A Drosophila Perspective

The so-called active zones at pre-synaptic terminals are the ultimate filtering devices, which couple between action potential frequency and shape, and the information transferred to the post-synaptic neurons, finally tuning behaviors. Within active zones, the release of the synaptic vesicle operates from specialized “release sites.” The (M)Unc13 class of proteins is meant to define release sites topologically and biochemically, and diversity between Unc13-type release factor isoforms is suspected to steer diversity at active zones. The two major Unc13-type isoforms, namely, Unc13A and Unc13B, have recently been described from the molecular to the behavioral level, exploiting Drosophila being uniquely suited to causally link between these levels. The exact nanoscale distribution of voltage-gated Ca2+ channels relative to release sites (“coupling”) at pre-synaptic active zones fundamentally steers the release of the synaptic vesicle. Unc13A and B were found to be either tightly or loosely coupled across Drosophila synapses. In this review, we reported recent findings on diverse aspects of Drosophila Unc13A and B, importantly, their nano-topological distribution at active zones and their roles in release site generation, active zone assembly, and pre-synaptic homeostatic plasticity. We compared their stoichiometric composition at different synapse types, reviewing the correlation between nanoscale distribution of these two isoforms and release physiology and, finally, discuss how isoform-specific release components might drive the functional heterogeneity of synapses and encode discrete behavior

Altered function in medial prefrontal cortex and nucleus accumbens links to stress-induced behavioral inflexibility

The medial prefrontal cortex (mPFC) and its output area, the nucleus accumbens (NAc), are implicated in mediating attentional set-shifting. Patients with posttraumatic stress disorder (PTSD) exhibit difficulties in the disengagement of attention from traumatic cues, which is associated with impairments in set shifting ability. However, unknown is whether alterations in corticostriatal function underlie deficits in this behavioral flexibility in individuals with PTSD. An animal model of single prolonged stress (SPS) has been partially validated as a model for PTSD, in which SPS rats recapitulate the pathophysiological abnormalities and behavioral characteristics of PTSD. In the present study, we firstly found that exposure to SPS impaired the ability in the shift from visual-cue learning to place response discrimination in rats. Conversely, SPS induced no effect on a place-to-cue set-shifting performance. Based on SPS-impaired set-shifting model, we used Western blot and immunofluorescent approaches to clarify SPS-induced alternations in synaptic plasticity and neuronal activation in the mPFC and NAc. Rats that were subjected to SPS exhibited a large increase in pSer845-G1uA1 and total G1uA1 levels in the mPFC, while no significant change in the NAc. We further found that exposure to SPS significantly decreased c-Fos expression in the NAc core but not the shell after set-shifting behavior. Whereas, enhanced c-Fos expression was observed in prelimbic and infralimbic cortices. Collectively, these findings suggest that abnormal hyperactivity in the mPFC and dysfunction in the NAc core underlie long-term deficits in executive function after traumatic experience, which might play an important role in the development of PTSD symptoms. (C) 2016 Elsevier B.V. All rights reserved.</p

A brain-wide form of presynaptic active zone plasticity orchestrates resilience to brain aging in Drosophila

The brain as a central regulator of stress integration determines what is threatening, stores memories, and regulates physiological adaptations across the aging trajectory. While sleep homeostasis seems to be linked to brain resilience, how age-associated changes intersect to adapt brain resilience to life history remains enigmatic. We here provide evidence that a brain-wide form of presynaptic active zone plasticity (“PreScale”), characterized by increases of active zone scaffold proteins and synaptic vesicle release factors, integrates resilience by coupling sleep, longevity, and memory during early aging of Drosophila. PreScale increased over the brain until mid-age, to then decreased again, and promoted the age-typical adaption of sleep patterns as well as extended longevity, while at the same time it reduced the ability of forming new memories. Genetic induction of PreScale also mimicked early aging-associated adaption of sleep patterns and the neuronal activity/excitability of sleep control neurons. Spermidine supplementation, previously shown to suppress early aging-associated PreScale, also attenuated the age-typical sleep pattern changes. Pharmacological induction of sleep for 2 days in mid-age flies also reset PreScale, restored memory formation, and rejuvenated sleep patterns. Our data suggest that early along the aging trajectory, PreScale acts as an acute, brain-wide form of presynaptic plasticity to steer trade-offs between longevity, sleep, and memory formation in a still plastic phase of early brain aging. This study shows that a brain-wide form of presynaptic plasticity ("PreScale") steers trade-offs between longevity, sleep and memory formation in a still plastic phase of early brain aging, illustrating how life strategy manifests at both circuit and synapse levels

N-methyl-D-aspartate receptor-mediated glutamate transmission in nucleus accumbens plays a more important role than that in dorsal striatum in cognitive flexibility

