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Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca 2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca 2+ concentration using fluorescence lifetime imaging microscopy (FLIM)., (© 2024. The Author(s).)

Sparse coding can improve discrimination of sensory stimuli by reducing overlap between their representations. Two factors, however, can offset sparse coding's benefits: similar sensory stimuli have significant overlap and responses vary across trials. To elucidate the effects of these 2 factors, we analyzed odor responses in the fly and mouse olfactory regions implicated in learning and discrimination-the mushroom body (MB) and the piriform cortex (PCx). We found that neuronal responses fall along a continuum from extremely reliable across trials to extremely variable or stochastic. Computationally, we show that the observed variability arises from noise within central circuits rather than sensory noise. We propose this coding scheme to be advantageous for coarse- and fine-odor discrimination. More reliable cells enable quick discrimination between dissimilar odors. For similar odors, however, these cells overlap and do not provide distinguishing information. By contrast, more unreliable cells are decorrelated for similar odors, providing distinguishing information, though these benefits only accrue with extended training with more trials. Overall, we have uncovered a conserved, stochastic coding scheme in vertebrates and invertebrates, and we identify a candidate mechanism, based on variability in a winner-take-all (WTA) inhibitory circuit, that improves discrimination with training., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Srinivasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Foraging animals must use decision-making strategies that dynamically adapt to the changing availability of rewards in the environment. A wide diversity of animals do this by distributing their choices in proportion to the rewards received from each option, Herrnstein's operant matching law. Theoretical work suggests an elegant mechanistic explanation for this ubiquitous behavior, as operant matching follows automatically from simple synaptic plasticity rules acting within behaviorally relevant neural circuits. However, no past work has mapped operant matching onto plasticity mechanisms in the brain, leaving the biological relevance of the theory unclear. Here, we discovered operant matching in Drosophila and showed that it requires synaptic plasticity that acts in the mushroom body and incorporates the expectation of reward. We began by developing a dynamic foraging paradigm to measure choices from individual flies as they learn to associate odor cues with probabilistic rewards. We then built a model of the fly mushroom body to explain each fly's sequential choice behavior using a family of biologically realistic synaptic plasticity rules. As predicted by past theoretical work, we found that synaptic plasticity rules could explain fly matching behavior by incorporating stimulus expectations, reward expectations, or both. However, by optogenetically bypassing the representation of reward expectation, we abolished matching behavior and showed that the plasticity rule must specifically incorporate reward expectations. Altogether, these results reveal the first synapse-level mechanisms of operant matching and provide compelling evidence for the role of reward expectation signals in the fly brain.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Memory guides behavior across widely varying environments and must therefore be both sufficiently specific and general. A memory too specific will be useless in even a slightly different environment, while an overly general memory may lead to suboptimal choices. Animals successfully learn to both distinguish between very similar stimuli and generalize across cues. Rather than forming memories that strike a balance between specificity and generality, Drosophila can flexibly categorize a given stimulus into different groups depending on the options available. We asked how this flexibility manifests itself in the well-characterized learning and memory pathways of the fruit fly. We show that flexible categorization in neuronal activity as well as behavior depends on the order and identity of the perceived stimuli. Our results identify the neural correlates of flexible stimulus-categorization in the fruit fly., Competing Interests: MM, AR, HR, YA, GT No competing interests declared, (© 2023, Modi et al.)

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability., Competing Interests: Declaration of interests A.S.A., L.D.L., and E.R.S. have filed for a patent on the chemigenetic voltage indicators. I.K. and C.R.F. are co-inventors on a patent describing pipette cleaning that is licensed by Sensapex. M.C. and L.C. have performed consulting services for Sensapex., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Calcium imaging with protein-based indicators 1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators 3-8 . The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation., (© 2023. The Author(s).)

