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Choline Transporter regulates olfactory habituation via a neuronal triad of excitatory, inhibitory and mushroom body neurons

['Runa Hamid', 'Centre For Cellular', 'Molecular Biology', 'Council Of Scientific', 'Industrial Research', 'Csir-Ccmb', 'Hyderabad', 'Hitesh Sonaram Sant', 'Mrunal Nagaraj Kulkarni']

Date: 2022-02 

Choline is an essential component of Acetylcholine (ACh) biosynthesis pathway which requires high-affinity Choline transporter (ChT) for its uptake into the presynaptic terminals of cholinergic neurons. Previously, we had reported a predominant expression of ChT in memory processing and storing region of the Drosophila brain called mushroom bodies (MBs). It is unknown how ChT contributes to the functional principles of MB operation. Here, we demonstrate the role of ChT in Habituation, a non-associative form of learning. Odour driven habituation traces are laid down in ChT dependent manner in antennal lobes (AL), projection neurons (PNs), and MBs. We observed that reduced habituation due to knock-down of ChT in MBs causes hypersensitivity towards odour, suggesting that ChT also regulates incoming stimulus suppression. Importantly, we show for the first time that ChT is not unique to cholinergic neurons but is also required in inhibitory GABAergic neurons to drive habituation behaviour. Our results support a model in which ChT regulates both habituation and incoming stimuli through multiple circuit loci via an interplay between excitatory and inhibitory neurons. Strikingly, the lack of ChT in MBs shows characteristics similar to the major reported features of Autism spectrum disorders (ASD), including attenuated habituation, sensory hypersensitivity as well as defective GABAergic signalling. Our data establish the role of ChT in habituation and suggest that its dysfunction may contribute to neuropsychiatric disorders like ASD.

Habituation is a conserved phenomenon that enables an organism to enhance attention only on salient stimuli in the surroundings and ignore stimuli without any positive or negative consequences. The circuitry, regulators, and molecular mechanisms involved in habituation are poorly understood. By using Drosophila model system, we demonstrate that Choline Transporter (ChT) regulates olfactory habituation and its central operational features. We show for the first time that ChT is localised in GABAergic neurons and demonstrate that reduced levels of ChT in mushroom body and GABAergic neurons lead to defective habituation, which is correlated to augmented sensory perception. Our data provides a new perspective of ChT in habituation and provides avenues for future research to investigate the molecular correlates of ChT in habituation-associated neuropsychiatric disorders.

Here, we show that ChT is required for olfactory habituation at multiple loci involving olfactory pathway and MB neurons. This study reports for the first time that ChT is also localised in GABAergic terminals of Drosophila larval brain suggesting that ChT is not unique to cholinergic neurons. We show that the olfactory PN terminals (majority of which are excitatory) and the inhibitory GABAergic neuron terminals express ChT and perhaps their teamwork regulates habituation and its central operational features: the response devaluation to an olfactory stimulus, spontaneous recovery on the removal of stimulus or dishabituation of response upon exposure to an unrelated stimulus. Knock-down of ChT in MBs was observed to be correlated with the hypersensitivity towards the incoming stimuli and defective habituation, suggesting that ChT bridges the link between upstream plasticity and downstream stimulus suppression. Our results demonstrate the role of a conserved protein, ChT, contributing to the dynamic nature of habituation and highlight that its dysfunction leads to sensory abnormalities. Thus, these findings add insight into habituation behaviour mechanisms at the neural circuit levels through choline metabolism.

