Prelimbic cortex-nucleus accumbens core projection positively regulates itch and itch-related aversion.

Itch is one of the most common clinical symptoms in patients with diseases of the skin, liver, or kidney, and it strongly triggers aversive emotion and scratching behavior. Previous studies have confirmed the role of the prelimbic cortex (Prl) and the nucleus accumbens core (NAcC), which are reward and motivation regulatory centers, in the regulation of itch. However, it is currently unclear whether the Prl-NAcC projection, an important pathway connecting these two brain regions, is involved in the regulation of itch and its associated negative emotions. In this study, rat models of acute neck and cheek itch were established by subcutaneous injection of 5-HT, compound 48/80, or chloroquine. Immunofluorescence experiments determined that the number of c-Fos-immunopositive neurons in the Prl increased during acute itch. Chemogenetic inhibition of Prl glutamatergic neurons or Prl-NAcC glutamatergic projections can inhibit both histaminergic and nonhistaminergic itch-scratching behaviors and rectify the itch-related conditioned place aversion (CPA) behavior associated with nonhistaminergic itch. The Prl-NAcC projection may play an important role in the positive regulation of itch-scratching behavior by mediating the negative emotions related to itch.

The Anterior Insula and its Projection to the Prelimbic Cortex are Involved in the Regulation of 5-HT-Induced Itch.

Itch is an unpleasant sensation that urges people and animals to scratch. Neuroimaging studies on itch have yielded extensive correlations with diverse cortical and subcortical regions, including the insular lobe. However, the role and functional specificity of the insular cortex (IC) and its subdivisions in itch mediation remains unclear. Here, we demonstrated by immunohistochemistry and fiber photometry tests, that neurons in both the anterior insular cortex (AIC) and the posterior insular cortex (PIC) are activated during acute itch processes. Pharmacogenetic experiments revealed that nonselective inhibition of global AIC neurons, or selective inhibition of the activity of glutaminergic neurons in the AIC, reduced the scratching behaviors induced by intradermal injection of 5-hydroxytryptamine (5-HT), but not those induced by compound 48/80. However, both nonselective inhibition of global PIC neurons and selective inhibition of glutaminergic neurons in the PIC failed to affect the itching-scratching behaviors induced by either 5-HT or compound 48/80. In addition, pharmacogenetic inhibition of AIC glutaminergic neurons effectively blocked itch-associated conditioned place aversion behavior, and inhibition of AIC glutaminergic neurons projecting to the prelimbic cortex significantly suppressed 5-HT-evoked scratching. These findings provide preliminary evidence that the AIC is involved, at least partially via aversive emotion mediation, in the regulation of 5-HT-, but not compound 48/80-induced itch.

The prelimbic cortex regulates itch processing by controlling attentional bias.

Itch is a complex and unpleasant sensory experience. Recent studies have begun to investigate the neural mechanisms underlying the modulation of sensory and emotional components of itch in the brain. However, the key brain regions and neural mechanism involved in modulating the attentional processing of itch remain elusive. Here, we showed that the prelimbic cortex (PrL) is associated with itch processing and that the manipulation of itch-responsive neurons in the PrL significantly disrupted itch-induced scratching. Interestingly, we found that increasing attentional bias toward a distracting stimulus could disturb itch processing. We also demonstrated the existence of a population of attention-related neurons in the PrL that drive attentional bias to regulate itch processing. Importantly, itch-responsive neurons and attention-related neurons significantly overlapped in the PrL and were mutually interchangeable in the regulation of itch processing at the cellular activity level. Our results revealed that the PrL regulates itch processing by controlling attentional bias.

Dysgranular retrosplenial cortex modulates histaminergic and nonhistaminergic itch processing.

