Compensatory cognition in neurological diseases and aging: A review of animal and human studies.

Specialized individual circuits in the brain are recruited for specific functions. Interestingly, multiple neural circuitries continuously compete with each other to acquire the specialized function. However, the dominant among them compete and become the central neural network for that particular function. For example, the hippocampal principal neural circuitries are the dominant networks among many which are involved in learning processes. But, in the event of damage to the principal circuitry, many times, less dominant networks compensate for the primary network. This review highlights the psychopathologies of functional loss and the aspects of functional recuperation in the absence of the hippocampus.

Compensatory Contextual Fear Memory Pathways Develop in the Infralimbic Cortex within 3 Days after the First Test in the Absence of the Dorsal Hippocampus.

The dorsal hippocampus (DH) is primarily involved in the formation of contextual fear-conditioned (CxFC) memory. However, CxFC memory can be formed even in the absence of the DH. In addition to the DH, the infralimbic cortex (IL), a sub-region of the medial prefrontal cortex (mPFC), also plays an important role in the consolidation of CxFC memory. However, role of IL in the development of compensatory CxFC memory is not known. Here, we have examined (a) the development of the compensatory circuitry of CxFC memory within 3 days after the first test in the absence of the DH and (b) the role of IL in the induction of compensatory CxFC memory in the absence of the DH. The DH-lesioned rats re-trained for CxFC 1 day after the first testing exhibited significantly less freezing compared to the control group. However, the DH-lesioned rats, re-trained for CxFC 3 days after the first testing, showed a robust freezing response. It suggests that the fully functional compensatory circuitry of contextual fear memory develops after multiple training separated by 3 days. Furthermore, we observed that reversible inactivation of the IL of the DH-lesioned rats during the first training waned the formation of compensatory CxFC. It suggests that (a) the IL receives contextual fear memory information during the first trial in the absence of the DH and (b) perturbation in fear memory information encoding in the IL during the first trial impairs the development of the compensatory network in the absence of the DH.

REM Sleep Deprivation Alters Learning-Induced Cell Proliferation and Generation of Newborn Young Neurons in the Dentate Gyrus of the Dorsal Hippocampus.

The hippocampus-dependent "trace-appetitive conditioning task" increases cell proliferation and the generation of newborn young neurons. Evidence suggests that adult hippocampal neurogenesis and rapid eye movement (REM) sleep play an essential role in memory consolidation. On the other hand, REM sleep deprivation (REM-SD) induces detrimental effects on training-induced cell proliferation in the hippocampus's dentate gyrus (DG). Nonetheless, the role of REM sleep in the trace-appetitive memory and fate determination of the newly proliferated cells is not known. Here, we have studied the following: (I) the effects of 24 h of REM-SD (soon after training) on trace- and delay-appetitive memory and cell proliferation in the adult DG and (II) the effects of chronic (96 h) REM-SD (3 days after the training, the period in which newly generated cells progressed toward the neuronal lineage) on trace-appetitive memory and the generation of newborn young neurons. We used a modified multiple platform method for the selective REM-SD without altering non-REM (NREM) sleep. We found that 24 h of REM-SD, soon after trace-conditioning, impaired the trace-appetitive memory and the training-induced cell proliferation. Nevertheless, 96 h of REM-SD (3 days after the training) did not impair trace memory. Interestingly, 96 h of REM-SD altered the generation of newborn young neurons. These results suggest that REM sleep plays an essential role in training-induced cell proliferation and the fate determination of the newly generated cells toward the neuronal lineage.

Proton Pump Inhibitor "Lansoprazole" Inhibits Locus Coeruleus's Neuronal Activity and Increases Rapid Eye Movement Sleep.

