Sushil K. Jha

Mechanisms of sleep-dependent consolidation of cortical plasticity.

Sleep is thought to consolidate changes in synaptic strength, but the underlying mechanisms are unknown. We investigated the cellular events involved in this process during ocular dominance plasticity (ODP)-a canonical form of in vivo cortical plasticity triggered by monocular deprivation (MD) and consolidated by sleep via undetermined, activity-dependent mechanisms. We find that sleep consolidates ODP primarily by strengthening cortical responses to nondeprived eye stimulation. Consolidation is inhibited by reversible, intracortical antagonism of NMDA receptors (NMDARs) or cAMP-dependent protein kinase (PKA) during post-MD sleep. Consolidation is also associated with sleep-dependent increases in the activity of remodeling neurons and in the phosphorylation of proteins required for potentiation of glutamatergic synapses. These findings demonstrate that synaptic strengthening via NMDAR and PKA activity is a key step in sleep-dependent consolidation of ODP.

A rodent model of sleep disturbances in posttraumatic stress disorder: The role of context after fear conditioning

BACKGROUND A prominent sleep disturbance, likely including a disruption of rapid eye movement sleep (REMS) continuity, characterizes posttraumatic stress disorder (PTSD). We set out to develop a fear conditioning paradigm in rats that displays alterations in sleep architecture analogous to those in PTSD. METHODS Baseline polysomnographic recordings of rats were performed in a neutral context to which the rats had been habituated for several days. Rats were then shock- or mock-trained in a distinctly different context, and their sleep was studied the following day in that context. A separate group of rats was shock-trained and studied in the neutral context on the following 2 days. RESULTS Rats that slept in the neutral context exhibited a REMS-selective increase in sleep 24 hours after training and increases in REMS and non-REMS 48 hours after training. In contrast, rats that slept in the presence of situational reminders of the training context exhibited a REMS-selective decrease in sleep 24 hours later. Animals that were mock-trained showed no changes in sleep. CONCLUSIONS Shock training induced days-long changes in sleep architecture that were disrupted when the animal was exposed to situational reminders of the training context.

Sleep-Dependent Plasticity Requires Cortical Activity

Recent findings in humans and animals suggest that sleep promotes synaptic plasticity, but the underlying mechanisms have not been identified. We have demonstrated recently an important role for sleep in ocular dominance (OD) plasticity, a classic form of in vivo cortical remodeling triggered by monocular deprivation (MD) during a critical period of development. The mechanisms responsible for the effects of sleep on OD plasticity are unknown but may depend on neuronal activity in the sleeping brain. We investigated the role of cortical activity in sleep-dependent plasticity by reversibly inactivating the sleeping visual cortex (V1) after a period of MD. Critical period cats were bilaterally implanted with cannulas in V1 and standard EEG/EMG electrodes for polysomnographic recording. After a period of MD, visual cortices were infused with the sodium channel blocker lidocaine in vehicle or vehicle only during sleep. A third group of cats served as sham controls and were infused with lidocaine outside of V1 (into the CSF). Both optical imaging of intrinsic cortical signals and microelectrode recordings showed that OD plasticity was significantly reduced in cats whose visual cortices were reversibly silenced during sleep. These findings demonstrate that the mechanisms governing this form of sleep-dependent plasticity require cortical activity. They provide an important insight into how sleep modifies synaptic circuitry by narrowing the range of possible candidate mechanisms to those that are activity dependent.

REM sleep: a sensitive index of fear conditioning in rats.

To examine the influence of conditioned fear stimuli on sleep‐wake states, we recorded sleep in Sprague–Dawley rats after exposure to tones previously paired with footshock. After habituation to a recording chamber and the recording procedure, a baseline sleep recording was obtained the next day. One day later, experimental animals were exposed to shock training designed to induce conditioned fear (FC), consisting of five tone‐footshock pairings. The 5‐s tones (conditioned stimuli; CS) co‐terminated with 1‐s footshocks (unconditioned stimuli; US). The next day sleep was recorded for 4 h in the recording chamber after presentation of five CSs alone. Sleep efficiency (total sleep time/recording period) and REM sleep (REM) and non‐REM (NREM) measures were determined. While sleep efficiency was not significantly changed after CS presentation, the percentage of total sleep time spent in REM (REM percentage) was reduced in the FC animals. The reduction in REM percentage in the FC animals was due to a decrease in the number of REM bouts. In a separate experiment, we repeated the procedures, except the tones and shocks were presented in an explicitly unpaired (UP) fashion. The next day, presentation of the tones increased REM percentage in the UP group. Results are discussed in terms of the decreases in REM as a response to conditioned fear, and the relevance of these findings to the sleep changes seen in post‐traumatic stress disorder (PTSD).

