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.

The modulation of orexin A on HCN currents of pyramidal neurons in mouse prelimbic cortex.

The hyperpolarization-activated/cyclic nucleotide (HCN)-gated channels make important contributions to neural excitability. In prefrontal cortex, HCN channels are localized on the distal dendrites of layer V pyramidal neurons and decrease neural excitability when they are open. In the present study, using whole-cell voltage clamp recordings, the effect of an arousal peptide, orexin A, on HCN currents in layer V pyramidal neurons from mouse prelimbic cortex (PL), the homolog of the prefrontal cortex was investigated. The results demonstrated that orexin A suppressed HCN currents and shifted their activation curve to a more negative direction. This action of orexin A was blocked by SB334867, an orexin receptor 1 (OXR1) blocker and bisindolylmaleimide, a protein kinase C (PKC) inhibitor, indicating the involvement of OXR1 and PKC. The excitatory effect of orexin A on PL pyramidal neurons was enhanced when HCN currents were diminished, while attenuated when HCN currents were enlarged. In summary, orexin A inhibits HCN currents and enhances excitability of pyramidal neurons in PL, which may contribute to arousal and cognition.

Specific stimulation of PV+ neurons at early stage ameliorates prefrontal ischemia-induced spatial working memory impairment.

Prefrontal ischemia can cause impairments in learning and memory, executive functions and cognitive flexibility. However, the related cellular mechanisms at the early stage are still elusive. The present study used ischemic stroke in medial prefrontal cortex and systemically investigated the electrophysiological changes of the parvalbumin (PV+) interneurons 12 h post ischemia. We found that Ih and the related voltage sags in PV+ interneurons are downregulated post ischemia, which correlates with hyperpolarization of the membrane potentials and increased input resistance in these interneurons. Consistent with the suppression of Ih, postischemic PV+ interneurons exhibited a reduction in excitability and exerted a less inhibitory control over the neighboring pyramidal excitatory neurons. Moreover, we found that specifically chemogenetic activation of PV+ neurons at early stage ameliorated prefrontal ischemia-induced spatial working memory dysfunction in T-maze without effects on the locomotor coordination and balance. In contrast, suppression of PV+ neurons by blockade of Ih leaded to further aggravate the damage of spatial memory. These findings indicate that dysfunctional Ih in the PV+ neuron postischemia induces the imbalance of excitation and inhibition, which might represent a novel mechanism underlying the prefrontal ischemia-induced cognitive impairment.

[Study on arousal effect of Orexin-A in rat in coma due to ischemic brain injury].

OBJECTIVE: To reproduce an ischemic brain injury coma model and explore the arousal effect of Orexin-A. METHODS: An ischemic brain injury coma model was reproduced in rats by partial four-vessel occlusion (4-VO with a needle of 0.60 mm in diameter in the lumen to create stenosis of the internal carotid arteries). One hundred and twenty minutes after the onset of coma, Orexin-A or its antagonist (SB-334867) was given intraventricularly, and the time of coma and changes in electroencephalogram (ECG) were observed, and the unit discharge of neurons in the prefrontal cortex was recorded. RESULTS: Partial occlusion of four internal carotid arteries, reducing the lumens to 0.60 mm, could prolong the time of coma to 6-8 hours with the rats still alive. The duration of coma showed a significant difference compared with that in rats who underwent 0.45 mm or 0.70 mm stenosis of the internal carotid arteries (F=344.43, P<0.01). Intraventricular Orexin-A in a dose of 4 nmol/10 microl could obviously decrease the duration of coma with a decrease in alpha wave and increase in unit discharge rate of neurons in coma rats (P<0.05 or P<0.01), but no significant change was observed when the dose was 2 nmol/10 microl. CONCLUSION: (1)Creating stenosis of all four internal carotid arteries is suitable to reproduce ischemic brain injury with coma in rats. (2)Intracerebroventricular injection of Orexin-A (4 nmol) has a potent arousal effect on ischemic brain injury coma in rats.

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.

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.