Innate behavioral biases and preferences can vary significantly among individuals of the same genotype. Though individuality is a fundamental property of behavior, it is not currently understood how individual differences in brain structure and physiology produce idiosyncratic behaviors. Here we present evidence for idiosyncrasy in olfactory behavior and neural responses in Drosophila We show that individual female Drosophila from a highly inbred laboratory strain exhibit idiosyncratic odor preferences that persist for days. We used in vivo calcium imaging of neural responses to compare projection neuron (second-order neurons that convey odor information from the sensory periphery to the central brain) responses to the same odors across animals. We found that, while odor responses appear grossly stereotyped, upon closer inspection, many individual differences are apparent across antennal lobe (AL) glomeruli (compact microcircuits corresponding to different odor channels). Moreover, we show that neuromodulation, environmental stress in the form of altered nutrition, and activity of certain AL local interneurons affect the magnitude of interfly behavioral variability. Taken together, this work demonstrates that individual Drosophila exhibit idiosyncratic olfactory preferences and idiosyncratic neural responses to odors, and that behavioral idiosyncrasies are subject to neuromodulation and regulation by neurons in the AL., Competing Interests: Conflict of interest statement: B.L.d.B. is a scientific advisor for FlySorter, LLC. The authors have no additional conflicts.

The Drosophila brain contains a relatively simple circuit for forming Pavlovian associations, yet it achieves many operations common across memory systems. Recent advances have established a clear framework for Drosophila learning and revealed the following key operations: a ) pattern separation, whereby dense combinatorial representations of odors are preprocessed to generate highly specific, nonoverlapping odor patterns used for learning; b ) convergence, in which sensory information is funneled to a small set of output neurons that guide behavioral actions; c ) plasticity, where changing the mapping of sensory input to behavioral output requires a strong reinforcement signal, which is also modulated by internal state and environmental context; and d ) modularization, in which a memory consists of multiple parallel traces, which are distinct in stability and flexibility and exist in anatomically well-defined modules within the network. Cross-module interactions allow for higher-order effects where past experience influences future learning. Many of these operations have parallels with processes of memory formation and action selection in more complex brains.

We compared performance of recently developed silicon photomultipliers (SiPMs) to GaAsP photomultiplier tubes (PMTs) for two-photon imaging of neural activity. Despite higher dark counts, SiPMs match or exceed the signal-to-noise ratio of PMTs at photon rates encountered in typical calcium imaging experiments due to their low pulse height variability. At higher photon rates encountered during high-speed voltage imaging, SiPMs substantially outperform PMTs.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila , the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.

Previously, we demonstrated that visual and olfactory associative memories of Drosophila share mushroom body (MB) circuits (Vogt et al., 2014). Unlike for odor representation, the MB circuit for visual information has not been characterized. Here, we show that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. We identified two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects.

Although associative learning has been localized to specific brain areas in many animals, identifying the underlying synaptic processes in vivo has been difficult. Here, we provide the first demonstration of long-term synaptic plasticity at the output site of the Drosophila mushroom body. Pairing an odor with activation of specific dopamine neurons induces both learning and odor-specific synaptic depression. The plasticity induction strictly depends on the temporal order of the two stimuli, replicating the logical requirement for associative learning. Furthermore, we reveal that dopamine action is confined to and distinct across different anatomical compartments of the mushroom body lobes. Finally, we find that overlap between sparse representations of different odors defines both stimulus specificity of the plasticity and generalizability of associative memories across odors. Thus, the plasticity we find here not only manifests important features of associative learning but also provides general insights into how a sparse sensory code is read out., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies have been an important model system for studying sparse coding in the olfactory system, where this format is important for accurate memory formation. In Drosophila, it has recently been shown that the 2,000 Kenyon cells of the mushroom body converge onto a population of only 34 mushroom body output neurons (MBONs), which fall into 21 anatomically distinct cell types. Here we provide the first, to our knowledge, comprehensive view of olfactory representations at the fourth layer of the circuit, where we find a clear transition in the principles of sensory coding. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit. Instead, at the population level, odour representations are reformatted so that positive and negative correlations arise between representations of different odours. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals, but that tuning of the same neuron across the two hemispheres of an individual fly was nearly identical. Thus, individualized coordination of tuning arises at this level of the olfactory circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to flexibly map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs. The reformatting of sensory representations we observe here may mark the beginning of this sensory-motor transition in the olfactory system.

Recent progress in intracellular calcium sensors and other fluorophores has promoted the widespread adoption of functional optical imaging in the life sciences. Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective. For many imaging applications a 3-axis motorized stage is critical, but commercially available motorization hardware (motorized translators, controller boxes, etc) are often very expensive. Furthermore, the firmware on commercial motor controllers cannot easily be altered and is not usually designed with a microscope stage in mind. Here we describe an open-source motorization solution that is simple to construct, yet far cheaper and more customizable than commercial offerings. The cost of the controller and motorization hardware are under $1000. Hardware costs are kept low by replacing linear actuators with high quality stepper motors. Electronics are assembled from commonly available hobby components, which are easy to work with. Here we describe assembly of the system and quantify the positioning accuracy of all three axes. We obtain positioning repeatability of the order of 1 μm in X/Y and 0.1 μm in Z. A hand-held control-pad allows the user to direct stage motion precisely over a wide range of speeds (10(-1) to 10(2) μm·s(-1)), rapidly store and return to different locations, and execute "jumps" of a fixed size. In addition, the system can be controlled from a PC serial port. Our "OpenStage" controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications.