Habituation enhances attention only on the salient features of the animal’s surroundings such as food, mate, danger, etc. Habituation has been observed in many organisms from as simple as a single cell protozoa to Aplysia californica, medicinal leech, territorial fish, Birds, and Drosophila to more complex life forms like rats, and humans suggesting its ubiquitous persistence [ 15 ]. Multiple habituation studies focused on Drosophila sensory systems such as olfactory, gustatory, visual, and proprioceptive systems have contributed to our understanding of the cellular and circuit basis of habituation [ 11 , 16 ]. At the synaptic level, habituation may result through the action of heterosynaptic modulation involving activation of inhibitory neurons or homosynaptic depression of excitatory neurons [ 17 ]. Studies in adult Drosophila suggest that potentiation of GABAergic inhibition onto PN terminals in AL cause olfactory habituation [ 18 ]. Habituation reflects the efficient sensory input processing, and defective or premature habituation may lead to sensory hyper-responsivity, which has been widely observed in individuals with Autism Spectrum Disorder (ASD) [ 19 – 22 ]. Recently, orthologs of 98 human intellectual disability (ID) genes were reported to be important for habituation in fruit flies and a large fraction of these genes were associated with ASD [ 23 ]. In view of our previous findings that report high expression of ChT in MBs, we study a putative role of ChT in ‘Habituation’ which is widely regarded as a prerequisite for more complex form of associative learning. Compilation of the previous information describing habituation in Drosophila reveals that most of the olfactory habituation paradigms engage olfactory circuitry [ 24 , 25 ]. Therefore, we mapped the function of ChT in MB neurons as well as in the olfactory pathway governing olfactory habituation in Drosophila larvae.

ACh synthesis for efficient neurotransmission at cholinergic synapses depends on the proteins of its metabolic cycle, namely, Choline acetyltransferase (ChAT), vesicular acetylcholine transferase (VAChT), Acetylcholine esterase (AChE) and ChT. The ACh is synthesized by enzyme ChAT from choline and acetylcoenzymeA. It is then transported into synaptic vesicles by VAChT. The ChT imports choline into the presynaptic terminal and is the rate-limiting step of ACh biosynthesis. The MBs in Drosophila CNS are bilateral neuropilar areas having evolutionary similarities with vertebrate cortex [ 5 ]. Drosophila MBs express ChAT and VAChT and require ACh for its function [ 6 , 7 ]. Based on immunostainings, we recently reported a preponderance of ChT in Drosophila MBs as compared to ChAT and VAChT [ 8 ]. This finding was intriguing which led us to explore why ChT has preferentially higher expression in MBs as compared to ChAT and VAChT. MBs receive olfactory input from the antennal lobes (AL) via projection neurons (PNs) and serve as the prime site for sensory integration and learning. MBs associate the memory with a reward or punishment pathway. However, before establishing such an association in MBs, an animal must evaluate each incoming stimulus, identify the salient stimuli and initiate the appropriate stimulus driven response. Therefore, to understand the mechanisms of complex associative learning, it is important to decipher the mechanisms in MBs that impart an animal the flexibility to choose only the salient incoming stimuli, ignore inconsequential ones and finally register this information in a context-dependent manner. ‘Habituation’ is one such behavioral process that enables an organism to evade inconsequential stimuli from the salient ones. Several reported Drosophila genes like dunce, rutabaga, turnip, radish, DCO, Leonardo, DAMB, Nmdar1 and 2 have high expression in MBs and contribute to Drosophila memory [ 9 ]. Many of the proteins expressed at elevated levels in the MBs are required for both associative learning and habituation [ 10 , 11 ], suggesting that the proteins contributing to associative learning also contribute to habituation. This implies the existence of an association between the two forms of plasticity. Flies that lack MBs display reduced habituation [ 10 , 12 , 13 ]. Also, habituation to repetitive footshock stimuli requires intact MBs [ 14 ]. Thus, these reports signify the importance of MB function in habituation behaviour.

Acetylcholine (ACh) is the fundamental neurotransmitter of cholinergic neurons. These neurons are widely distributed throughout the central nervous system (CNS) in the vertebrate and invertebrate brain. In vertebrates, all pre-ganglionic sympathetic neurons, part of post-ganglionic sympathetic neurons, pre and post ganglionic parasympathetic neurons are cholinergic [ 1 ]. Also, in invertebrates like Drosophila, almost all major types of sensory neurons, including chemosensory, chordotonal, olfactory neurons and most regions of the central brain and interneurons are cholinergic [ 2 – 4 ]. Given the widespread distribution of cholinergic neurons in the vertebrate and invertebrate brain, ACh mediated neurotransmission is crucial for neural functions that include varied sensory modalities.