Itch is an unpleasant sensation followed by an intense desire to scratch. Previous researches have advanced our understanding about the role of anterior cingulate cortex and prelimbic cortex in itch modulation, whereas little is known about the effects of retrosplenial cortex (RSC) during this process. Here we firstly confirmed that the neuronal activity of dysgranular RSC (RSCd) is significantly elevated during itch-scratching processing through c-Fos immunohistochemistry and fiber photometry recording. Then with designer receptors exclusively activated by designer drugs approaches, we found that pharmacogenetic inhibition of global RSCd neurons attenuated the number of scratching bouts as well as the cumulative duration of scratching bouts elicited by both 5-HT or compound 48/80 injection into rats' nape or cheek; selective inhibition of the pyramidal neurons in RSCd, or of the excitatory projections from caudal anterior cingulate cortex (cACC) to RSCd, demonstrated the similar effects of decreasing itch-related scratching induced by both 5-HT or compound 48/80. Pharmacogenetic intervention of the neuronal or circuitry activities did not affect rats' motor ability. This study presents direct evidence that pyramidal neurons in RSCd, and the excitatory projection from cACC to RSCd are critically involved in central regulation of both histaminergic and nonhistaminergic itch.

Itch-specific neurons in the ventrolateral orbital cortex selectively modulate the itch processing.

Itch is a cutaneous sensation that is critical in driving scratching behavior. The long-standing question of whether there are specific neurons for itch modulation inside the brain remains unanswered. Here, we report a subpopulation of itch-specific neurons in the ventrolateral orbital cortex (VLO) that is distinct from the pain-related neurons. Using a Tet-Off cellular labeling system, we showed that local inhibition or activation of these itch-specific neurons in the VLO significantly suppressed or enhanced itch-induced scratching, respectively, whereas the intervention did not significantly affect pain. Conversely, suppression or activation of pain-specific neurons in the VLO significantly affected pain but not itch. Moreover, fiber photometry and immunofluorescence verified that these itch- and pain-specific neurons are distinct in their functional activity and histological location. In addition, the downstream targets of itch- and pain-specific neurons were different. Together, the present study uncovers an important subpopulation of neurons in the VLO that specifically modulates itch processing.

Infralimbic cortex-medial striatum projections modulate the itch processing.

Itch is an unpleasant sensation that induces the desire to scratch. Except for a sketchy map focusing on neural mechanisms underlying itch processing being drawn at the peripheral and spinal level over the past decades, the brain mechanisms remain poorly understood. Several previous studies indicated that anterior cingulate cortex (ACC) and prelimbic cortex (PrL), two subregions of the medial prefrontal cortex (mPFC) play an important role in regulating itch processing. However, the knowledge about whether infralimbic cortex (IL), another subregion of mPFC, is involved in modulating itch processing remains unclear. Here, we showed that the activity of IL excitatory pyramidal neurons was significantly elevated during itch-related scratching, and pharmacogenetic inhibition of IL pyramidal neurons significantly impaired itch-related scratching. Moreover, IL-medial striatum (MS) projections were verified as a critical neural pathway for modulating itch processing. Therefore, the present study firstly presents the regulatory function of IL pyramidal neurons during itch processing and also reveals that IL-MS projections are involved in modulating the itch processing.

Differential roles of prelimbic and anterior cingulate cortical region in the modulation of histaminergic and non-histaminergic itch.

Itch is an unpleasant sensation that evokes a desire to scratch. Itch processing in the peripheral and spinal cord has been studied extensively, but the mechanism of itch in the central nervous system is still unclear. Anterior cingulate cortex (ACC) and prelimbic cortex (Prl), two subregions of the prefrontal cortex closely related to emotion and motivation, have been reported to be activated during itching in a series of functional imaging studies. However, the exact role of Prl and the differences between ACC and Prl in itch modulation remains unknown. To directly test the differential roles of ACC and Prl in itch processing, we chemogeneticlly inhibited the caudal ACC and Prl, respectively. We found that inhibition of caudal ACC reduced histaminergic but not non-histaminergic itch-induced scratching behaviors. In contrast, inhibition of Prl reduced both histaminergic and non-histaminergic itch-induced scratching behaviors. Our study provided direct evidence of Prl involvement in itch modulation and revealed the differential roles of caudal ACC and Prl in regulating histaminergic and non-histaminergic itch.

Optogenetic Neuronal Stimulation Promotes Functional Recovery After Spinal Cord Injury.