In the olfactory system, sensory inputs are arranged in different glomerular channels, which respond in combinatorial ensembles to the various chemical features of an odor. We investigated where and how this combinatorial code is read out deeper in the brain. We exploited the unique morphology of neurons in the Drosophila mushroom body, which receive input on large dendritic claws. Imaging odor responses of these dendritic claws revealed that input channels with distinct odor tuning converge on individual mushroom body neurons. We determined how these inputs interact to drive the cell to spike threshold using intracellular recordings to examine mushroom body responses to optogenetically controlled input. Our results provide an elegant explanation for the characteristic selectivity of mushroom body neurons: these cells receive different types of input and require those inputs to be coactive to spike. These results establish the mushroom body as an important site of integration in the fly olfactory system.

The brain represents sensory information in the coordinated activity of neuronal ensembles. Although the microcircuits underlying olfactory processing are well characterized in Drosophila, no studies to date have examined the encoding of odor identity by populations of neurons and related it to the odor specificity of olfactory behavior. Here we used two-photon Ca(2+) imaging to record odor-evoked responses from >100 neurons simultaneously in the Drosophila mushroom body (MB). For the first time, we demonstrate quantitatively that MB population responses contain substantial information on odor identity. Using a series of increasingly similar odor blends, we identified conditions in which odor discrimination is difficult behaviorally. We found that MB ensemble responses accounted well for olfactory acuity in this task. Kenyon cell ensembles with as few as 25 cells were sufficient to match behavioral discrimination accuracy. Using a generalization task, we demonstrated that the MB population code could predict the flies' responses to novel odors. The degree to which flies generalized a learned aversive association to unfamiliar test odors depended upon the relative similarity between the odors' evoked MB activity patterns. Discrimination and generalization place different demands on the animal, yet the flies' choices in these tasks were reliably predicted based on the amount of overlap between MB activity patterns. Therefore, these different behaviors can be understood in the context of a single physiological framework.

Sensory stimuli are represented in the brain by the activity of populations of neurons. In most biological systems, studying population coding is challenging since only a tiny proportion of cells can be recorded simultaneously. Here we used two-photon imaging to record neural activity in the relatively simple Drosophila mushroom body (MB), an area involved in olfactory learning and memory. Using the highly sensitive calcium indicator GCaMP3, we simultaneously monitored the activity of >100 MB neurons in vivo (∼5% of the total population). The MB is thought to encode odors in sparse patterns of activity, but the code has yet to be explored either on a population level or with a wide variety of stimuli. We therefore imaged responses to odors chosen to evaluate the robustness of sparse representations. Different odors activated distinct patterns of MB neurons; however, we found no evidence for spatial organization of neurons by either response probability or odor tuning within the cell body layer. The degree of sparseness was consistent across a wide range of stimuli, from monomolecular odors to artificial blends and even complex natural smells. Sparseness was mainly invariant across concentrations, largely because of the influence of recent odor experience. Finally, in contrast to sensory processing in other systems, no response features distinguished natural stimuli from monomolecular odors. Our results indicate that the fundamental feature of odor processing in the MB is to create sparse stimulus representations in a format that facilitates arbitrary associations between odor and punishment or reward.

Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells. Although recent studies have linked gap junctions to memory formation, it remains unclear how they contribute to this process. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In both insect and vertebrate olfactory systems only two synapses separate the sensory periphery from brain areas required for memory formation and the organisation of behaviour. In the Drosophila olfactory system, which is anatomically very similar to its vertebrate counterpart, there has been substantial recent progress in understanding the flow of information from experiments using molecular genetic, electrophysiological and optical imaging techniques. In this review, we shall focus on how olfactory information is processed and transformed in order to extract behaviourally relevant information. We follow the progress from olfactory receptor neurons, through the first processing area, the antennal lobe, to higher olfactory centres. We address both the underlying anatomy and mechanisms that govern the transformation of neural activity. We emphasise our emerging understanding of how different elementary computations, including signal averaging, gain control, decorrelation and integration, may be mapped onto different circuit elements.