Results

Decrement of odour-specific chemotactic response in naïve Drosophila larvae conforms to habituation parameters Attraction towards an odour is referred to as chemotaxis, which is essential for a diversity of insects to navigate for food sources, potential mating partners, assessing danger, search for egg-laying sites, etc. The olfactory system of insects has evolved to impart them great discriminatory power for behaviourally relevant odours, decipher this message in CNS and finally exhibit appropriate behaviours. Thus, chemotaxis-based behavioural responses towards an odour are significant for their survival. Foraging Drosophila larvae are in constant search of food for their development. The larval olfactory system is similar but numerically simpler than the adult flies, discern a wide range of odours and learn to discriminate between different odours and concentrations [26–29]. Thus, it provides a system with a genetically accessible, well-defined neural circuit relevant to our study. We first tested and standardised the habituation assay in wildtype 3rd instar foraging larvae to affirm if our assay accedes to habituation parameters. Naïve wildtype larvae were attracted by Ethyl acetate (ETA) and Amyl acetate (AMA) as assessed by calculating response index (Pre-hab R.I) (Fig 1A and 1B). Continuous exposure of naïve wild type larvae for 5 min to AMA and ETA, evoked significant avoidance of these odours (Post-hab R.I) (Fig 1A and 1B). However, among both the odours tested, ETA elicited a more robust chemotactic response and showed stronger avoidance after prior exposure. Therefore, we used the attractant ETA for subsequent experiments. We further investigated whether the decrement of chemotactic response conforms to classical habituation parameters [30], which means animals that habituate to a stimulus should regain the response after a time-lapse, if the stimulus is withheld. This phenomenon is termed as ‘spontaneous recovery’ (Fig 1C). Indeed, we observed a spontaneous recovery of the chemotactic response to the naïve levels after 15 and 30 min rest time (Fig 1B). To confirm the initial decrement as habituation and not a fatigue or sensory adaptation, we tested another classical habituation feature called ‘Dishabituation’. It means if the habituated animal is exposed to an unrelated strong stimulus, the naïve response is fully or partially restored. We attempted dishabituation using a 1 min exposure to cold shock on ice (Schematics, Fig 1D). This significantly reverses the chemotactic response of larvae pre-exposed to 5’ ETA (Fig 1B). Notably, 1 min exposure to cold shock given to naïve larvae did not affect R.I towards ETA as compared to pre-hab R.I, suggesting that cold shock does not affect the general perception of olfactory stimuli or cause sensitisation (Fig 1B). We also assessed effect of 1 min cold shock on larval motility before and after the cold shock and observed that 100 percent larvae had left the choice point (Fig 1E). Under both conditions (with cold shock and without cold shock), 28 percent larvae moved to zone 1 and 71 percent larvae moved to zone 2 (Fig 1E), suggesting that 1 min cold shock does not have any effect on larval motility. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Olfactory habituation assay in wild-type Drosophila larvae. (A) Schematics represent the specific time segments to induce olfactory habituation in wild-type (W1118) larvae. Naïve larvae were exposed to the odour and response index (R.I) was calculated after 3 min (Pre-hab R.I). The same set of larvae were left for additional 2 min in presence of odour for habituation (This makes total odour exposure time to 5 min). The odour exposed larvae were brought back in the middle of the petriplate and the R.I was again calculated (Post-hab R.I). (B) Scatter plot shows response indices (R.I, black circles) of naïve wild type larvae towards amyl acetate (AMA) or ethyl acetate (ETA) (pre-hab R.I) and after 5 min of odour exposure (post-hab R.I). Larvae pre-exposed to odour for 5 min shows spontaneous recovery after 15 or 30 min in the absence of stimulus (see spontaneous recovery schematics). Larvae pre-exposed to odour for 5 min shows recovery by 1min cold shock (5’ETA+cold shock), the phenomenon called dishabituation (see dishabituation schematics). R.I of naïve larvae without odour pre-exposure but exposed to cold shock (Naïve+cold shock) is not altered due to cold shock. (C) Schematics represent the specific time segments to induce spontaneous recovery, (D) Schematics represent the specific time segments to induce dishabituation. (E) The scatter plot shows percent larval motility of 1 min cold shock treated (+) and untreated larvae (-), in a petriplate with ETA and water on opposite ends. As shown in the schematics on right, the larvae were kept in the centre and after 3 min, the total number of larvae that moved to different zones were counted. The data represented here shows percentage of total larvae moved, larvae moved to zone1, and zone 2, N = 8. Data is represented as scatter plot with error bars showing SEM and N≥16, unless mentioned. Each N represent one experiment performed with a group of 30–40 larvae. Each data group was analysed for normal distribution using Shapiro-Wilk test. Statistical significance was determined by two-tailed unpaired t-test (parametric) with Welchs correction. *** represent p≤0.0001, n.s means statistical non-significance when p≥0.05. For statistical details and numerical data values in the scatter plot refer to S1 and S2 Data. https://doi.org/10.1371/journal.pgen.1009938.g001 Taken together, the decrement of chemotaxis in wildtype larvae on continuous exposure to an odorant stimulus, the recovery of the response when the stimulus was withheld, and the dishabituation conforms to the habituation parameters and demonstrates that the response attenuation is attributed to olfactory habituation. We have used these olfaction-based paradigms to demonstrate the functional relevance of ChT in regulating habituation in our subsequent experiments.