Although spinal cord injury (SCI) is the main cause of disability worldwide, there is still no definite and effective treatment method for this condition. Our previous clinical trials confirmed that the increased excitability of the motor cortex was related to the functional prognosis of patients with SCI. However, it remains unclear which cell types in the motor cortex lead to the later functional recovery. Herein, we applied optogenetic technology to selectively activate glutamate neurons in the primary motor cortex and explore whether activation of glutamate neurons in the primary motor cortex can promote functional recovery after SCI in rats and the preliminary neural mechanisms involved. Our results showed that the activation of glutamate neurons in the motor cortex could significantly improve the motor function scores in rats, effectively shorten the incubation period of motor evoked potentials and increase motor potentials' amplitude. In addition, hematoxylin-eosin staining and nerve fiber staining at the injured site showed that accurate activation of the primary motor cortex could effectively promote tissue recovery and neurofilament growth (GAP-43, NF) at the injured site of the spinal cord, while the content of some growth-related proteins (BDNF, NGF) at the injured site increased. These results suggested that selective activation of glutamate neurons in the primary motor cortex can promote functional recovery after SCI and may be of great significance for understanding the neural cell mechanism underlying functional recovery induced by motor cortex stimulation.

Prepulse inhibition and acoustic startle response in young healthy Chinese.

Prepulse inhibition (PPI) and habituation of the acoustic startle response (ASR) are considered to be effective neurobiological measures of sensorimotor gating and information processing. The deficit of PPI and habituation of ASR has been proposed to be candidate endophenotypes of schizophrenia spectrum disorders. However, there has been little information on PPI and ASR measures in Chinese. The present study aimed to provide more information about the characteristics of PPI and ASR in young healthy Chinese and investigate their sensitivity to experimental parameters and characteristics of population. In this study, we examined the PPI and habituation of ASR in 41 young healthy adults (21 males and 20 females), using an acoustic startle stimulus of 115 dB and a prepulse of 75 dB at a lead interval (LI) of 60 ms and 120 ms, respectively. The behavioral performance demonstrated that the PPI and habituation of ASR in all the young participants were robust. The significant difference was not observed in PPI and habituation between male and female. The block effect on PPI was significant; PPI reduces with increasing training. Latency facilitation was observed under prepulse conditions, with a significant effect of LI. Compared to previous studies in Caucasians, Chinese in this study shows a higher habituation and PPI. In conclusion, this research provides more data of behavioral characteristics of PPI and ASR in young healthy Chinese. Chinese in this study shows a higher habituation and PPI than Caucasians in previous studies.

Reevaluating the ability of cerebellum in associative motor learning.

It has been well established that the cerebellum and its associated circuitry constitute the essential neuronal system for both delay and trace classical eyeblink conditioning (DEC and TEC). However, whether the cerebellum is sufficient to independently modulate the DEC, and TEC with a shorter trace interval remained controversial. Here, we used direct optogenetic stimulation of mossy fibers in the middle cerebellar peduncle (MCP) as a conditioned stimulus (CS) replacement for the peripheral CS (eg, a tone CS or a light CS) paired with a periorbital shock unconditioned stimulus (US) to examine the ability of the cerebellum to learn the DEC and the TEC with various trace intervals. Moreover, neural inputs to the pontine nucleus (PN) were pharmacological blocked to limit the associative motor learning inside the cerebellum. We show that all rats quickly acquired the DEC, indicating that direct optogenetic stimulation of mossy fibers in the left MCP is a very effective and sufficient CS to establish DEC and to limit the motor learning process inside the cerebellum. However, only five out of seven rats acquired the TEC with a 150-ms trace interval, three out of nine rats acquired the TEC with a 350-ms trace interval, and none of the rats acquired the TEC with a 500-ms trace interval. Moreover, pharmacological blocking glutamatergic and GABAergic inputs to the PN from the extra-cerebellar and cerebellar regions has no significant effect on the DEC and TEC learning with the optogenetic CS. These results indicate that the cerebellum has the ability to independently support both the simple DEC, and the TEC with a trace interval of 150 or 350 ms, but not the TEC with a trace interval of 500 ms. The present results are of great importance in our understanding of the mechanisms and ability of the cerebellum in associative motor learning and memory.

Optogenetic inhibition of ventral hippocampal neurons alleviates associative motor learning dysfunction in a rodent model of schizophrenia.