Alteration of the bodily CO2 concentration and proton pump activity affects the sleep architecture. The brainstem locus coeruleus (LC) area plays an essential role in rapid eye movement (REM) sleep generation and chemoregulation. Previously, we reported that lansoprazole injections (intraperitoneal) increased REM sleep in the rats. However, it is not known if proton pumps in the LC influence REM sleep. Here, we studied the effects of lansoprazole in the LC on the neuronal activity and REM sleep expression. Male Wistar rats (250-300 g) were surgically prepared for sleep recording and drug microinjections into the LC. We determined the localization of proton pumps and expression levels of cFOS in the LC neurons immunohistochemically. Sleep-wake was recorded before and after the microinjections of drugs/vehicles. Our results demonstrate (i) the presence of proton pumps in the LC neurons, (ii) that the microinjection of lansoprazole into the LC reduced the number of cFOS+ve-TH+ve double-labeled neurons in the LC by 52.6% (p < 0.001) compared to the vehicle and (iii) that low and high doses of lansoprazole significantly increased REM sleep by 32% (p < 0.001) and 60% (p < 0.001), respectively, compared to the vehicle. Our results suggest that the proton pumps modulate the LC's noradrenergic (NE-ergic) neuronal activity and REM sleep. The increased amount of REM sleep can be attributed to the inhibition of the LC NE-ergic activity. Further, the REM sleep amount increased after the lansoprazole microinjections into the LC with a significant increase in the REM sleep episode numbers. Overall, our results suggest that proton pumps in the LC may be involved in REM sleep generation.

Rapid tumor inhibition via magnetic hyperthermia regulated by caspase 3 with time-dependent clearance of iron oxide nanoparticles.

Among conventional cancer therapies, radio-frequency magnetic hyperthermia (MHT) has widely been investigated for use with magnetic nanoparticles (MNPs). However, the majority of in vivo biodistribution studies have tested very low MNP dosages (equivalent to magnetic resonance imaging (MRI) applications) to check for clearance rate; which is far below the clinical dose of MHT. Due to this poor validation in preclinical scenarios, quite a few MNPs already in clinical use were later discontinued, on grounds of unexpected clinical outcomes in terms of inflammation, and prolonged clearance in vivo. By exploiting an economical method of synthesis, we have developed chitosan-coated Fe3O4 nanoparticles with high heating efficiency performance. Their anti-tumor response was evaluated in an ectopic tumor model of C6 glioblastoma by MHT. The intratumoral injection of MNPs on days 1 and 7 resulted in rapid tumor inhibition rate of 69.4% within 8 days, with complete inhibition within 32 days, and no recurrence recorded over a 5-month follow-up. Notably, the MNP-mediated MHT therapy achieved the highest degree of therapeutic efficacy required for complete tumor ablation by combining controlled temperature range (<44 °C), reduced MNP dosage; much lower than in most reported studies, and AMF parameters (time of exposure and frequency) within the clinical safety limit. Periodic body weight measurements confirmed negligible adverse side effects in rats. The anti-tumor activity was validated by severe apoptosis (TUNEL, cleaved Caspase-3), reduced proliferation (Ki 67) and disrupted vasculature (CD 31) in the Fe3O4-MHT-treated group. Real-time gene expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1α, IL-1ß) confirmed the intratumoral activation of IL-6, suggesting the role of immunomodulation in triggering the adaptive immune response for faster tumor regression in the treated group. In addition, the biodistribution and clearance rate of MNPs monitored using ICP-OES confirmed their time-dependent biodegradation via excretion (urine, feces), phagocytosis (liver) and circulatory system (blood), with negligible deposition in other major organs (kidney, heart, lungs). Although we could not show complete clearance of our MNPs within the time frame tested, future studies should focus on combining MHT with immunotherapy, and target tumors at a much-reduced iron dose, consequently improving in vivo clearance rate, and hence overcoming the limitations of MHT in clinical therapy.

Locus Coeruleus Acid-Sensing Ion Channels Modulate Sleep-Wakefulness and State Transition from NREM to REM Sleep in the Rat.