Cognitive flexibility is a critical ability for adapting to an ever-changing environment in humans and animals. Deficits in cognitive flexibility are observed in most schizophrenia patients. Previous studies reported that the medial prefrontal cortex-to-ventral striatum and orbital frontal cortex-to-dorsal striatum circuits play important roles in extra- and intra-dimensional strategy switching, respectively. However, the precise function of striatal subregions in flexible behaviors is still unclear. N-methyl-D-aspartate receptors (NMDARs) are major glutamate receptors in the striatum that receive glutamatergic projections from the frontal cortex. The membrane insertion of Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) depends on NMDAR activation and is required in learning and memory processes. In the present study, we measured set-shifting and reversal learning performance in operant chambers in rats and assessed the effects of blocking NMDARs and Ca(2+)-permeable AMPARs in striatal subregions on behavioral flexibility. The blockade of NMDARs in the nucleus accumbens (NAc) core by AP5 impaired set-shifting ability by causing a failure to modify prior learning. The suppression of NMDAR-mediated transmission in the NAc shell induced a deficit in set-shifting by disrupting the learning and maintenance of novel strategies. During reversal learning, infusions of AP5 into the NAc shell and core impaired the ability to learn and maintain new strategies. However, behavioral flexibility was not significantly affected by blocking NMDARs in the dorsal striatum. We also found that the blockade of Ca(2+)-permeable AMPARs by NASPM in any subregion of the striatum did not affect strategy switching. These findings suggest that NMDAR-mediated glutamate transmission in the NAc contributes more to cognitive execution compared with the dorsal striatum

Region-specific roles of the prelimbic cortex, the dorsal CA1, the ventral DG and ventral CA1 of the hippocampus in the fear return evoked by a sub-conditioning procedure in rats

The return of learned fear is an important issue in anxiety disorder research since an analogous process may contribute to long-term fear maintenance or clinical relapse. A number of studies demonstrate that mPFC and hippocampus are important in the modulation of post-extinction re-expression of fear memory. However, the region-specific role of these structures in the fear return evoked by a sub-threshold conditioning (SC) is not known. In the present experiments, we first examined specific roles of the pre limbic cortex (PL), the dorsal hippocampus (DH, the dorsal CA1 area in particular), the ventral hippocampus (the ventral dentate gyrus (vDG) and the ventral CA1 area in particular) in this fear return process. Then we examined the role of connections between PL and vCA1 with this behavioral approach. Rats were subjected to five tone-shock pairings (1.0-mA shock) to induce conditioned fear (freezing), followed by three fear extinction sessions (25 tone-alone trials each session). After a post-test for extinction memory, some rats were retrained with the SC procedure to reinstate tone-evoked freezing. Rat groups were injected with low doses of the GABAA agonist muscimol to selectively inactivate PL, DH, vDG, or vCA1 120 min before the fear return test. A disconnection paradigm with ipsilateral or contralateral muscimol injection of the PL and the vCA1 was used to examine the role of this pathway in the fear return. We found that transient inactivation of these areas significantly impaired fear return (freezing): inactivation of the prelimbic cortex blocked SC-evoked fear return in particular but did not influence fear expression in general; inactivation of the DH area impaired fear return, but had no effect on the extinction retrieval process; both ventral DG and ventral CA1 are required for the return of extinguished fear whereas only ventral DG is required for the extinction retrieval. These findings suggest that PL, DH, vDG, and vCA1 all contribute to the fear return and connections between PL and vCA1 may be involved in the modulation of this process. (c) 2016 Elsevier Inc. All rights reserved

PreScale promotes survival over memory formation during early aging.

(A) Direct curve-fitting comparisons between PreScale during early aging from 1d to 30d (Fig 1) and genetic brp titration from 1xBRP to 4xBRP at age 5d [33]. (B and C) Lifespan analysis of flies with BRP titration from 1xBRP to 4xBRP. For male flies (B), n = 394 for 1xBRP (p n = 396 for 3xBRP (p n = 395 for 4xBRP (p wt (n = 393). For female flies (C), n = 400 for 1xBRP (p = 0.017), n = 399 for 3xBRP (ns) and n = 397 for 4xBRP (p wt (n = 399). (D and E) An independent experiment of the lifespan of 2xBRP and 3xBRP flies. For male flies (D), n = 94 for 3xBRP compared to 2xBRP wt (n = 36, p E), n = 98 for 3xBRP compared to 2xBRP wt (n = 59, p F and G) Lifespan analysis of flies with 3xBRP in wake mutant background. For male flies (F), n = 234 for 2xBRP;wake compared to 2xBRP wt control (n = 233, p wake (n = 155, p G), n = 235 for 2xBRP;wake compared to 2xBRP wt control (n = 230, p wake (n = 231, ns). (H and I) Lifespan analysis of flies with 3xBRP in inc mutant background. For male flies (H), n = 236 for inc;2xBRP compared to 2xBRP wt (n = 232, p = 0.009) or compared to inc;3xBRP (n = 212, p I), n = 235 for inc;2xBRP compared to 2xBRP wt (n = 235, p wt is also shown in Fig 1A) or compared to inc;3xBRP (n = 182, ns). (J and K) Lifespan analysis of flies with 3xBRP in atg7 mutant background. For male flies (J), n = 108 for atg7,2xBRP compared to 2xBRP wt (n = 195, p atg7;3xBRP (n = 87, ns). For female flies (K), n = 109 for atg7,2xBRP compared to 2xBRP wt (n = 208, p atg7;3xBRP (n = 87, p L and M) Lifespan analysis of flies with 3xBRP in Alzheimer’s disease model flies (elav>Aβ). For male flies (L), n = 203 for 2xBRP;Aβ compared to elav>mCD8-GFP control (n = 238, p Aβ (n = 151, p = 0.006). For female flies (M), n = 249 for 2xBRP;Aβ compared to elav>mCD8-GFP control (n = 228, p Aβ (n = 241, p = 0.002). Gehan–Breslow–Wilcoxon test with Bonferroni correction for multiple comparisons is shown for all longevity experiments. (N and O) STM for 5d (N) and 30d (O) 2xBRP and 3xBRP flies. n = 19 for 5d 2xBRP, n = 12 for 5d 3xBRP. n = 7–8 for 30d for both groups at 30d. (P and Q) MTM tested 3 h after training for 5d (P) and 30d (Q) 2xBRP and 3xBRP flies. n = 21 for 5d 2xBRP, n = 16 for 5d 3xBRP. n = 8–9 for 30d for both groups at 30d. Student t test is shown. *p S3 Data. BRP, Bruchpilot; MTM, middle-term memory; STM, short-term memory; wt, wild type.</p