Schizophrenia (SZ) is a serious and incurable mental disorder characterized by clinical manifestations of positive and negative symptoms and cognitive dysfunction. High-frequency deep brain stimulation (DBS) of the ventral hippocampus (VHP) has been recently applied as a therapeutic approach for SZ in both experimental and clinical studies. However, little is known about the precise mechanism of VHP-DBS treatment for SZ and the role of hippocampal cell activation in the pathogenesis of SZ. With optogenetic technology in this study, we tried to inhibit neuronal activity in the VHP which has dense projections to the prefrontal cortex, before measuring long stumulus-induced delay eyeblink conditioning (long-dEBC) in a rodent model of SZ. Rats were administrated with phencyclidine (PCP, 3 mg/kg, 1/d, ip) for successive 7 days before optogenetic intervention. The current data show that PCP administration causes significant impairment in the acquisition and timing of long-dEBC; the inhibition of bilateral VHP neurons alleviates the decreased acquisition and impaired timing of longd-dEBC in PCP-administered rats. The results provide direct evidence at the cellular level that the inhibition of VHP neuronal cells may be a prominent effect of hippocampal DBS intervention, and increased activity in the hippocampal network play a pivotal role in SZ.

Modulation of eyeblink conditioning through sensory processing of conditioned stimulus by cortical and subcortical regions.

Classical eyeblink conditioning (EBC) is one of the simplest forms of associative learning that depends critically on the cerebellum. Using delay EBC (dEBC), a standard paradigm in which the unconditioned stimulus (US) is delayed and co-terminates with the conditioned stimulus (CS), converging lines of evidence has been accumulated and shows that the essential neural circuit mediating EBC resides in the cerebellum and brainstem. In addition to this essential circuit, multiple cerebral cortical and subcortical structures are required to modulate dEBC with suboptimal training parameters, and trace EBC (tEBC) in which a trace-interval separates the CS and US. However, it remains largely unclear why and how so many brain regions are involved for modulation of EBC. Previous research has suggested that the forebrain regions, such as medial prefrontal cortex (mPFC) and hippocampus, may be required to process weak CSs, or to realize temporal overlap between the CS and US signal inputs when the two stimuli were separated in time (i.e. during tEBC). Here, we proposed a multi-level network model for EBC modulation which focuses on sensory processing of CS. The model explains how different neural pathways projecting to pontine nucleus (PN) are involved to amplify or extend CS through heterosynaptic facilitation mechanism or "substitution effect" under different circumstances to achieve EBC. As such, our model can serve as a general framework to explain the modulating mechanism of EBC in a variety of conditions and to help understand the interaction among cerebellum, brainstem, cortical and subcortical regions in EBC modulation.

Absence of associative motor learning and impaired time perception in a rare case of complete cerebellar agenesis.

Primary cerebellar agenesis (PCA), a brain disease where the cerebellum does not develop, is an extremely rare congenital disease with only eleven living cases reported thus far. Studies of the PCA case will thus provide valuable insights into the necessity of cerebellar development for controlling and modulating cognitive functions of the brain. In this follow-up study, we further investigated the performance of associative learning and time perception of a 26-year-old female complete PCA case. We assessed whether delayed eyeblink conditioning (EBC), which represents prototypical associative motor learning function of the cerebellum, could be partially compensated by the extracerebellar brain regions in complete absence of the cerebellum. We also assessed whether the cerebellum, a critical brain region for millisecond-range interval timing, is essential for perception of the second-range time interval. Twelve neurotypical age-matched individuals were used as controls. We found that although the complete PCA patient had only mild to moderate motor deficits, she was unable to perform the delayed EBC even after 1-week of extensive training. Additionally, the PCA patient also performed poorly during time reproduction experiments in which she overproduced the millisecond-range time intervals, while underproduced the second-range time intervals. The PCA patient also failed to perform the temporal eyeblink conditioning with a 5 s fixed interval as the conditioned stimulus. These results indicate that the cerebellum is indispensable for associative motor learning and involved in timing of sub-second intervals, as well as in the perception of second-range intervals.

Time-limited involvement of caudal anterior cingulate cortex in trace eyeblink conditioning retrieval is dependent on conditioned stimulus intensity.