The locus coeruleus (LC) is one of the essential chemoregulatory and sleep-wake (S-W) modulating centers in the brain. LC neurons remain highly active during wakefulness, and some implicitly become silent during rapid eye movement (REM) sleep. LC neurons are also involved in CO2-dependent modulation of the respiratory drive. Acid-sensing ion channels (ASICs) are highly expressed in some brainstem chemosensory breathing regulatory areas, but their localization and functions in the LC remain unknown. Mild hypercapnia increases the amount of non-REM (NREM) sleep and the number of REM sleep episodes, but whether ASICs in the LC modulate S-W is unclear. Here, we investigated the presence of ASICs in the LC and their role in S-W modulation and the state transition from NREM to REM sleep. Male Wistar rats were surgically prepared for chronic polysomnographic recordings and drug microinjections into the LC. The presence of ASIC-2 and ASIC-3 in the LC was immunohistochemically characterized. Microinjections of amiloride (an ASIC blocker) and APETx2 (a blocker of ASIC-2 and -3) into the LC significantly decreased wakefulness and REM sleep, but significantly increased NREM sleep. Mild hypercapnia increased the amount of NREM and the number of REM episodes. However, APETx2 microinjection inhibited this increase in REM frequency. These results suggest that the ASICs of LC neurons modulate S-W, indicating that ASICs could play an important role in vigilance-state transition. A mild increase in CO2 level during NREM sleep sensed by ASICs could be one of the determinants of state transition from NREM to REM sleep.

Training on an Appetitive Trace-Conditioning Task Increases Adult Hippocampal Neurogenesis and the Expression of Arc, Erk and CREB Proteins in the Dorsal Hippocampus.

Adult hippocampal neurogenesis (AHN) plays an essential role in hippocampal-dependent memory consolidation. Increased neurogenesis enhances learning, whereas its ablation causes memory impairment. In contrast, few reports suggest that neurogenesis reduces after learning. Although the interest in exploring the role of adult neurogenesis in learning has been growing, the evidence is still limited. The role of the trace- and delay-appetitive-conditioning on AHN and its underlying mechanism are not known. The consolidation of trace-conditioned memory requires the hippocampus, but delay-conditioning does not. Moreover, the dorsal hippocampus (DH) and ventral hippocampus (VH) may have a differential role in these two conditioning paradigms. Here, we have investigated the changes in: (A) hippocampal cell proliferation and their progression towards neuronal lineage; and (B) expression of Arc, Erk1, Erk2, and CREB proteins in the DH and VH after trace- and delay-conditioning in the rat. The number of newly generated cells significantly increased in the trace-conditioned but did not change in the delay-conditioned animals compared to the control group. Similarly, the expression of Arc protein significantly increased in the DH but not in the VH after trace-conditioning. Nonetheless, it remains unaltered in the delay-conditioned group. The expression of pErk1, pErk2, and pCREB also increased in the DH after trace-conditioning. Whereas, the expression of only pErk1 pErk2 and pCREB proteins increased in the VH after delay-conditioning. Our results suggest that appetitive trace-conditioning enhances AHN. The increased DH neuronal activation and pErk1, pErk2, and pCREB in the DH may be playing an essential role in learning-induced cell-proliferation after appetitive trace-conditioning.

RF hyperthermia by encapsulated Fe3O4 nanoparticles induces cancer cell death via time-dependent caspase-3 activation.

Aim: To explore the optimum temperature for cancer cell death using magnetic hyperthermia (MH), which in turn will affect the mode of cell death. Method: The focus of this study is to improve upon the existing methodology for the synthesis of chitosan encapsulated Fe3O4. MH was done at different temperatures. The cell death pathway was explored using flow cytometry and western blot. Results: Coated Fe3O4 exhibited low cytotoxicity, high stability and heating efficiency. MH at 43°C was the optimum temperature for robust cell death. Cell death pathway suggested that during the initial stages of recovery, apoptosis was the main mode of cell death. While at later stages, major apoptosis and minor necrosis were observed. Conclusion: It is important to find out the long-term effect of hyperthermia treatment on cancer cells and their consequences on surrounding healthy cells.

The formation of compensatory contextual fear memory in the absence of dorsal hippocampus does not change sleep architecture.