Signaling pathways of hypocretin-1 actions on pyramidal neurons in the rat prefrontal cortex.

We have investigated the direct excitatory effects of hypocretin-1 on acutely isolated prefrontal cortical pyramidal neurons and explored the signaling mechanisms of these actions. Puff application of hypocretin-1 caused an excitation in the recorded neurons. These effects of hypocretin-1 were abolished by a phospholipase C inhibitor D609, demonstrating that phospholipase C mediates the actions of hypocretin-1. A specific protein kinase C inhibitor, bisindolylmaleimide II, blocked the excitatory actions of hypocretin-1, suggesting that protein kinase C plays a key role. Finally, protein kinase A inhibitor applied intracellularly did not affect the responses. These results indicate that hypocretin-1 excites prefrontal neurons by activation of phospholipase C and protein kinase C pathways, but not protein kinase A.

Progesterone alleviates neural behavioral deficits and demyelination with reduced degeneration of oligodendroglial cells in cuprizone-induced mice.

Demyelination occurs widely in neurodegenerative diseases. Progesterone has neuroprotective effects, is known to reduce the clinical scores and the inflammatory response. Progesterone also promotes remyelination in experimental autoimmune encephalomyelitis and cuprizone-induced demyelinating brain. However, it still remains unclear whether progesterone can alleviate neural behavioral deficits and demyelination with degeneration of oligodendroglial cells in cuprizone-induced mice. In this study, mice were fed with 0.2% cuprizone to induce demyelination, and treated with progesterone to test its potential protective effect on neural behavioral deficits, demyelination and degeneration of oligodendroglial cells. Our results showed noticeable alleviation of neural behavioral deficits following progesterone treatment as assessed by changes in average body weight, and activity during the open field and Rota-rod tests when compared with the vehicle treated cuprizone group. Progesterone treatment alleviated demyelination as shown by Luxol fast blue staining, MBP immunohistochemical staining, and electron microscopy. There was an obvious decrease in TUNEL and Caspase-3-positive apoptotic cells, and an increase in the number of oligodendroglial cells staining positive for PDGFRα, Olig2, Sox10 and CC-1 antibody in the brains of cuprizone-induced mice after progesterone administration. These results indicate that progesterone can alleviate neural behavioral deficits and demyelination against oligodendroglial cell degeneration in cuprizone-induced mice.

Developmental changes and subcellular location in inhibitor of DNA binding 2 (Id2) immunoreactivity in the rat Corpus callosum.

The mechanisms underlying oligodendrocyte differentiation and myelination are still unclear, but understanding them will be critical for the development of therapies for multiple sclerosis. Inhibitor of DNA binding 2 (Id2) is a transcription factor thought to inhibit oligodendrocyte differentiation, however, it is not known whether the developmental changes and subcellular localization of Id2 are related to myelination. Therefore, we investigated the developmental changes in and the subcellular localization of Id2 immunoreactivity in the rat Corpus callosum, at post-natal developmental stages P0, P7, P14, P21, P42 and P90, by immunohistochemistry. Id2 expression increased from P0 to a peak at P42, the late stage of myelination in the Corpus callosum. Id2 immunostaining decreased slightly, but still remained high at P90. Subcellular localization of Id2 changed from presence in cytoplasm at P14 to the nuclei at P42. Moreover, Id2 was mainly co-localized with CC-1-immunopositive mature oligodendrocytes at P42. These results may be consistent with Id2 inhibitory function in oligodendrocyte differentiation, at the end of myelination or in compaction of myelin in the Corpus callosum of postnatal rat brain.

Arousal effects of orexin A on acute alcohol intoxication-induced coma in rats.

