Encoding of self-initiated actions in axon terminals of the mesocortical pathway.

Significance: The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim: To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach: We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results: Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions: We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior.

Functional diversity of dopamine axons in prefrontal cortex during classical conditioning.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

Functional Diversity of Dopamine Axons in Prefrontal Cortex During Classical Conditioning.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Quantitative study of the somatosensory sensitization underlying cross-modal plasticity.

Loss of one sensory modality can cause other types to become more perceptive (cross-modal plasticity). To test the hypothesis that the loss of vision changes the perceptual threshold in the somatosensory system, we applied optogenetics to directly manipulate the afferent inputs involved in the whisker-barrel system using a transgenic rat (W-TChR2V4) that expresses channelrhodopsin-2 (ChR2) selectively in the large mechanoreceptive neurons in the trigeminal ganglion (TG) and their peripheral nerve terminals. The licking behavior of W-TChR2V4 rat was conditioned to a blue LED light cue on the whisker area while the magnitude and duration of light pulses were varied. The perceptual threshold was thus quantitatively determined for each rat according to the relationship between the magnitude/duration of light and the reaction time between the LED light cue and the first licking event after it. We found that the perceptual threshold was more significantly reduced than the control non-deprived rats when the rats were visually deprived at postnatal 26-30 days (P26-30, early VD group), but not at P58-66 (late VD group). However, the sensory threshold of a late VD animal was similar to that of a control. Our results suggest the presence of cross-modal plasticity by which the loss of vision at the juvenile period increased the sensitivity of the somatosensory system involved in the touch of whiskers.

Optogenetic conditioning of paradigm and pattern discrimination in the rat somatosensory system.

The rodent whisker-barrel cortical system is a model for studying somatosensory discrimination at high spatiotemporal precision. Here, we applied optogenetics to produce somatosensory inputs in the whisker area using one of transgenic rat lines, W-TChR2V4, which expresses channelrhodopsin-2 (ChR2) in the mechanoreceptive nerve endings around whisker follicles. An awake W-TChR2V4 rat was head-fixed and irradiated by blue LED light on the whisker area with a paradigm conditioned with a reward. The Go task was designed so the rat is allowed to receive a reward, when it licked the nozzle within 5 s after photostimulation. The No-go task was designed so as the rat has to withhold licking for at least 5 s to obtain a reward after photostimulation. The Go-task conditioning was established within 1 hr of training with a reduction in the reaction time and increase of the success rate. To investigate the relationship between the spatiotemporal pattern of sensory inputs and the behavioral output, we designed a multi-optical fiber system that irradiates the whisker area at 9 spots in a 3×3 matrix. Although the Go-task conditioning was established using synchronous irradiation of 9 spots, the success rate was decreased with an increase of the reaction time for the asynchronous irradiation. After conditioning to the Go task, the rat responded to the blue LED flash irradiated on the barrel cortex, where many neurons also express ChR2, or photostimulation of the contralateral whisker area with a similar reaction time and success rate. Synchronous activation of the peripheral mechanoreceptive nerves is suggested to drive a neural circuit in the somatosensory cortex that efficiently couples with the decision. Our optogenetic system would enable the precise evaluation of the psychophysical values, such as the reaction time and success rate, to gain some insight into the brain mechanisms underlying conditioned behaviors.

Ultrafast hydrogen-bonding dynamics in the electronic excited state of photoactive yellow protein revealed by femtosecond stimulated Raman spectroscopy.

The ultrafast structural dynamics in the electronic excited state of photoactive yellow protein (PYP) is studied by femtosecond stimulated Raman spectroscopy. Stimulated Raman spectra in the electronic excited state, S(1), can be obtained by using a Raman pump pulse in resonance with the S(1)-S(0) transition. This is confirmed by comparing the experimental results with numerical calculations based on the density matrix treatment. We also investigate the hydrogen-bonding network surrounding the wild-type (WT)-PYP chromophore in the ground and excited states by comparing its stimulated Raman spectra with those of the E46Q-PYP mutant. We focus on the relative intensity of the Raman band at 1555 cm(-1), which includes both vinyl bond C═C stretching and ring vibrations and is sensitive to the hydrogen-bonding network around the phenolic oxygen of the chromophore. The relative intensity for the WT-PYP decreases after actinic excitation within the 150 fs time resolution and reaches a similar intensity to that for E46Q-PYP. These observations indicate that the WT-PYP hydrogen-bonding network is immediately rearranged in the electronic excited state to form a structure similar to that of E46Q-PYP.

Femtosecond stimulated Raman spectroscopy of the dark S1 excited state of carotenoid in photosynthetic light harvesting complex.

Vibrational dynamics of the excited state in the light-harvesting complex (LH1) have been investigated by femtosecond stimulated Raman spectroscopy (FSRS). The native and reconstituted LH1 complexes have same dynamics. The ν(1) (C=C stretching) vibrational mode of spirilloxanthin in LH1 shows ultrafast high-frequency shift in the S(1) excited state with a time constant of 0.3 ps. It is assigned to the vibrational relaxation of the S(1) state following the internal conversion from the photoexcited S(2) state.