Although the dorsal hippocampus (DH) plays an essential role in the consolidation of contextual fear-conditioned (CxFC) memory, this consolidation may also occur in the absence of DH. It is, however, not known if the development of a compensatory circuit for CxFC memory is time-dependent. The DH-dependent contextual fear memory influences sleep architecture, but whether the compensatory fear memory can influence sleep, is not known. Here, we have studied (a) the temporal progression of compensatory contextual fear memory in the absence of DH and (b) the influence of compensatory contextual fear memory on sleep architecture. Rats were surgically prepared for chronic polysomnographic recordings and drug injections in the DH. They were divided into four groups: DH-non-lesioned and fear-conditioned, DH-non-lesioned and non-fear-conditioned, DH-lesioned and fear-conditioned and DH-lesioned and non-fear-conditioned groups. The DH was lesioned with ibotenic acid. The animals were conditioned to contextual fear twice: 1st training on Day 5 and testing on Day 6; 2nd training on Day 10 and testing on Day 11. The DH-lesioned and fear-conditioned animals did not exhibit freezing response during the first testing but showed a robust freezing response when re-trained after a gap of three days. In addition, wakefulness and NREM sleep amount did not change, but REM sleep significantly decreased in the DH-dependent CxFC memory group. Interestingly, REM sleep did not decrease in the DH-independent CxFC memory group. Our findings suggest that the development of compensatory CxFC memory is a time-dependent process and the compensatory CxFC memory may not influence sleep architecture.

Training on an Appetitive (Delay)-Conditioning Task Enhances Oscillatory Waves During Sleep in the Cortical and Amygdalar Network.

Oscillating waves during sleep play an essential role in memory consolidation. The cortical slow wave activity (SWA) and sigma waves during NREM sleep and theta waves during REM sleep increase after a variety of memory tasks including declarative, procedural and associative learning tasks. These oscillatory waves during sleep help to promote neural dialog between circuitries, which possibly plays a causal role in memory consolidation. However, the role of sleep-associated oscillating waves in a complex appetitive-conditioning paradigm is not clear. The parietal cortex and amygdala are involved in the cognitive evaluation of the environmental stimuli, and appetitive conditioning. Here, we have studied the changes in sleep architecture and oscillatory waves during NREM and REM sleep in the parietal cortices and amygdalar-local field potential (A-LFP) after appetitive-conditioning in the rat. We observed that REM sleep increased significantly after appetitive conditioning, which significantly positively correlated with performance on the appetitive-conditioning task. Further, the cortical SWA (0.1-4.5 Hz), and sigma (12-14.25 Hz) waves during NREM sleep, theta (6-9 Hz) waves during REM sleep, the amygdalar SWA (0.1-3.75 Hz) during NREM sleep and theta (6-8.25 Hz) waves during REM sleep significantly increased after appetitive conditioning. Interestingly, the augmented oscillatory waves significantly positively correlated with the performances on the appetitive-conditioning task. Our results suggest that the augmented REM sleep after conditioning may be required for the consolidation of appetitive-conditioned memory. Further, a significant correlation between augmented power in oscillatory waves during sleep and performance suggesting that these waves may be playing a crucial role in the consolidation of appetitive-conditioned memory.

Localized cancer treatment by radio-frequency hyperthermia using magnetic nanoparticles immobilized on graphene oxide: from novel synthesis to in vitro studies.

We have produced an innovative, theranostic hybrid nanocomposite of graphene oxide and iron oxide (GO-Fe3O4) for radio-frequency hyperthermia therapy. A new electrochemical synthesis route for the GO-Fe3O4 nanocomposite is employed. Superparamagnetic nanoparticles used for magnetic hyperthermia for biomedical application face longstanding obstacles, including the large number of nanoparticles required to achieve the desired therapeutic temperature, poor colloidal stability in aqueous suspension or physiological media, poor biocompatibility and, most importantly, low specific absorption rate (SAR). To limit the dosage of nanoparticles for therapeutic use, efforts are being made to increase the heating efficiency of nanoparticles. We have introduced an alternative way to increase the SAR value by improving the colloidal stability of magnetic nanoparticles. It is necessary to immobilize these nanoparticles on a support to prevent their agglomeration and precipitation in aqueous suspension. To address these issues, we report a reproducible electrochemical synthesis route for the GO-Fe3O4 nanocomposite. Our nanocomposite demonstrated good colloidal stability and low cytotoxicity in vitro. Due to its good colloidal stability, the nanocomposite had a high SAR of 543 W g-1 and corresponding intrinsic loss power of 5.98 nH m2 kg-1, which is 46% better than the best commercial equivalents. In vitro cytotoxicity studies demonstrated almost 70% cell viability at 200 µg mL-1 GO-Fe3O4 nanocomposite, a comparable concentration for clinical use according to FDA standards. We also showed the therapeutic potential of the nanocomposite using magnetic hyperthermia. We observed cancer cell (A549 human lung epithelial adenocarcinoma) ablation at 41, 42 and 43 °C for 30, 45, and 60 min. A maximum cancer cell death rate of 80.5% was observed at 43 °C for 60 min under alternating magnetic field exposure. Thus, the nanocomposites could be used in the efficient treatment of cancer.