The medial prefrontal cortex (mPFC) has been widely investigated for its roles in learning and memory. The present study investigated the time-limited involvement of the caudal anterior cingulate cortex (cACC) of the mPFC in the retrieval process for a simple associative motor learning, trace eyeblink conditioning (tEBC), using a 75 dB or 100 dB tone as the conditioned stimulus (CS). The GABAA receptor agonist muscimol was injected into the cACC of guinea pigs at 1 day or 4 weeks after tEBC acquisition. When muscimol was administered 1 day after tEBC acquisition, the conditioned response (CR) of the 75 dB group was severely impaired, whereas the CR of the 100 dB group exhibited no significant change relative to the control. When muscimol was administered 4 weeks after tEBC acquisition, the CR was impaired in both the 75 dB and 100 dB groups. This study indicate that the cACC of the mPFC is necessary for recent retrieval of tEBC with a low-intensity CS but not of tEBC with a high-intensity CS, whereas for remote retrieval of tEBC, the cACC of the mPFC is essential regardless of whether the CS intensity is high or low. These results support a conditional role for the mPFC in modulating recent retrieval of tEBC and a persistent role for its involvement in remote retrieval of tEBC.

Medial Prefrontal Cortex-Pontine Nuclei Projections Modulate Suboptimal Cue-Induced Associative Motor Learning.

Diverse and powerful mechanisms have evolved to enable organisms to modulate learning and memory under a variety of survival conditions. Cumulative evidence has shown that the prefrontal cortex (PFC) is closely involved in many higher-order cognitive functions. However, when and how the medial PFC (mPFC) modulates associative motor learning remains largely unknown. Here, we show that delay eyeblink conditioning (DEC) with the weak conditioned stimulus (wCS) but not the strong CS (sCS) elicited a significant increase in the levels of c-Fos expression in caudal mPFC. Both optogenetic inhibition and activation of the bilateral caudal mPFC, or its axon terminals at the pontine nucleus (PN) contralateral to the training eye, significantly impaired the acquisition, recent and remote retrieval of DEC with the wCS but not the sCS. However, direct optogenetic activation of the contralateral PN had no significant effect on the acquisition, recent and remote retrieval of DEC. These results are of great importance in understanding the elusive role of the mPFC and its projection to PN in subserving the associative motor learning under suboptimal learning cue.

Optogenetic Inhibition of Medial Prefrontal Cortex-Pontine Nuclei Projections During the Stimulus-free Trace Interval Impairs Temporal Associative Motor Learning.

The medial prefrontal cortex (mPFC) is closely involved in many higher-order cognitive functions, including learning to associate temporally discontiguous events (called temporal associative learning). However, direct evidence for the role of mPFC and the neural pathway underlying modulation of temporal associative motor learning is sparse. Here, we show that optogenetic inhibition of the mPFC or its axon terminals at the pontine nuclei (PN) during trace intervals or whole trial period significantly impaired the trace eyeblink conditioning (TEC), but had no significant effects on TEC during the conditioned stimulus or intertrial interval period. Our results suggest that activities associated with the mPFC-PN projection during trace intervals is crucial for trace associative motor learning. This finding is of great importance in understanding the mechanisms and the relevant neural pathways underlying mPFC modulation of temporal associative motor learning.

Establishment and transfer of classical eyeblink conditioning using electrical microstimulation of the hippocampus as the conditioned stimulus.

The present experiment was designed to determine whether classical eyeblink conditioning (EBC) can be established by using electrical microstimulation of the hippocampus as a conditioned stimulus (CS) paired with an air-puff unconditioned stimulus (US). We intended to examine whether EBC transfer could occur when a CS was shifted between microstimulation of the hippocampus as a CS (Hip-CS) and tone as a CS (tone-CS) and to compare the difference in transfer effectiveness between delay EBC (dEBC) and trace EBC (tEBC). Eight groups of guinea pigs, including 4 experimental groups and 4 control groups, were included in the study. First, the experimental groups received either a Hip-CS or a tone-CS paired with a US; then, these groups were exposed to a shifted CS (tone-CS or Hip-CS) paired with the US. The control groups received the corresponding Hip-CS or tone-CS, which was, however, pseudo-paired with the US. The control groups were then shifted to the tone-CS (or Hip-CS) paired with the US. The results show that EBC can be successfully established when using microstimulation of the hippocampus as a CS paired with an air-puff US, and that the acquisition rates of EBC are higher in the experimental groups than in the control groups after switching from the Hip-CS to the tone-CS or vice versa, indicating the occurrence of learning transfer between EBC established with the Hip-CS and tone-CS. The present study also demonstrated that the EBC re-acquisition rates were remarkably higher in dEBC than in tEBC with both types of transfer, which suggests that the saving effect was more evident in dEBC than tEBC. These results significantly expand our knowledge of EBC transfer as well as the functional neural circuit underlying EBC transfer.

