Effects Of Short-term Prolonged Wakefulness On The Medial Prefrontal Cortical Activity And Behavior Of Rats

Prolonged wakefulness is commonplace in modern society. The homeostatic pressurefor sleep, which accumulates with longer durations of wakefulness, often fights against theindividual's motivation to remain awake, and leads to deficits in cognition as well asinstability of wake state. Prefrontal cortex (PFC), a critical area for cognitive function, isparticularly sensitive to sleep loss. Therefore, understanding the changes in PFC activityafter short-term sleep deprivation will be helpful to counteracting the impairment inperformance during prolonged wakefulness.Growing evidence has indicated that prolonged wakefulness leads to an increase incortical excitability. This alteration has been attributed to experience-dependentpotentiation in excitatory synaptic activity. Nevertheless, the propensity of a neuron to fireaction potentials when exposed to an input signal, i.e. the intrinsic excitability of a neuron,is also an important determinant of the whole cortical excitability. Previous studies in ratshave revealed that72h of paradoxical sleep deprivation results in a decrease in the intrinsicexcitability of hippocampal neurons. On the other hand, it has been found that the intrinsicexcitability of a neuron also undergoes experience-dependent modification as the synapticplasticity. However, little is known about what happens to the intrinsic excitability of PFCneurons after short-term sleep deprivation.PFC receives afferents from many components of the ascending arousal system,including lateral hypothalamus (orexin) and dorsal raphe nucleus (5-HT). Theseneurotransmitters are not only critical for maintaining the excitability of PFC, but also formodulating the network function. A deficit in the response of PFC neurons to orexin and5-HT may weaken the regulation by arousal system during wakefulness. It has beenreported that restraint stress produces a decrement in both orexin-and5-HT-inducedexcitatory postsynaptic currents (EPSCs) in neurons from rat medial prefrontal cortex (mPFC). Moreover, after2h of sleep deprivation, photostimulation of orexin neurons failsto increase the neural activity in locus ceruleus and tuberomammilary nucleus, and thus theorexin-mediated sleep-to-wake transitions are diminished. However, it is unclear whetherthe effects of orexin and5-HT on PFC neurons will change during prolonged wakefulness.In the present study, to assess the neural activity in rat mPFC after4h of sleepdeprivation (SD), we examined the expression of c-Fos, a biomarker of active neurons.Subsequently, we investigated the effects of4h of SD on the electrophysiologicalproperties of layer5mPFC pyramidal neurons by whole-cell patch-clamp recordings,observing their EPSCs, intrinsic excitability, and responses to orexin-A and5-HT. Then, weexplored the behavior of rats following4and8h of SD, in order to reveal the relationbetween behavioral alteration and changes in neural activity. The main results were asfollows:1. The c-Fos expression in mPFC was increased after4-h SDThe c-Fos-positive cells in the mPFC of rats exposed to SD (286.85±11.18/mm~2, n=5rats) were significantly more than in controls allowed to sleep (221.86±16.64/mm~2, n=5rats,p<0.05), suggesting an increase in neural activity.2. The excitatory synaptic activity of mPFC neurons was potentiated after4-h SDIn the presence of100μM picrotoxin, an antagonist of GABAAreceptor, we recordedthe EPSCs in mPFC neurons. The amplitude and frequency of EPSCs were notably higherin rats kept awake for4h than in control rats (SD:13.98±0.14pA,1.86±0.19Hz, n=9cellsfrom5rats, control:11.28±0.13pA,0.98±0.09Hz, n=9cells from5rats, p<0.01),suggesting a potentiation in excitatory synaptic efficacy.3. The intrinsic excitability of mPFC neurons was enhanced after4-h SDWe compared the resting membrane potentials and input resistances in mPFC neuronsfrom control rats (n=22cells from12rats), SD rats (n=23cells from11rats) and ratsobtaining recovery sleep (RS) after SD (n=20cells from10rats), finding no significantdifference in these parameters (p>0.05). Moreover, no significant differences were observedacross groups in the spike threshold, rheobase and amplitude of action potential (p>0.05).Nevertheless, the number of action potential that could be elicited by1-s depolarizingcurrent steps (from+80pA to+300pA) were markedly higher in SD neurons (n=18) ascompared to control and RS neurons (n=18and18, respectively, p<0.05). Subsequently, we assessed the input-output relation for each neuron by determining the initial slope offrequency-current curve. The initial slope in SD neurons (131.84±4.16) was significantlyincreased compared with in control and RS neurons (99.89±4.15and102.91±5.40,respectively, p<0.01). In addition, CNQX (10μM) and APV (50μM), antagonists forglutamate receptors, were applied to slices to eliminate the effect of background excitatorysynaptic activity on input-output relation. In this condition, a higher initial slope was stillobserved in SD neurons (control:91.25±9.03, n=6, SD:120.35±6.38, n=7, RS:93.71±10.37,n=6,p<0.05).To quantified frequency adaptation, the ratios of the first interspike interval (ISI) to the10th ISI (ISIfirst/ISI10th) and to the last ISI (ISIfirst/ISIlast) were determined during repetitivespiking evoked by a+200pA current step. The ISIfirst/ISI10thand ISIfirst/ISIlastwere bothelevated in SD neurons (0.40±0.04and0.35±0.04, respectively, n=18) compared with incontrol neurons (0.29±0.03and0.26±0.03, n=18) and RS neurons (0.29±0.03and0.24±0.02, n=18,p<0.05), indicating a decrease in frequency adaptation following SD. Theabove results together suggested that the intrinsic excitability in