Multiple pathways link expression of PTR2, the transporter of di- and tripeptides in the yeast Saccharomyces cerevisiae, to the availability and quality of nitrogen sources. Previous work has shown that induction of PTR2 by extracellular amino acids requires, in particular, SSY1 and PTR3. SSY1 is structurally similar to amino acid transporters but functions as a sensor of amino acids. PTR3 acts downstream of SSY1. Expression of the PTR2 peptide transporter is induced not only by amino acids but also by dipeptides with destabilizing N-terminal residues. These dipeptides bind to UBR1, the ubiquitin ligase of the N-end rule pathway, and allosterically accelerate the UBR1-dependent degradation of CUP9, a transcriptional repressor of PTR2. UBR1 targets CUP9 through its internal degron. Here we demonstrate that the repression of PTR2 by CUP9 requires TUP1 and SSN6, the corepressor proteins that form a complex with CUP9. We also show that the induction of PTR2 by amino acids is mediated by the UBR1-dependent acceleration of CUP9 degradation that requires both SSY1 and PTR3. The acceleration of CUP9 degradation is shown to be attained without increasing the activity of the N-end rule pathway toward substrates with destabilizing N-terminal residues. We also found that GAP1, a general amino acid transporter, strongly contributes to the induction of PTR2 by Trp. Although several aspects of this complex circuit remain to be understood, our findings establish new functional links between the amino acids-sensing SPS system, the CUP9-TUP1-SSN6 repressor complex, the PTR2 peptide transporter, and the UBR1-dependent N-end rule pathway.

Learning and memory has been studied extensively in Drosophila using behavioral, molecular, and genetic approaches. These studies have identified the mushroom body as essential for the formation and retrieval of olfactory memories. We investigated odor responses of the principal neurons of the mushroom body, the Kenyon cells (KCs), in Drosophila using whole cell recordings in vivo. KC responses to odors were highly selective and, thus sparse, compared with those of their direct inputs, the antennal lobe projection neurons (PNs). We examined the mechanisms that might underlie this transformation and identified at least three contributing factors: excitatory synaptic potentials (from PNs) decay rapidly, curtailing temporal integration, PN convergence onto individual KCs is low ( approximately 10 PNs per KC on average), and KC firing thresholds are high. Sparse activity is thought to be useful in structures involved in memory in part because sparseness tends to reduce representation overlaps. By comparing activity patterns evoked by the same odors across olfactory receptor neurons and across KCs, we show that representations of different odors do indeed become less correlated as they progress through the olfactory system.

Molecular genetics has revealed a precise stereotypy in the projection of primary olfactory sensory neurons onto secondary neurons. A major challenge is to understand how this mapping translates into odor responses in these second-order neurons. We investigated this question in Drosophila using whole-cell recordings in vivo. We observe that monomolecular odors generally elicit responses in large ensembles of antennal lobe neurons. Comparison of odor-evoked activity from afferents and postsynaptic neurons in the same glomerulus revealed that second-order neurons display broader tuning and more complex responses than their primary afferents. This indicates a major transformation of odor representations, implicating lateral interactions within the antennal lobe.

In the insect olfactory system, oscillatory synchronization is functionally relevant and reflects the coherent activation of dynamic neural assemblies. We examined the role of such oscillatory synchronization in information transfer between networks in this system. The antennal lobe is the obligatory relay for olfactory afferent signals and generates oscillatory output. The mushroom body is responsible for formation and retrieval of olfactory and other memories. The format of odor representations differs significantly across these structures. Whereas representations are dense, dynamic, and seemingly redundant in the antennal lobe, they are sparse and carried by more selective neurons in the mushroom body. This transformation relies on a combination of oscillatory dynamics and intrinsic and circuit properties that act together to selectively filter and synthesize the output from the antennal lobe. These results provide direct support for the functional relevance of correlation codes and shed some light on the role of oscillatory synchronization in sensory networks.