Short-Term Total Sleep-Deprivation Impairs Contextual Fear Memory, and Contextual Fear-Conditioning Reduces REM Sleep in Moderately Anxious Swiss Mice.

The conditioning tasks have been widely used to model fear and anxiety and to study their association with sleep. Many reports suggest that sleep plays a vital role in the consolidation of fear memory. Studies have also demonstrated that fear-conditioning influences sleep differently in mice strains having a low or high anxiety level. It is, therefore, necessary to know, how sleep influences fear-conditioning and how fear-conditioning induces changes in sleep architecture in moderate anxious strains. We have used Swiss mice, a moderate anxious strain, to study the effects of: (i) sleep deprivation on contextual fear conditioned memory, and also (ii) contextual fear conditioning on sleep architecture. Animals were divided into three groups: (a) non-sleep deprived (NSD); (b) stress control (SC); and (c) sleep-deprived (SD) groups. The SD animals were SD for 5 h soon after training. We found that the NSD and SC animals showed 60.57% and 58.12% freezing on the testing day, while SD animals showed significantly less freezing (17.13% only; p < 0.001) on the testing day. Further, we observed that contextual fear-conditioning did not alter the total amount of wakefulness and non-rapid eye movement (NREM) sleep. REM sleep, however, significantly decreased in NSD and SC animals on the training and testing days. Interestingly, REM sleep did not decrease in the SD animals on the testing day. Our results suggest that short-term sleep deprivation impairs fear memory in moderate anxious mice. It also suggests that NREM sleep, but not REM sleep, may have an obligatory role in memory consolidation.

Influence of cued-fear conditioning and its impairment on NREM sleep.

Many studies suggest that fear conditioning influences sleep. It is, however, not known if the changes in sleep architecture after fear conditioning are essentially associated with the consolidation of fearful memory or with fear itself. Here, we have observed that within sleep, NREM sleep consistently remained augmented after the consolidation of cued fear-conditioned memory. But a similar change did not occur after impairing memory consolidation by blocking new protein synthesis and glutamate transmission between glial-neuronal loop in the lateral amygdala (LA). Anisomycin (a protein synthesis inhibitor) and DL-α-amino-adipic acid (DL- α -AA) (a glial glutamine synthetase enzyme inhibitor) were microinjected into the LA soon after cued fear-conditioning to induce memory impairment. On the post-conditioning day, animals in both the groups exhibited significantly less freezing. In memory-consolidated groups (vehicle groups), NREM sleep significantly increased during 2nd to 5th hours after training compared to their baseline days. However, in memory impaired groups (anisomycin and DL- α -AA microinjected groups), similar changes were not observed. Our results thus suggest that changes in sleep architecture after cued fear-conditioning are indeed a consolidation dependent event.

Short-term total sleep deprivation alters delay-conditioned memory in the rat.

