Anja Friedrich

A subset of dopamine neurons signals reward for odour memory in Drosophila

Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.

The Mushroom Body of Adult Drosophila Characterized by GAL4 Drivers

Abstract: The mushroom body is required for a variety of behaviors of Drosophila melanogaster. Different types of intrinsic and extrinsic mushroom body neurons might underlie its functional diversity. There have been many GAL4 driver lines identified that prominently label the mushroom body intrinsic neurons, which are known as “Kenyon cells.” Under one constant experimental condition, we analyzed and compared the the expression patterns of 25 GAL4 drivers labeling the mushroom body. As an internet resource, we established a digital catalog indexing representative confocal data of them. Further more, we counted the number of GAL4-positive Kenyon cells in each line. We found that approximately 2,000 Kenyon cells can be genetically labeled in total. Three major Kenyon cell subtypes, the γ, α′/β′, and α/β neurons, respectively, contribute to 33, 18, and 49% of 2,000 Kenyon cells. Taken together, this study lays groundwork for functional dissection of the mushroom body.

Catalytic Dehydrocoupling/Dehydrogenation of N-Methylamine-Borane and Ammonia-Borane: Synthesis and Characterization of High Molecular Weight Polyaminoboranes

The catalytic dehydrocoupling/dehydrogenation of N-methylamine-borane, MeNH(2)·BH(3) (7), to yield the soluble, high molecular weight poly(N-methylaminoborane) (8a), [MeNH-BH(2)](n) (M(W) > 20 000), has been achieved at 20 °C using Brookharts Ir(III) pincer complex IrH(2)POCOP (5) (POCOP = [μ(3)-1,3-(OPtBu(2))(2)C(6)H(3)]) as a catalyst. The analogous reaction with ammonia-borane, NH(3)·BH(3) (4), gave an insoluble product, [NH(2)-BH(2)](n) (8d), but copolymerization with MeNH(2)·BH(3) gave soluble random copolymers, [MeNH-BH(2)](n)-r-[NH(2)-BH(2)](m) (8b and 8c). The structures of polyaminoborane 8a and copolymers 8b and 8c were further analyzed by ultrahigh resolution electrospray mass spectrometry (ESI-MS), and 8a, together with insoluble homopolymer 8d, was also characterized by (11)B and (1)H solid-state NMR, IR, and wide-angle X-ray scattering (WAXS). The data indicate that 8a-8c are essentially linear, high molecular weight materials and that the insoluble polyaminoborane 8d possesses a similar structure but is of lower molecular weight (ca. 20 repeat units), presumably due to premature precipitation during its formation. The yield and molecular weight of polymer 8a was found to be relatively robust toward the influence of different temperatures, solvents, and adduct concentrations, while higher catalyst loadings led to higher molecular weight materials. It was therefore unexpected that the polymerization of 7 using 5 was found to be a chain-growth rather than a step-growth process, where high molecular weights were already attained at about 40% conversion of 7. The results obtained are consistent with a two stage polymerization mechanism where, first, the Ir catalyst 5 dehydrogenates 7 to afford the monomer MeNH═BH(2) and, second, the same catalyst effects the subsequent polymerization of this species. A wide range of other catalysts based on Ru, Rh, and Pd were also found to be effective for the transformation of 7 to polyaminoborane 8a. For example, polyaminoborane 8a was even isolated from the initial stage of the dehydrocoupling/dehydrogenation of 7 with [Rh(μ-Cl)(1,5-cod)](2) (2) as the catalyst at 20 °C, a reaction reported to give the N,N,N-trimethyl borazine, [MeN-BH](3), under different conditions (dimethoxyethane, 45 °C). The ability to use a variety of catalysts to prepare polyaminoboranes suggests that the synthetic strategy should be applicable to a broad range of amine-borane precursors and is a promising development for this new class of inorganic polymers.

Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability

Animals acquire predictive values of sensory stimuli through reinforcement. In the brain of Drosophila melanogaster, activation of two types of dopamine neurons in the PAM and PPL1 clusters has been shown to induce aversive odor memory. Here, we identified the third cell type and characterized aversive memories induced by these dopamine neurons. These three dopamine pathways all project to the mushroom body but terminate in the spatially segregated subdomains. To understand the functional difference of these dopamine pathways in electric shock reinforcement, we blocked each one of them during memory acquisition. We found that all three pathways partially contribute to electric shock memory. Notably, the memories mediated by these neurons differed in temporal stability. Furthermore, combinatorial activation of two of these pathways revealed significant interaction of individual memory components rather than their simple summation. These results cast light on a cellular mechanism by which a noxious event induces different dopamine signals to a single brain structure to synthesize an aversive memory.