Data underlying Fig 3.

(XLSX)</p

Synaptic plasticity across the fly lifespan.

(A) A typical survival curve of wt female flies. n = 235. (B-H) Representative western blots (B) and relative levels of a spectrum of synaptic proteins, including BRP (C), Unc13A (D), Dlg1 (E), Syn (F), and Syx (G), and the relative ratios of active lipidated and unlipidated autophagy-related protein Atg8a (Atg8a-II:Atg8-I; H) in wt female flies across the lifespan. n = 6–8. One-way ANOVA with Bonferroni multiple comparisons test is shown. (I and J) Confocal images (I) and whole-mount brain staining analysis (J) of BRP in wt female flies at different ages. n = 15 for 8d, n = 9 for 30d, n = 14–15 for 50d, and n = 9 for 60d. Student t test is shown. *p p p K) Nonlinear line fitting quadratic regression of the protein levels of BRP (C), Unc13A (D), Dlg1 (E), and Syn (F) levels in western blot across the fly lifespan. Underlying data can be found in S1 Data. Raw images of this figure are provided in S1 Raw Images. BRP, Bruchpilot; Dlg1, Discs large; Syn, Synapsin; Syx, Syntaxin; wt, wild type.</p

BRP promotes sleep in a dosage-dependent manner in male flies, similar to females.

(A-E) Sleep structure of 1xBRP-4xBRP male flies averaged from measurements over 2–4 days, including sleep profile plotted in 30-min bins (A), daily sleep amount (B), number and duration of sleep episodes (C and D), and sleep latencies (E). n = 63–80. One-way ANOVA with Bonferroni multiple comparisons test is shown. (F) Linear regression analysis of daily sleep amount in flies with different brp copies in both male (R2 = 0.81) and female (R2 = 0.95) flies. n = 63–80 per group for male, n = 123–128 per group for female. (G-K) Sleep structure of 5d female and male wt flies from measurements over 2–4 days, including sleep profile plotted in 30-min bins (G), daily sleep amount (H), number and duration of sleep episodes (I and J), and sleep latencies (K). n = 62–64 per group. (L) Genomic mapping and sequence of the integration site of the brp P[acman] transgenic construct. Red letters indicate brp P[acman] sequence, and black letters indicate genomic CG11357 gene sequence. (M) Simplified gene span of CG11357 and the integration site of the brp P[acman] and another P-element mediated allele EY12484 that are both localized at the 5′ UTR region of CG11357. (N-R) Sleep structure of EY12484 female flies averaged from measurements over 2–4 days, including sleep profile plotted in 30-min bins (N), daily sleep amount (O), number and duration of sleep episodes (P and Q), and sleep latencies (R). n = 55 for wt control and n = 32 for EY12484. Student t test is shown. (S and T) An independent experiment of the lifespan analysis of 2xBRP and 4xBRP flies. For male flies (S), n = 134 for 4xBRP compared to 2xBRP (n = 132, p T), n = 134 for 4xBRP compared to 2xBRP wt control flies (n = 141, p U and V) Lifespan analysis of 2xBRP wt and EY12484 flies. For male flies (U), n = 128 for EY12484 compared to 2xBRP (n = 127, ns). For female flies (V), n = 129 for EY12484 compared to 2xBRP wt control flies (n = 127, ns). Gehan–Breslow–Wilcoxon test is shown for all longevity experiments. *p p p S1 Data Sheet. BRP, Bruchpilot; CDS, coding DNA sequence; wt, wild type. (TIF)</p