Short-term sleep deprivation soon after training may impair memory consolidation. Also, a particular sleep stage or its components increase after learning some tasks, such as negative and positive reinforcement tasks, avoidance tasks, and spatial learning tasks, and so forth. It suggests that discrete memory types may require specific sleep stage or its components for their optimal processing. The classical conditioning paradigms are widely used to study learning and memory but the role of sleep in a complex conditioned learning is unclear. Here, we have investigated the effects of short-term sleep deprivation on the consolidation of delay-conditioned memory and the changes in sleep architecture after conditioning. Rats were trained for the delay-conditioned task (for conditioning, house-light [conditioned stimulus] was paired with fruit juice [unconditioned stimulus]). Animals were divided into 3 groups: (a) sleep deprived (SD); (b) nonsleep deprived (NSD); and (c) stress control (SC) groups. Two-way ANOVA revealed a significant interaction between groups and days (training and testing) during the conditioned stimulus-unconditioned stimulus presentation. Further, Tukey post hoc comparison revealed that the NSD and SC animals exhibited significant increase in performances during testing. The SD animals, however, performed significantly less during testing. Further, we observed that wakefulness and NREM sleep did not change after training and testing. Interestingly, REM sleep increased significantly on both days compared to baseline more specifically during the initial 4-hr time window after conditioning. Our results suggest that the consolidation of delay-conditioned memory is sleep-dependent and requires augmented REM sleep during an explicit time window soon after training. (PsycINFO Database Record

Proton pump inhibition increases rapid eye movement sleep in the rat.

Increased bodily CO2 concentration alters cellular pH as well as sleep. The proton pump, which plays an important role in the homeostatic regulation of cellular pH, therefore, may modulate sleep. We investigated the effects of the proton pump inhibitor "lansoprazole" on sleep-wakefulness. Male Wistar rats were surgically prepared for chronic polysomnographic recordings. Two different doses of lansoprazole (low: 1 mg/kg; high: 10 mg/kg) were injected intraperitoneally in the same animal (n = 7) and sleep-wakefulness was recorded for 6 hrs. The changes in sleep-wakefulness were compared statistically. Percent REM sleep amount in the vehicle and lansoprazole low dose groups was 9.26 ± 1.03 and 9.09 ± 0.54, respectively, which increased significantly in the lansoprazole high dose group by 31.75% (from vehicle) and 34.21% (from low dose). Also, REM sleep episode numbers significantly increased in lansoprazole high dose group. Further, the sodium-hydrogen exchanger blocker "amiloride" (10 mg/kg; i.p.) (n = 5) did not alter sleep-wake architecture. Our results suggest that the proton pump plays an important role in REM sleep modulation and supports our view that REM sleep might act as a sentinel to help maintain normal CO2 level for unperturbed sleep.

The effect of glial glutamine synthetase inhibition on recognition and temporal memories in the rat.

The glutamate neurotransmitter is intrinsically involved in learning and memory. Glial glutamine synthetase enzyme synthesizes glutamine, which helps maintain the optimal neuronal glutamate level. However, the role of glutamine synthetase in learning and memory remains unclear. Using associative trace learning task, we investigated the effects of methionine sulfoximine (MSO) (glutamine synthetase inhibitor) on recognition and temporal memories. MSO and vehicle were injected (i.p.) three hours before training in separate groups of male Wistar rats (n=11). Animals were trained to obtain fruit juice after following a set of sequential events. Initially, house-light was presented for 15s followed by 5s trace interval. Thereafter, juice was given for 20s followed by 20s inter-presentation interval. A total of 75 presentations were made over five sessions during the training and testing periods. The average number of head entries to obtain juice per session and during individual phases at different time intervals was accounted as an outcome measure of recognition and temporal memories. The total head entries in MSO and vehicle treated animals were comparable on training and testing days. However, it was 174.90% (p=0.08), 270.61% (p<0.05), 143.20% (p<0.05) more on training day and 270.33% (p<0.05), 157.94% (p<0.05), 170.42% (p<0.05) more on testing day, during the house-light, trace-interval and inter-presentation interval phases in MSO animals. Glutamine synthetase inhibition did not induce recognition memory deficit, while temporal memory was altered, suggesting that glutamine synthetase modulates some aspects of mnemonic processes.

Sleep alterations in mammals: did aquatic conditions inhibit rapid eye movement sleep?