The mechanism of borane-amine dehydrocoupling with bifunctional ruthenium catalysts.

Borane-amine adducts have received considerable attention, both as vectors for chemical hydrogen storage and as precursors for the synthesis of inorganic materials. Transition metal-catalyzed ammonia-borane (H3N-BH3, AB) dehydrocoupling offers, in principle, the possibility of large gravimetric hydrogen release at high rates and the formation of B-N polymers with well-defined microstructure. Several different homogeneous catalysts were reported in the literature. The current mechanistic picture implies that the release of aminoborane (e.g., Ni carbenes and Shvos catalyst) results in formation of borazine and 2 equiv of H2, while 1 equiv of H2 and polyaminoborane are obtained with catalysts that also couple the dehydroproducts (e.g., Ir and Rh diphosphine and pincer catalysts). However, in comparison with the rapidly growing number of catalysts, the amount of experimental studies that deal with mechanistic details is still limited. Here, we present a comprehensive experimental and theoretical study about the mechanism of AB dehydrocoupling to polyaminoborane with ruthenium amine/amido catalysts, which exhibit particularly high activity. On the basis of kinetics, trapping experiments, polymer characterization by (11)B MQMAS solid-state NMR, spectroscopic experiments with model substrates, and density functional theory (DFT) calculations, we propose for the amine catalyst [Ru(H)2PMe3{HN(CH2CH2PtBu2)2}] two mechanistically connected catalytic cycles that account for both metal-mediated substrate dehydrogenation to aminoborane and catalyzed polymer enchainment by formal aminoborane insertion into a H-NH2BH3 bond. Kinetic results and polymer characterization also indicate that amido catalyst [Ru(H)PMe3{N(CH2CH2PtBu2)2}] does not undergo the same mechanism as was previously proposed in a theoretical study.

Acceptorless Dehydrogenation of Alcohols: Perspectives for Synthesis and H2 Storage.

The oxidation of alcohols represents an important synthetic route towards carbonyl compounds, such as aldehydes, ketones, or carbonic acid derivatives. Obtaining high selectivities for these reactions can be challenging, owing to the risk of substrate over-oxidation. Metalcatalyzed oxidation permits the use of mild, inexpensive, and environmentally benign oxidizing agents, such as O2 or H2O2. [2] In particular, late transition metal catalysts were successfully utilized for aerobic alcohol oxidation. 3] Other mild oxidizing agents used as hydrogen acceptors have included ketones, olefins, and amine-N-oxides. However, with respect to atom economy it is desirable to dehydrogenate without a hydrogen acceptor by release of H2. Furthermore, such acceptorless alcohol dehydrogenations (AADs) are of great current interest for hydrogen storage. The thermodynamics of AAD demand elevated temperatures and removal of H2 from the equilibrium. [8, 9] However, based on the principle of microscopic reversibility, catalyst systems used in ketone hydrogenation could be suitable for the reverse reaction, as well. Accordingly, several late metal homogeneous and colloidal catalysts have been successfully used for thermal AAD. Generally, secondary alcohols are converted to ketones by AAD (Scheme 1, A). Likewise, with catalysts, such as [Ru(OC(O)CF3)2(CO) ACHTUNGTRENNUNG(PPh3)2]/HOC(O)CF3 or metal nanoparticles, AAD of primary alcohols to aldehydes was reported (Scheme 1, A, R’= H). 14a, 15] However, formation of esters (Scheme 1, B) took place in the presence of ruthenium catalysts, such as [RuCl2ACHTUNGTRENNUNG(PPh3)3] or [RuH2 ACHTUNGTRENNUNG(PPh3)4] , 13a, 16a–b] , and AAD of diols usually gives the corresponding lactones. Whereas ester formation could be attributed to a Tishchenko reaction of the initially formed aldehyde, Murahashi and co-workers proposed a pathway via a hemiacetal from the addition of alcohol to the aldehyde. 18b] In fact, formaldehyde represents an intermediate in ruthenium-catalyzed AAD of methanol to methylformate. However, whereas various AAD catalysts have been reACHTUNGTRENNUNGported, systems without acid or base cocatalysts and with high selectivities in primary alcohol AAD remain scarce. Side products, from aldol reactions, for example, must be suppressed by neutral reaction conditions. Furthermore, CO, a strong fuel-cell catalyst poison, stemming from aldehyde decarbonylation is particularly detrimental for H2 storage applications. In this context, Milstein and co-workers recently presented ruthenium AAD catalysts with pyridine-based pincer ligands (Figure 1). Complex 1 a, activated with base, is a moderately active catalyst for secondary alcohols, but failed to dehydrogenate primary alcohols. A catalytic cycle was proposed, based on [Ru(H)2]/ ACHTUNGTRENNUNG[Ru0]. Modification of the ligand sphere resulted in highly selective AAD of primary alcohols to esters by 1 b and 2 with equimolar amounts of KOH. Furthermore, benzylic deprotonation of 2 with KOtBu gave amido complex 3, as was recently described for other aromatic and aliphatic PNP pincer complexes. Complex 3 exhibited high activity and selectivity in the dehydrocoupling of primary alcohols to esters under base-free conditions and ester hydrogenation to the corresponding alcohols. Owing to the reversible addition of H2 to 3 to give dihydride 4, this equilibrium was suggested to be the crucial step in H2 activation/release. Complex 3 did not catalyze the Tishchenko reaction of benzaldehyde, which supports Murahashi’s mechanistic proposal for ester formation. Furthermore, this methodology could be extended towards amide synthesis by dehydrogenative coupling of alcohols and amines (Scheme 1, C). 21] In a recent contribution, Milstein and co-workers described AAD with a new acridine-based pincer complex (Figure 1, 5). 22] Complex 5 has an interesting molecular structure with a particularly long Ru N bond and the heterocycle [a] A. Friedrich, Dr. S. Schneider Department Chemie, Technische Universit t M nchen, Lichtenbergstr. 4, 85748 Garching (Germany) Fax: (+ 49) 89-289-13473 E-mail : sven.schneider@ch.tum.de Scheme 1. Products from metal-catalyzed AAD. Figure 1. Ru pincer complexes used for AAD.

