino acids and to sugars. The response properties and the morphologies of the neurons were unique to each type. Responses of Type I cells were stimulus-dependent excitatory postsynaptic potentials and superimposed impulse trains; those in Type II were stimulus-dependent inhibitory postsynaptic potentials; those in Type III were compound responses consisting of short latency hyperpolarizations, followed by depolarizing post-synaptic potentials and impulses. All three cell types had extensive, multiglomerular dendritic arbors in the olfactory lobe, but each of their respective branching pattern morphologies was distinctive. Two types had additional dendrite branches in the lateral antennular neuropil and the olfactory-globular tract neuropil. We conclude that these broad-spectrum neurons are part of a parallel olfactory pathway that is separate from the putative quality coding circuitry in the crayfish olfactory system.Abbreviations AL accessory lobe - LAN lateral antennular neuropil - OGT olfactory globular tract - OGTN olfactory globular tract neuropil - OL olfactory lobe     

Dual inhibitory pathways mediated by GABA- and histaminergic interneurons in the lobster olfactory lobe

Antisera to GABA and histamine (HA) label distinct populations of interneurons that innervate glomeruli in the olfactory lobe (OL) of the spiny lobster. GABA-immunoreactive interneurons branch most heavily in the cap of the glomeruli, while HA-immunoreactive interneurons branch mostly in the glomerular subcap. Perfusing GABA or HA into the isolated brain increases the intensity of electrical stimulation of the antennular nerve necessary to elicit action potentials in OL projection neurons. The GABA receptor antagonist picrotoxin (30–100 μmol?·?l^?1) and the HA receptor antagonist cimetidine (1–5 mmol?·?l^?1) both reduce the stimulus intensity needed to elicit action potentials. However, cimetidine also eliminates the hyperpolarizing phase of the evoked response and reveals a delayed, prolonged excitation of up to 10 s, whereas picrotoxin enhances the hyperpolarization and, at higher concentrations, transiently suppresses all phases of the evoked response. We conclude that GABA- and HA-ergic interneurons constitute two overlapping, yet functionally distinct inhibitory pathways in the OL, an organizational feature which may be fundamental to processing at this level of the olfactory pathway.     

Glutamate receptor changes in brain synaptic membranes from human alcoholics

Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membraneenriched fraction and the study ofl-[³H]glutamic acid and 3-((±)-2-carboxypiperazin-4-yl)-[1,2³H]propyl-l-phosphonic acid ([³H]CPP) binding sites. The pharmacological characteristics ofl-[³H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-d-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population ofl-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates orl-[³H]glutamate binding to synaptic membranes. In order to determine whether some of the changes inl-[³H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [³H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [³H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics. These observations suggest selective changes among different glutamate receptor subtypes in human brain under conditions of chronic alcohol intake.     

Responses of protocerebral neurons in Manduca sexta to sex-pheromone mixtures

Male Manduca sexta moths are attracted to a mixture of two components of the female’s sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male’s antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.