Intrinsic neurons of MB require ChT for olfactory habituation and incoming odour stimulus suppression There are three types of MB intrinsic neurons, i.e., α/β, α’/β’ and γ neurons, also termed as Kenyon cells (KC). These are distinctly implicated in olfactory learning and memory [31,32]. To understand the role of ChT in MBs, we depleted it in independent domains of MB intrinsic neurons with the help of UAS-GAL4 binary expression system [33]and investigated if these neurons require ChT function in habituation. Our immunostainings show ChT colocalization in MB calyx and different domains of MB neurons as assessed by driving expression of UAS-mCD8GFP with MB247 (α/β + γ class of KC) and C739 (α/β class of KC), C305a (α’/β’ class of KC), and NP1131 (γ class of KC) (S2 Fig). We used two RNAi fly stocks [ChTRNAi1 (V101485) and ChTRNAi2 (BL28613)] to knock down ChT in MB intrinsic neurons. The knock-down efficiency of ChTRNAi2 was assessed by immunostaining (S1 Fig), while that of ChTRNAi1 was described previously by us [8]. First, we evaluated the naïve chemotactic response of 3rd instar foraging larvae towards ETA upon knock-down of ChT in each of the neuronal domains of MBs. Interestingly, we observed a significant increase in naïve chemotactic response upon knock-down of ChT in γ lobe neurons (NP1131> ChTRNAi1 or ChTRNAi2) as well as in α’/β’ (C305a> ChTRNAi1 or ChTRNAi2) and α/β + γ (MB247> ChTRNAi1 or ChTRNAi2) class of KC as compared to their genetic controls (Fig 2A–2C). To test if chemotactic enhancement is specific to the knock-down of ChT, we expressed ChT transgene on UAS-ChTRNAi1 background in all the three neuronal domains of MBs (MBGAL4s> ChTRNAi1;UAS-ChT) and observed a reversal of the response index in γ and α’/β’ class of KC but not in α/β+ γ class of KC when compared with respective genotypes of MBGAL4s> + (Fig 2A–2C). Additionally, we compared response index of genotype MBGAL4s> ChTRNAi1;UAS-ChT with MBGAL4s> ChTRNAi1 but observed statistically significant reversal of the response index only in γ lobe but not in α’/β’ and α/β+ γ class of KC (Fig 2A–2C). Next, we tested the effect of ChT knock-down in MBs on olfactory habituation. A significant reduction in habituation index was observed in the group of larvae upon knock-down of ChT with both ChTRNAi1 and ChTRNAi2 fly lines in γ, α’/β’ and α/β+ γ class of KC as compared to their genetic controls (Fig 2D–2F). Transgenic expression of ChT in UAS-ChTRNAi1 background significantly enhanced the habituation index of the larvae in α’/β’ and α/β+ γ class of KC but not in γ KC (Fig 2D–2F). This difference in the rescue of response index and habituation index in different KC may be due to the differences in the expression levels of UAS-ChTRNAi1 and UAS-ChT transgenes driven by the different GAL4s. To clarify, if the levels of ChT determine the extent of habituation and chemotaxis, we over-expressed ChT in all the three classes of KC neurons using specific GAL4 drivers (MBGAL4’s>UAS-ChT). The extent of the naïve olfactory response, as well as the habituation, remains unaffected by overexpression of ChT (Fig 2A–2F). Next, we confirmed if larvae’s enhanced response towards ETA and decreased habituation is specific to the knock-down of ChT and not specific to the kind of pre-exposed odour. For this, we knocked down ChT in γ, α’/β’ and α/β+ γ KC using UAS-ChTRNAi1 (MBGAl4s>ChTRNAi1) and exposed these group of larvae to amyl acetate (AMA) and the alcoholic class of odour, 3-Octanol. We observed an enhancement of chemotaxis and reduced habituation for both the pre-exposed odours, suggesting it to be ChT specific phenotype and not to the class of odour (S3A–S3D Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Knock-down of ChT in MB intrinsic neurons enhances chemotaxis towards odour but suppresses habituation. (A-C) Response index of naïve larvae (also referred to as chemotaxis) towards ETA, (D-E) habituation index of larvae exposed to ETA, of genotypes: NP1131GAL4 was used as a driver line for expression in MB γ-lobe. Scatter plot represents R.I (A) and H.I (D) of genotypes NP1131> UAS-ChTRNAi1, NP1131>UAS-ChTRNAi1;UAS-ChT, NP1131> UAS-ChT as compared to their controls NP1131> +. NP1131> UAS-ChTRNAi2 as compared to UAS-ChTRNAi2>+. C305aGAL4 was used as a driver line for expression