mPFC neurons wasincreased after4h of SD, and this alteration could be counteracted by a period of sleep.4. A reduction in post-burst afterhyperpolarization (AHP) might be responsiblefor the increased intrinsic excitabilityThe peak amplitude and integrated area of post-burst AHP were both significantlylower in SD neurons (3.05±0.23mV and1708.66±330.9mV·ms, n=22) than in controlneurons (6.07±0.26mV and3085.02±290.02mV·ms, n=19) and RS neurons (5.61±0.26mV,3212.51±515.45mV·ms, n=15, p<0.01). Consistently, decreases in the peak amplitudeand integrated area of Ca2+-dependent AHP currents were also observed in SD neurons(12.84±3.24pA and9685.89±1983.34pA·ms, n=7) as compared to control neurons(50.95±14.79pA and41200.98±12429.05pA·ms, n=6) and RS neurons (52.86±10.78pAand40990.15±7786.88pA·ms, n=6, p<0.05). These observations suggested a reduction inthe post-burst AHP in mPFC neurons after4h of SD. This alteration was in line with thechange in intrinsic excitability. Additionally, in the presence of200μM CdCl2, whichinhibited post-burst AHP and eliminated the difference in post-burst AHP across groups(control: n=11, SD: n=12, RS: n=8, p>0.05), the initial slope of frequency-current curve inSD neurons (n=7) was similar to those in control neurons (n=7) and RS neurons (n=8, p>0.05). Thus, the alteration in post-burst AHP seemed to contribute to the SD-inducedincrease in intrinsic excitability.We also assessed the fast AHP (fAHP) following single action potential, but found nosignificant difference across groups (control: n=16, SD: n=18, RS: n=15, p>0.05).Moreover, to examine the property of hyperpolarization-activated cation (HCN) channels,we measured the voltage sag evoked by a hyperpolarizing current pulse (-200pA), and theHCN currents elicited by a serious of hyperpolarizing voltage steps (from-65to-125mV).Neither the sag ratio (control: n=13, SD: n=10, RS: n=8, p>0.05) nor HCN currents (control:n=4, SD: n=5, RS: n=6, p>0.05) differed across groups. These results suggested that thefAHP or HCN currents in mPFC neurons were not affected by4h of SD.5. The effects of4-h SD on the responses of mPFC neurons to orexin-A and5-HTIn the presence of0.5μM TTX, which blocks synaptic transmission, application of400μM orexin-A caused a depolarization in the layer5mPFC neurons from both controlrats (n=5rats) and SD rats (n=5rats). There was no significant difference in theresponsivity to orexin-A between control neurons (6/16) and SD neurons (5/14, p>0.05).The amplitude of orexin-A-caused depolarization was also similar between the two groups(control:8.25±0.39mV, SD:7.60±1.71mV, p>0.05). Moreover, in both control and SDneurons, orexin-A and5-HT could increase the amplitude of EPSCs (p<0.01), and decreasethe interevent interval of EPSCs (p<0.01). The orexin-A-or5-HT-induced changes in thefrequency of EPSCs did not differ between control and SD groups (orexin-A: control147.25±8.05%, n=5, SD138.10±6.16%, n=5, p>0.05;5-HT: control154.34±12.09%, n=6,SD136.06±8.29%, n=5, p>0.05). The above results suggested that the responses toorexin-A or5-HT observed here did not change after4h of SD.In addition, we also observed a depolarizing effect of orexin-A on layer2/3mPFCneurons in normal rats. The depolarization induced by orexin-A persisted in the presence of0.5μM TTX or Ca2+-free ACSF, but was inhibited by1μM SB-334867, a selective OX1Rantagonist (16.11±4.31%of control, n=5,p<0.01). Moreover,[Ala11,D-Leu15]-orexin-B(400nM and1μM), a selective OX2R agonist, did not cause a depolarization in neurons(n=4), which showed response to orexin-A. These results suggested that orexin-A excitedlayer2/3mPFC neurons in a postsynaptic manner via OX1R. 6. The effects of short-term SD on rat behaviorAfter4-h SD, a significant decrease of the average of sleep latency trials was observedcomparing the baseline day values (baseline:721.21±81.67s, SD:489.77±73.45s, n=8,p<0.05), suggesting an increase in sleepiness. In the T-maze delayed alternation task, theperformance of rats was maintained at the baseline level after4h of SD (n=8, p>0.0.5).However, after prolonged wakefulness for8h, the accuracy of the task on30s delay wasnotably reduced compared with the baseline level (baseline:75.00±3.27%, SD:61.25±2.95%, n=8,p<0.01). Thus,4h of SD seemed not to affect the working memory ofrats, but if SD was extended to a longer time (e.g.8h), the working memory would beimpaired.In the open field test,4h of SD led to an increase in the number of grid crossing(control:81.88±14.75, n=8, SD:120.63±10.05, n=8, p<0.05). The numbers of rearing andvisiting center area, as well as the time spent in center area was slightly higher in the ratsexposed to4-h SD than control rats, but the differences were not significant (p>0.05). Thenumber of grooming was slightly lower in the rats exposed to4-h SD than controls, butthere was no significant difference between the two groups (p>0.05). The rats exposed to8-h SD showed tendencies of lower number of grooming, more time spent in center area, aswell as higher numbers of grid crossing, rearing and visiting center area compared withtheir controls, but no significant difference was found between groups (control: n=8, SD:n=8, p>0.05). These results might suggest that short-term SD could increase the exploratorymotivation of rats to a new environment. Moreover,4h of SD seemed to result in higherlocomotor activity, and this alternation would be eliminated by longer wakefulness.In summery, the observations here suggested an enhanced neural activity in mPFCfollowing4h of SD, due to an increase in both excitatory synaptic input and intrinsicexcitability. These alterations seemed to lead to a serious of changes in rat behavior,including sleepiness, working memory, and exploratory activity.