Highly stereoselective proton/hydride exchange: assistance of hydrogen bonding for the heterolytic splitting of H2.

The dihydrido amine complex [Ru(H)(2)PMe(3){HN(CH(2)CH(2)P(i)Pr(2))(2)}] and H(2)O exhibit highly unusual, stereoselective H(+)/H(-) exchange, as derived using (1)H 2D EXSY NMR spectroscopy. While H(RuA) rapidly exchanges with H(2)O [k = 337(20) L mol(-1) s(-1)], no direct H(RuB)/H(2)O proton exchange was detected. Methylation of the pincer amine nitrogen results in unselective slow exchange of both hydrides with H(2)O. These results emphasize the important role of hydrogen bonding of N with Brønsted acids (e.g., water) for heteroloytic H(2) activation with Ru-amide hydrogenation catalysts, which was confirmed computationally.

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Significance A biologically relevant event such as finding food under starvation conditions or being poisoned can drive long-term memory (LTM) in a single training session. Neuronal mechanisms by which such a strong reward or punishment induces stable memory are poorly understood. Here we show that distinct subsets of dopamine neurons signal reward for short- and long-term appetitive memories in Drosophila. The temporal dynamics of memory components triggered by the distinct reward signals are complementary, and together contribute to a temporally stable memory retention. Two subsets of dopamine neurons could signal different reward properties: sweet taste and nutritional value of sugar. Sugar reward is thus intricately encoded in the fly brain, given the importance of long-lasting food-related memory in survival. Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.

Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx

To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial‐section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear‐ or dense‐core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2–14) than nonribbon (1–10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses. J. Comp. Neurol. 520:2185–2201, 2012.

Two Pairs of Mushroom Body Efferent Neurons Are Required for Appetitive Long-Term Memory Retrieval in Drosophila

One of the challenges facing memory research is to combine network- and cellular-level descriptions of memory encoding. In this context, Drosophila offers the opportunity to decipher, down to single-cell resolution, memory-relevant circuits in connection with the mushroom bodies (MBs), prominent structures for olfactory learning and memory. Although the MB-afferent circuits involved in appetitive learning were recently described, the circuits underlying appetitive memory retrieval remain unknown. We identified two pairs of cholinergic neurons efferent from the MB α vertical lobes, named MB-V3, that are necessary for the retrieval of appetitive long-term memory (LTM). Furthermore, LTM retrieval was correlated to an enhanced response to the rewarded odor in these neurons. Strikingly, though, silencing the MB-V3 neurons did not affect short-term memory (STM) retrieval. This finding supports a scheme of parallel appetitive STM and LTM processing.