Acoustic transduction in air from two bulk-micromachined silicon structures is investigated. Both contain silicon diaphragms of the order of 2 mm2 in close proximity to a metallized substrate. One diaphragm is mass-loaded; the other is not. Their resonant frequencies (70 and 360 kHz) are dominated by squeeze film trapping of ambient air, and the Q of each device is about 8. The lower frequency (LF) device is characterized by electrical and acoustic measurements using a calibrated microphone. Novel diagnostic methods that exploit the non-linear nature of the transducer are described. The adequacy of calibration by reciprocity is confirmed at 70 kHz and applied to the high frequency device. An insertion loss of 19 dB is measured, which compares well with reports of other silicon-based transducers. Observed losses are accounted for by squeeze-film damping applied to the diaphragm-substrate gap. The ability to control the bandwidth by the squeeze film effect, the good efficiency, and the relatively standard method of construction could make such ultrasonic transducers useful in specialist applications.

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.

Protein degradation by the ubiquitin system controls the intracellular concentrations of many regulatory proteins. A protein substrate of the ubiquitin system is conjugated to ubiquitin through the action of three enzymes, E1, E2 and E3, with the degradation signal (degron) of the substrate recognized by E3 (refs 1-3). The resulting multi-ubiquitylated substrate is degraded by the 26S proteasome. Here we describe the physiological regulation of a ubiquitin-dependent pathway through allosteric modulation of its E3 activity by small compounds. Ubr1, the E3 enzyme of the N-end rule pathway (a ubiquitin-dependent proteolytic system) in Saccharomyces cerevisiae mediates the degradation of Cup9, a transcriptional repressor of the peptide transporter Ptr2 (ref. 5). Ubr1 also targets proteins that have destabilizing amino-terminal residues. We show that the degradation of Cup9 is allosterically activated by dipeptides with destabilizing N-terminal residues. In the resulting positive feedback circuit, imported dipeptides bind to Ubr1 and accelerate the Ubr1-dependent degradation of Cup9, thereby de-repressing the expression of Ptr2 and increasing the cell's capacity to import peptides. These findings identify the physiological rationale for the targeting of Cup9 by Ubr1, and indicate that small compounds may regulate other ubiquitin-dependent pathways.

Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.

Delta agent is a small RNA virus which depends on hepatitis B virus for replication. Infection with delta virus in Britain and other countries in Northern Europe and North America is largely confined to drug abusers and their social contacts. In Merseyside, between 1982 and 1987, we found evidence of delta virus infection in 10 (4.4%) of 224 cases of acute hepatitis B in drug abusers. Among carriers of hepatitis B surface antigen (HBsAg) with a history of drug abuse, however, the proportion with delta antibody rose from 10% in 1984 to 30.8% in 1987. Among all HBsAg carriers identified in 1986 and 1987, delta antibody was detected in 11 (32.4%) of 34 with a history of drug abuse but in only 3 (1.6%) of 183 in whom drug abuse was not admitted. Long-term delta infection in these carriers is often associated with development of chronic active hepatitis and represents a major problem not only in countries where hepatitis B and delta virus infections are endemic but also potentially among drug abusers in Britain. Control of delta virus infection depends on social action to curb drug abuse and on the use of hepatitis B vaccine where appropriate to prevent hepatitis B in those at risk. In order to monitor the prevalence of delta virus infection in the community, priority should be given to testing for delta antibody HBsAg carriers with a history of drug abuse.

After the 1976 outbreak of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections had been controlled, less than 1 per cent of cases of gonorrhoea in Liverpool in 1977 and 1978 were caused by PPNG. Thereafter the steady increase in PPNG infections to 5.6 per cent of all cases in 1982 was associated with marked changes in epidemiological pattern, plasmids and auxotypes. In 1976 nearly all PPNG infections were acquired by young black males living in the inner city from women frequenting clubs; the PPNG were all of the African 3.2 megadalton (MD) plasmid type and of arginine-requiring auxotype. Between 1977 and 1982 female patients were increasingly ship girl prostitutes associating with seamen who constituted more than 50 per cent of the male patients. These men and other travellers introduced PPNG into Liverpool from the Far East and West Africa. In 1978 PPNG of the Asian type with 4.4 MD plasmid with or without 24.5 MD transfer plasmids were isolated in Liverpool where in 1979 all PPNG carried 4.4 MD and 24.5 MD plasmids. In 1982 strains of the 'new' African type with 3.2 and 24.5 MD plasmids were isolated as were PPNG of the Asian type that had been acquired in West Africa. Auxotyping of the 1982 isolates showed that none were arginine-requiring but three other types were identified: proline-requiring: proline-arginine-requiring; non-requiring. For the control of PPNG, a strategy based on constant vigilance, appropriate diagnostic procedures, rapidly effective treatment and determined contact tracing is needed.

