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.

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.

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.

Prefrontal control of cerebellum-dependent associative motor learning.

Behavioral studies have demonstrated that both medial prefrontal cortex (mPFC) and cerebellum play critical roles in trace eyeblink conditioning. However, little is known regarding the mechanism by which the two brain regions interact. By use of electrical stimulation of the caudal mPFC as a conditioned stimulus, we show evidence that persistent outputs from the mPFC to cerebellum are necessary and sufficient for the acquisition and expression of a trace conditioned response (CR)-like response. Specifically, the persistent outputs of caudal mPFC are relayed to the cerebellum via the rostral part of lateral pontine nuclei. Moreover, interfering with persistent activity by blockade of the muscarinic Ach receptor in the caudal mPFC impairs the expression of learned trace CRs. These results suggest an important way for the caudal mPFC to interact with the cerebellum during associative motor learning.

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.

Reevaluating the role of the medial prefrontal cortex in delay eyeblink conditioning.

It has been proposed that the medial prefrontal cortex (mPFC) is not necessary for delay eyeblink conditioning (DEC). Here, we investigated the involvement of the mPFC in DEC with a soft or loud tone as the conditioned stimulus (CS) by using electrolytic lesions or muscimol inactivation of guinea pig mPFC. Interestingly, when a soft tone was used as a CS, electrolytic lesions of the mPFC significantly retarded acquisition of the conditioned response (CR), and muscimol infusions into mPFC distinctly inhibited the acquisition and expression of CR, but had no significant effect on consolidation of well-learned CR. In contrast, both electrolytic lesions and muscimol inactivation of mPFC produced no significant deficits in the CR when a loud tone was used as the CS, or in the unconditioned response (UR) when a soft or loud tone was used as the CS. These results demonstrate that the mPFC is essential for the DEC with the soft tone CS but not for the DEC with the loud tone CS.

Classical eyeblink conditioning using electrical stimulation of caudal mPFC as conditioned stimulus is dependent on cerebellar interpositus nucleus in guinea pigs.

AIM: To determine whether electrical stimulation of caudal medial prefrontal cortex (mPFC) as conditioned stimulus (CS) paired with airpuff unconditioned stimulus (US) was sufficient for establishing eyeblink conditioning in guinea pigs, and whether it was dependent on cerebellar interpositus nucleus. METHODS: Thirty adult guinea pigs were divided into 3 conditioned groups, and trained on the delay eyeblink conditioning, short-trace eyeblink conditioning, and long-trace eyeblink conditioning paradigms, respectively, in which electrical stimulation of the right caudal mPFC was used as CS and paired with corneal airpuff US. A pseudo conditioned group of another 10 adult guinea pigs was given unpaired caudal mPFC electrical stimulation and the US. Muscimol (1 µg in 1 µL saline) and saline (1 µL) were infused into the cerebellar interpositus nucleus of the animals through the infusion cannula on d 11 and 12, respectively. RESULTS: The 3 eyeblink conditioning paradigms have been successfully established in guinea pigs. The animals acquired the delay and short-trace conditioned responses more rapidly than long-trace conditioned responses. Muscimol infusion into the cerebellar interpositus nucleus markedly impaired the expression of the 3 eyeblink conditioned responses. CONCLUSION: Electrical stimulation of caudal mPFC is effective CS for establishing eyeblink conditioning in guinea pigs, and it is dependent on the cerebellar interpositus nucleus.

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.

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.

Detection of CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood of patients with chronic autoimmune urticaria.

BACKGROUND/OBJECTIVES: Compelling evidence indicates a significant role for a population of CD4(+) T regulatory cells in suppressing immune responses and in maintaining immunological homeostasis. This study aims to investigate the potential role of CD4(+) CD25(HIGH) FOXP3(+) T regulatory cells in patients with chronic autoimmune urticaria and to define the characteristics of CD4(+) CD25(HIGH) FOXP3(+) cells in chronic urticaria. METHODS: We used flow cytometry to assess the expression of CD4(+) CD25(HIGH) FOXP3(+) cells in the peripheral blood mononuclear cells of patients with chronic autoimmune urticaria. RESULTS: In this study, we found that patients with chronic autoimmune urticaria have a significantly reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.39 ± 0.27% vs 2.09 ± 0.34%; P = 0.001) in their peripheral blood, accompanied by a decreased intensity of FOXP3 expression (50.13 ± 9.79 vs 68.19 ± 6.40; P < 0.001). Notably, although patients with chronic idiopathic urticaria had a reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.85 ± 0.46% vs 3.64 ± 0.48%; P < 0.001), their FOXP3 expression levels did not differ from those in healthy controls. CONCLUSIONS: Patients with chronic autoimmune urticaria displayed a reduced percentage of CD4(+) CD25(+) FOXP3(+) regulatory T cells. The results imply CD4(+) CD25(+) FOXP3(+) regulatory T cells may contribute to the autoimmune pathological process of chronic autoimmune urticaria.

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.

Involvement of the ipsilateral and contralateral cerebellum in the acquisition of unilateral classical eyeblink conditioning in guinea pigs.

AIM: The aim of this study was to evaluate the relative contributions of the ipsilateral and contralateral cerebellum to the acquisition of unilateral classical eyeblink conditioning (EBCC). METHODS: The unilateral EBCC was achieved using a binaural tone conditioned stimulus (CS) paired with a left airpuff unconditioned stimulus (US). A high-resolution potentiometer was used to monitor eyeblink responses. Guinea pigs received one CS-US session followed by three CS-US sessions (sessions 2 to 4), during which microinjections of muscimol, a GABA(A) receptor agonist, were performed to reversibly inactivate the cerebellum unilaterally prior to training. To test whether any learning had occurred during these inactivation sessions, training was continued for six more CS-US sessions (sessions 5 to 10) without any inactivation. RESULTS: Animals with inactivation of the left cerebellum had no signs of left conditioned response (CR) during sessions 2 to 4, and their CR acquisition during sessions 5 to 10 was not distinguishable from that of control animals during sessions 2 to 7. In contrast, animals with inactivation of the right cerebellum acquired left CRs during sessions 2 to 4, although their CR acquisition was significantly retarded during session 2. In addition, microinjections of muscimol into the right cerebellum did not affect left neuro-behavioral activity. Finally, microinjections of muscimol into either the left or the right cerebellum did not affect the performance of tone-airpuff evoked unconditioned response (UR). CONCLUSION: In contrast to the essential role of the ipsilateral cerebellum, the contralateral cerebellum is potentially involved in the acquisition of unilateral EBCC during the early stage of training.

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.

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.

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.

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.