in MB α’/β’-lobe. Scatter plot represents R.I (B) and H.I (E) of genotypes C305a> UAS-ChTRNAi1, C305a> UAS-ChTRNAi1;UAS-ChT, C305a> UAS-ChT as compared to their controls C305a> +. C305a> UAS-ChTRNAi2 as compared to ChTRNAi2>+. MB247GAL4 was used as a driver line for expression in MB α/β+ γ lobe. Scatter plot represents R.I (C) and H.I (F) of genotypes MB247> UAS-ChTRNAi1, MB247> UAS-ChTRNAi1;UAS-ChT, MB247> UAS-ChT as compared to their controls MB247> +. MB247> UAS-ChTRNAi2 as compared to UAS-ChTRNAi2>+. Pink circles represent knockdown using UAS-ChTRNAi1 and UAS-ChTRNAi2, Blue circles represent rescue, and yellow triangles represent transgenic over-expression of UAS-ChT as compared to genetic controls (black circles). Data are represented as scatter plot with error bars showing SEM and N≥16. Each N represent one experiment performed with a group of 40 larvae. Each data group was analysed for normal distribution using Shapiro-Wilk test. Statistical significance was determined by two-tailed unpaired t-test (parametric) with Welchs correction. *** represent p≤0.0001, n.s means statistical non-significance when p≥0.05. For more statistical details and numerical data values in the scatter plot refer to S1 and S2 Data. https://doi.org/10.1371/journal.pgen.1009938.g002 To investigate if the synaptic transmission from MBs is required to regulate incoming odour stimulus and habituation, we expressed the temperature-sensitive mutant of Dynamin orthologue, Shibire, in MB neurons using a UAS-Shits transgene. Shibirets mutant causes a block of synaptic vesicle recycling at non-permissible temperature (29°C), leading to a rapid decline of neurotransmitter release and synaptic transmission [34]. The neural activity was perturbed specifically in α’β’, γ and αβ+ γ class of KC intrinsic neurons using C305a-GAL4, NP1131-GAL4 and MB247-GAL4, respectively, and their consequent effect on the naïve chemotactic response and habituating ability of the larvae at non-permissible temperature was assessed. A significantly enhanced chemotaxis towards ETA and reduced habituation were observed as compared to their genetics controls (GAL4s>+) (S4A and S4B Fig). Inactivation of synaptic vesicle endocytosis in MB intrinsic neurons resulted in enhanced chemotaxis and attenuated habituation similar to those observed with knock-down of ChT in these neurons. This suggests a need for neurotransmitter release from MBs for incoming odour stimulus suppression and habituation. Previously, we reported attenuated neuromuscular junctions (NMJ) in third instar larvae due to knock-down of ChT in α/β and γ intrinsic lobes of MB [8]. To ascertain if the observed reduction in habituation index is the acute functional change in the neurons during habituation or has resulted from changes in the NMJ, we employed the TARGET system [35]. TARGET system uses a Tubulin promotor to express temperature-sensitive repressor of GAL4 (TubGAL80ts) where expression of GAL80ts allows RNAi expression by GAL4 at 29°C but not at 18°C. Using this system, we limited the expression of UAS-ChTRNAi1(MBGAL4>UAS-ChTRNAi1;tubGAL80ts) to the developmental window of foraging 3rd instar larvae during which the assay was performed (S5A Fig). We observed a significant enhancement in response index towards the odour (S5B Fig) and a drastic reduction in habituation index (S5C Fig) upon knock-down of ChT in α/β and γ lobe neurons at 29°C but not at 18°C (S5B and S5C Fig). Reportedly, MB247GAL4 has expression domain in both γ and α/β domain [36]. Therefore, we validated our results obtained with MB247GAL4 by performing experiments with additional GAL4 line, C739GAL4, which preferentially expresses only in α/β domain [36]. We observed an enhanced response index and reduced habituation index as compared to their genetic controls at 29°C but not at 18°C (S5D and S5E Fig). These results confirm that ChT is acutely required in MB intrinsic neurons to facilitate habituation and the observed habituation defects are not due to changes in neurons during development. Altogether, our results indicate that knock-down of ChT in MB does not affect the odour perception. It has a function in MB intrinsic neurons that support devaluation of the incoming stimuli and regulates sensitivity towards incoming olfactory stimuli.