Prolonged deficits of associative motor learning in cynomolgus monkeys after long-term administration of phencyclidine.

Phencyclidine (PCP) is a potent drug of abuse that induces sustained schizophrenia-like symptoms in humans by blocking neurotransmission at N-methyl-d-aspartate (NMDA)-type glutamate receptors. Alterations in NMDA receptor function have been linked to numerous behavioral deficits and cognitive dysfunction. Classical eye-blink conditioning (EBC), including delay (dEBC) and trace (tEBC) paradigms, provides an effective means to study the neurobiology of associative motor learning in rodents, mammals and primates. To assess whether administration of low-dosage PCP for extended periods has prolonged effect to alter associative motor learning, in this study 19 adult cynomolgus monkeys were administered PCP (0.3mg/kg, intramuscularly) or saline twice a day for 14days. Twelve-fifteen months after PCP or saline injection, monkeys received dEBC, tEBC, or pseudo-paired training for 6 or 12 successive daily sessions, respectively. The results of this study show that percentage of conditioned response (CR) in dEBC increased as a function of training sessions in both PCP-treated and control monkeys and there was no significant CR% difference between the two groups. However, the CR timing in dEBC of PCP-treated monkeys was significantly impaired, as manifested by shorter CR peak latencies than those of the control group. PCP-treated animals showed significantly lower percentage of CR in tEBC compared to controls. PCP-treated animals were also more sensitive to outside stimuli in tEBC because the UR peak latency of PCP-treated group was significantly lower than the control group. These results indicated that cynomolgus monkeys manifested prolonged deficits in associative motor learning after long-term administration of phencyclidine.

Theta synchronization between medial prefrontal cortex and cerebellum is associated with adaptive performance of associative learning behavior.

Associative learning is thought to require coordinated activities among distributed brain regions. For example, to direct behavior appropriately, the medial prefrontal cortex (mPFC) must encode and maintain sensory information and then interact with the cerebellum during trace eyeblink conditioning (TEBC), a commonly-used associative learning model. However, the mechanisms by which these two distant areas interact remain elusive. By simultaneously recording local field potential (LFP) signals from the mPFC and the cerebellum in guinea pigs undergoing TEBC, we found that theta-frequency (5.0-12.0 Hz) oscillations in the mPFC and the cerebellum became strongly synchronized following presentation of auditory conditioned stimulus. Intriguingly, the conditioned eyeblink response (CR) with adaptive timing occurred preferentially in the trials where mPFC-cerebellum theta coherence was stronger. Moreover, both the mPFC-cerebellum theta coherence and the adaptive CR performance were impaired after the disruption of endogenous orexins in the cerebellum. Finally, association of the mPFC -cerebellum theta coherence with adaptive CR performance was time-limited occurring in the early stage of associative learning. These findings suggest that the mPFC and the cerebellum may act together to contribute to the adaptive performance of associative learning behavior by means of theta synchronization.

Optogenetic stimulation of mPFC pyramidal neurons as a conditioned stimulus supports associative learning in rats.

It is generally accepted that the associative learning occurs when a behaviorally neutral conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (US) in close temporal proximity. Eyeblink conditioning (EBC) is a simple form of associative learning for motor responses. Specific activation of a population of cells may be an effective and sufficient CS for establishing EBC. However, there has been no direct evidence to support this hypothesis. Here, we show in rats that optogenetic activation of the right caudal mPFC pyramidal neurons as a CS is sufficient to support the acquisition of delay eyeblink conditioning (DEC). Interestingly, the associative memory was not stably expressed during the initial period of daily conditioning session even after the CR acquisition reached the asymptotic level. Finally, the intensity and consistency of the CS were found to be crucial factors in regulating the retrieval of the associative memory. These results may be of importance in understanding the neural cellular mechanisms underlying associative learning and the mechanisms underlying retrieval process of memory.