The key role of the hypothalamic neuropeptides orexins in maintenance and promotion of arousal has been well established in normal mammalian animals, but whether orexins exert arousal effects under pathological condition such as coma was little studied. In this study, a model of unconscious rats induced by acute alcohol intoxication was used to examine the effects of orexins through intracerebroventricular injection. The results revealed that either orexin A or orexin B induced decrease of duration of loss of right reflex in alcohol-induced unconscious rats. In the presence of the selective orexin receptor 1 antagonist SB 334867 and orexin receptor 2 antagonist TCS OX2 29, the excitatory action of orexin A was completely blocked. Our data further presented that orexin A also induced reduction of delta power in EEG in these rats. Single-unit recording experiment in vivo demonstrated that orexin A could evoke increase of firing activity of prefrontal cortex neurons in unconscious rats. This excitation was completely inhibited by an H(1) receptor antagonist, pyrilamine, whereas application of α(1)-adrenoreceptor antagonist prazosin or 5-HT(2) selective receptor antagonist ritanserin partially attenuated the excitatory effects of orexin A on these neurons. Consistently, the results of EEG recordings showed that microinjection of pyrilamine, prazosin, or ritanserin suppressed reduction of delta power in EEG induced by orexin A on unconscious rats. Thus, these data suggest that orexins exert arousal effects on alcohol-induced unconscious rats by the promotion of cortical activity through activation of histaminergic, noradrenergic and serotonergic systems. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

[Effect of orexin-A on recovery from ketamine anesthesia in aged rats].

OBJECTIVE: To observe the effect of orexin-A on the recovery and cognitive function of aged rats after ketamine anesthesia. METHODS: Fifty-five aged rats were divided randomly into control group, model control group, 1 nmol/L Orexin-A group, and 4 nmol/L Orexin-A group. In the latter 3 groups, the rats received an intraperitoneal injection of ketamine at 100 mg/kg, and normal saline was injected in the control group. Ten minutes after the injections, the rats received intraventricular injections of artificial cerebrospinal fluid (control and model control group) or of 10 microl 1 or 4 nmol/L Orexin-A as indicated. The behavioral changes of the rats were assessed by the duration of loss of righting reflex (LORR). Electroencephalogram (EEG) recordings were used to evaluate the changes in rat brain activity by comparison of the percent of sigma wave in EEG before and after the intraventricular injections. Morris water maze was used to test the learning and spatial localization abilities of the rats. RESULTS: Ketamine resulted in obvious impairment of learning and memory abilities of the aged rats. Orexin-A at 4 nmol/L induced significant decrease in the duration of LORR and marked reduction of sigma activities in anesthetic rats (P<0.05), and obviously improved the learning and spatial localization abilities of the rats after anesthesia (P<0.05). CONCLUSION: Orexin-A can promote the recovery and improve the cognitive function of aged rats after ketamine anesthesia.

Postsynaptic excitation of prefrontal cortical pyramidal neurons by hypocretin-1/orexin A through the inhibition of potassium currents.

Hypocretins are crucial for the regulation of wakefulness by the excitatory actions on multiple subcortical arousal systems. To date, there is little information about the direct postsynaptic excitatory effects of hypocretins on the neurons in prefrontal cortex (PFC), which is important for higher cognitive functions and is correlated with level of wakefulness. In this study, we tested the excitatory effects of hypocretin-1 on acutely isolated PFC pyramidal neurons of rats and studied the possible ionic mechanisms by using whole-cell patch-clamp techniques. Puff application of hypocretin-1 caused a dose-dependent excitation. Further observations that perfusion of Ca2+-free artificial cerebrospinal fluid did not influence the depolarizing effects of hypocretin-1, in conjunction with the findings that hypocretin-1 could decrease net whole-cell K+ currents, demonstrate that the excitatory effects of hypocretin-1 on PFC neurons are mediated by the inhibition of K+ currents but not Ca2+ influx. Finally, the decrease in K+ currents induced by hypocretin-1 was abolished by a protein kinase C (PKC) inhibitor (BIS II) or a phospholipase C (PLC) inhibitor (D609), suggesting that PKC and PLC appear to be involved in mediating the inhibitory effects of hypocretin-1 on K+ currents. These results indicate that hypocretin-1 exerts a postsynaptic excitatory action on PFC neurons through the inhibition of K+ currents, which probably results from activation of PKC and PLC signaling pathways.