ChT is essential in intrinsic neurons of MB for the maintenance of key characteristics of habituation A habituated animal should re-establish the response towards stimuli partially or wholly within a specific time frame if the stimulus is withheld [30]. To ascertain that the decrement in response due to knock down in ChT is indeed habituation phenotype, we assessed the response recovery (spontaneous recovery), after a rest period of 15 min and 30 min, in the habituated larvae when the stimulus was withheld. The knock-down of ChT using the expression of UAS-ChTRNAi1 and UAS-ChTRNAi2 lines driven by NP1131GAL4 (γ lobe, Fig 3A), C305aGAL4 (α’/β’lobe, Fig 3B) and MB247GAL4 (α/β+ γ lobe, Fig 3C) intrinsic lobes caused a defect in the larvae to spontaneously recover. On the other hand, larvae from their genetic controls (MBGAL4s>+ or UAS-ChTRNAi2>+) significantly recovered to the naïve levels after just 15 min (Fig 3A–3C). The defect in recovery due to depletion of ChT was restored partially or completely when ChT was transgenically expressed on a ChTRNAi1 background in α’/β’, α/β+ γ and γ class of KC neurons (Fig 3A–3C). However, we noticed a significant rescue in spontaneous recovery in γ lobe only after 60 min whereas in α’/β’ and α/β+ γ, the recovery was achieved within 30 min. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Knock-down of ChT in MB intrinsic neurons impairs spontaneous recovery and dishabituation of habituated larvae. Scatter plot shows response indices towards ETA of larvae pre-exposed to 5 min ETA (Post-hab). For spontaneous recovery, the larvae were pre-exposed to 5’ETA and R.I was calculated after 15, 30 or 60 min rest time in absence of stimulus. For dishabituation, the larvae were pre-exposed to 5’ETA and R.I was calculated after 1 min cold shock (5’ETA+coldshock). Scatter plot also shows R.I of naïve larvae presented to 1 min cold shock (naïve+ cold shock). Scatter plot shows R.I in the described conditions in genotypes: (A) NP1131> UAS-ChTRNAi1 (pink circles), NP1131> UAS-ChTRNAi1;UAS-ChT (blue circles) as compared to their controls NP1131> + (black circles). NP1131> UAS-ChTRNAi2 (pink circles) as compared to UAS-ChTRNAi2>+ (black circles). (B) C305a> UAS-ChTRNAi1 (pink circles), C305a> UAS-ChTRNAi1;UAS-ChT (blue circles) as compared to their controls C305a> +(black circles). C305a> ChTRNAi2 (pink circles) as compared to UAS-ChTRNAi2>+ (black circles). (C) MB247> UAS-ChTRNAi1 (pink circles), MB247> UAS-ChTRNAi1;UAS-ChT (blue circles) as compared to their controls MB247> + (black circles). MB247> UAS-ChTRNAi2 (pink circles) as compared to UAS-ChTRNAi2>+ (black circles). Both spontaneous recovery and dishabituation was impaired in ChT knocked-down group of larvae (data represented by pink circles). Each N represent one experiment performed with a group of 30 larvae. Each data group was analysed for normal distribution using Shapiro-Wilk test. Statistical significance was determined by two-tailed unpaired t-test (parametric) with Welchs correction. *** represent p≤0.0001, ** represent p≤0.001, n.s means statistical non-significance when p≥0.05. For more statistical details and numerical data values in the scatter plot refer to S1 and S2 Data. https://doi.org/10.1371/journal.pgen.1009938.g003 Next, we induced dishabituation by exposing the habituated larvae to cold shock for 1 min. The knock-down of ChT in the γ, α’/β’ and α/β+ γ class of KC neurons of larvae using NP1131GAL4 (Fig 3A), C305aGAL4 (Fig 3B) and MB247GAL4 (Fig 3C) driver lines, respectively, were unable to dishabituate upon cold shock. On the other hand, exposure of habituated larvae to 1 min cold shock reverses chemotaxis decrement to naïve levels in the control group of larvae (MBsGAL4>+, Fig 3A–3C). The dishabituating capability was restored in these larvae when ChT was transgenically expressed on a ChTRNAi background (UAS-ChTRNAi1;UAS-ChT) in γ, α’/β’, and α/β class of KC neurons (Fig 3A–3C). The exposure of naïve larvae to similar cold shock did not affect the chemotaxis both in control and ChT-depleted group of larvae, suggesting that the observed enhancement of response index is not the sensitisation of olfactory receptors due to cold shock. The control group of larvae (MBsGAL4>+) are capable of re-establishing baseline response levels that follow an unrelated dishabituating stimulus as well as after a prolonged lapse of time in the absence of stimulus while larvae deficit of ChT function in MB neurons are incapable of resuming the initial response levels. This suggests that in the absence of ChT, the synaptic capability to re-establish the response is compromised, leading to defective spontaneous recovery and dishabituation.

[END]

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