In a study of the distribution of e-antigen and anti-e in subjects whose blood was positive for hepatitis B surface antigen (HBsAg), patients with acute hepatitis B who were tested during the incubation period were all e-antigen-positive but after the onset of illness e-antigen was detected in only 11%. Persistence, and in some instances reappearance of e-antigen in those who became long-term carriers of HBsAg was associated with high titres of HBsAg. There was a high incidence of e-antigen in those conditions in which cell-mediated immunity may be depressed, including Down's syndrome and chronic renal failure. The majority of HBsAg carries identified as sources of infection were e-antigen-positive. A postive reaction for e-antigen is evidently associated with a defective immune response to hepatitis B virus infection which permits continued replication of virus in liver cells accompanied by high titres of HBsAg, numerous Dane particles and detectable DNA polymerase in the blood with consequently a greater likelihood of transmitting infection. Although it cannot be assumed that anti-e positive carriers of HBsAg are not infective, it may be necessary, in the assessment of passive or active immunization for the control of hepatitis B, to take into account the e-antigen/antibody status of possible sources of infection.

44% of 63 British patients with either haemophilia A or B were HTLV-III antibody positive (HTLV-VIII+). HTLV-III+ was more frequent in high factor VIII concentrate users and 75% of severely affected haemophilia A patients were HTLV-III+. All eight patients who were exposed to factor IX concentrate were HTLV-III-. 17 haemophilia A patients who received only British made factor VIII concentrate (average 12,000 units/year) were HTLV-III-. Two of 63 patients had evidence of a pre-AIDS type symptom complex and both were HTLV-III+. Information from a cohort of 21 Liverpool haemophiliacs suggests that HTLV-III was first introduced into this country in 1981. OKT4/T8 ratios were abnormal in 52% of 21 patients studied but this finding was not confined to either HTLV-III+ or HTLV-III- individuals. The spouses of 14 HTLV-III+ haemophiliacs were all HTLV-III-.

In order to develop a rapid and reliable method for screening large numbers of urines for leucocytes and bacteria a dipstick method was used with colorimetric reading of colour changes with a reflectance spectrophotometer. One thousand consecutive urine specimens were tested by that method using a single dipstick for leucocyte esterase, nitrite, blood and protein in parallel with routine methods for enumeration of leucocytes by microscopy and quantitative culture for bacteria. Results of the four dipstick tests taken together had predictive values of 83.7% for a positive test for evidence of infection and 97.2% for a negative result. The false negative rate was 1%. Of the 191 urines with a significant bacteriuria (greater than 10(5) organisms/ml), 10 were false negatives by dipstick testing, representing 5% of all infected urines. Elimination of the need for culture on specimens that were negative by all four tests would reduce the workload by 36%.

A study of the distribution of subtypes ad and ay among sera from hepatitis B antigen-positive subjects in North West England and North Wales revealed a marked contrast between symptomless carriers among whom ad predominated and patients with acute hepatitis the majority of whom were ay. Those with hepatitis associated with drug addiction or other forms of "needle transmission" were almost all of subtype ay. On the other hand in cases of "sporadic" hepatitis without evidence of parenteral exposure subtypes ad and ay are about equally distributed. These findings are similar to those reported from other countries in Northern Europe and North America. Although geographical and social factors clearly affect the distribution of the two subtypes it is suggested that the virus of subtype ay may be more readily transmitted than subtype ad by parenteral routes involving small amounts of blood.

The epidemiological features are described of an outbreak of gonorrhoea caused by penicillinase-producing strains of gonococci in 76 patients in Liverpool between February and November 1976. Initially infections were confined to a socially deprived inner city area with a large immigrant population, and subsequent spread of infection remained circumscribed. The characteristics of those patients acquiring these infections were similar to those infected by gonococci of diminished sensitivity to benzylpenicillin, and showed a strong association with adverse social factors. A comparison of the clinical features of patients harbouring sensitive, less sensitive, and penicillinase-producing strains showed severe symptoms and signs in men and a greater involvement of multiple sites in women infected with penicillinase-producing gonococci. Treatment with penicillins failed. Tetracycline was satisfactory in men but was less so in women in whom gonococci persisted in the rectum. Cefuroxime and spectinomycin were effective.