Sleep has been studied widely in mammals and to some extent in other vertebrates. Higher vertebrates such as birds and mammals have evolved an inimitable rapid eye movement (REM) sleep state. During REM sleep, postural muscles become atonic and the temperature regulating machinery remains suspended. Although REM sleep is present in almost all the terrestrial mammals, the aquatic mammals have either radically reduced or completely eliminated REM sleep. Further, we found a significant negative correlation between REM sleep and the adaptation of the organism to live on land or in water. The amount of REM sleep is highest in terrestrial mammals, significantly reduced in semi-aquatic mammals and completely absent or negligible in aquatic mammals. The aquatic mammals are obligate swimmers and have to surface at regular intervals for air. Also, these animals live in thermally challenging environments, where the conductive heat loss is approximately ~90 times greater than air. Therefore, they have to be moving most of the time. As an adaptation, they have evolved unihemispheric sleep, during which they can rove as well as rest. A condition that immobilizes muscle activity and suspends the thermoregulatory machinery, as happens during REM sleep, is not suitable for these animals. It is possible that, in accord with Darwin's theory, aquatic mammals might have abolished REM sleep with time. In this review, we discuss the possibility of the intrinsic role of aquatic conditions in the elimination of REM sleep in the aquatic mammals.

Sleep deprivation impairs consolidation of cued fear memory in rats.

Post-learning sleep facilitates negative memory consolidation and also helps preserve it over several years. It is believed, therefore, that sleep deprivation may help prevent consolidation of fearful memory. Its effect, however, on consolidation of negative/frightening memories is not known. Cued fear-conditioning (CuFC) is a widely used model to understand the neural basis of negative memory associated with anxiety disorders. In this study, we first determined the suitable circadian timing for consolidation of CuFC memory and changes in sleep architecture after CuFC. Thereafter, we studied the effect of sleep deprivation on CuFC memory consolidation. Three sets of experiments were performed in male Wistar rat (n=51). In experiment-I, animals were conditioned to cued-fear by presenting ten tone-shock paired stimuli during lights-on (7 AM) (n=9) and lights-off (7 PM) (n=9) periods. In experiment-II, animals were prepared for polysomnographic recording (n=8) and changes in sleep architecture after CuFC was determined. Further in experiment-III, animals were cued fear-conditioned during the lights-off period and were randomly divided into four groups: Sleep-Deprived (SD) (n=9), Non-Sleep Deprived (NSD) (n=9), Stress Control (SC) (n=9) and Tone Control (n=7). Percent freezing amount, a hallmark of fear, was compared statistically in these groups. Rats trained during the lights-off period exhibited significantly more freezing compared to lights-on period. In CuFC trained animals, total sleep amount did not change, however, REM sleep decreased significantly. Further, out of total sleep time, animals spent proportionately more time in NREM sleep. Nevertheless, SD animals exhibited significantly less freezing compared to NSD and SC groups. These data suggest that sleep plays an important role in the consolidation of cued fear-conditioned memory.

A Moderate Increase of Physiological CO(2) in a Critical Range during Stable NREM Sleep Episode: A Potential Gateway to REM Sleep.

Sleep is characterized as rapid eye movement (REM) and non-rapid eye movement (NREM) sleep. Studies suggest that wake-related neurons in the basal forebrain, posterior hypothalamus and brainstem, and NREM sleep-related neurons in the anterior-hypothalamic area inhibit each other, thus alternating sleep-wakefulness. Similarly, pontine REM-ON and REM-OFF neurons reciprocally inhibit each other for REM sleep modulation. It has been proposed that inhibition of locus coeruleus (LC) REM-OFF neurons is pre-requisite for REM sleep genesis, but it remains ambiguous how REM-OFF neurons are hyperpolarized at REM sleep onset. The frequency of breathing pattern remains high during wake, slows down during NREM sleep but further escalates during REM sleep. As a result, brain CO(2) level increases during NREM sleep, which may alter REM sleep manifestation. It has been reported that hypocapnia decreases REM sleep while hypercapnia increases REM sleep periods. The groups of brainstem chemosensory neurons, including those present in LC, sense the alteration in CO(2) level and respond accordingly. For example, one group of LC neurons depolarize while other hyperpolarize during hypercapnia. In another group, hypercapnia initially depolarizes but later hyperpolarizes LC neurons. Besides chemosensory functions, LC REM-OFF neurons are an integral part of REM sleep executive machinery. We reason that increased CO(2) level during a stable NREM sleep period may hyperpolarize LC neurons including REM-OFF, which may help initiate REM sleep. We propose that REM sleep might act as a sentinel to help maintain normal CO(2) level for unperturbed sleep.