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.

Sleep-related neurons in the central nucleus of the amygdala of rats and their modulation by the dorsal raphe nucleus

The amygdala (AMY) plays an important role in initiating appropriate neurobehavioral responses to emotionally arousing events. Its major efferents from the central nucleus (Ace) to the basal forebrain, hypothalamus and brainstem permit it to influence sleep mechanisms. To characterize further the neuronal activity of AMY during sleep and wakefulness, we recorded single neuronal activity in Ace across behavioral states in freely moving, normally behaving rats. Of the 49 neurons recorded from Ace, 24 neurons had firing patterns related to sleep-wakefulness (S-W). Of these, 50% (n = 12) had a high firing frequency during wakefulness (W) or both W and REM sleep (REM), 12% (n = 3) were non-REM (NREM)-related, 17% (n = 4) had a high firing rate in REM (REM-ON), and 20% (n = 5) fired at a low rate during REM. Because serotonin introduced into AMY during REM induces short-latency changes of state, we also studied the effects of low frequency (1 Hz) electrical stimulation of the dorsal raphe nucleus (DRN) on Ace neurons. All REM-ON neurons recorded from Ace were inhibited by DRN stimulation, and other cell types were unaffected. Thus, we found that the majority of cells in Ace related to S-W fired slowly during NREM and increased their discharge during W and/or REM, and that the DRN has the potential for modulating the spontaneous activity of REM-ON cells in rats.

Presence of α-1 adrenoreceptors on thermosensitive neurons in the medial preoptico-anterior hypothalamic area in Rats

Earlier microinjection studies showed that norepinephrine in the medial preoptico-anterior hypothalamic area (mPOAH) regulates body temperature and the action is mediated through alpha-1 adrenoceptors. This study was conducted to confirm if the thermosensitive neurons in the mPOAH of rats possess alpha-1 adrenoceptors. First, the thermosensitivity of mPOAH neurons was tested and then the effects of microiontophoretic application of prazosin, alpha 1 adrenoceptor antagonist, on the firing rate of both the thermosensitive as well as the insensitive neurons were recorded. Prazosin significantly inhibited the firing rate of the thermosensitive neurons suggesting that most of the cold and warm sensitive neurons in the mPOAH possess alpha-1 adrenoceptors. These results at the single neuronal level confirm our earlier hypothesis that in the mPOAH, norepinephrine regulates body temperature by acting on alpha-1 adrenoceptors. The data also suggest that sensitivity of the mPOAH neurons to norepinephrine alter with changes in body temperature. The detailed physiological significance of the results with special reference to thermoregulation at the single neuronal level has been discussed.

ROLE OF GABA-A RECEPTOR IN THE PREOPTIC AREA IN THE REGULATION OF SLEEP-WAKEFULNESS AND RAPID EYE MOVEMENT SLEEP

The role of GABA in medial preoptico-anterior hypothalamic area in the regulation of spontaneous sleep-wakefulness and rapid eye movement sleep was investigated in this study. Local microinjection of picrotoxin, a GABA-A antagonist, into this area increased quiet wakefulness but significantly reduced deep sleep and rapid eye movement sleep. Both the frequency of generation and duration per episode of the latter were significantly reduced. It is concluded that GABA-ergic neurotransmission in the medial preoptic area is spontaneously active in modulating the hypnogenic function including rapid eye movement sleep and the action is mediated by GABA-A receptor.

Paradoxical effects of the hypnotic Zolpidem in the neonatal ferret.

Hypnotic drugs designed to treat insomnia in adults are now increasingly used in children, but the effects of these compounds on neonatal sleep are poorly understood. We investigated the hypnotic effects of the commonly prescribed non-benzodiazepine sleep agent Zolpidem (Ambien) on sleep architecture and electroencephalographic (EEG) activity in the neonatal ferret. Six ferret kits were surgically prepared for EEG/electromyographic (EMG) recordings using techniques adopted for use in neonatal animals. They were then administered in a counter-balanced design vehicle, or Zolpidem (2 mg/kg or 20 mg/kg) via intraperitoneal injection (1x/day over three days at 1 p.m.). Zolpidem did not increase non-rapid-eye-movement (NREM) or total sleep time. Instead Zolpidem reduced REM sleep and total sleep amounts and increased NREM sleep bout duration. Zolpidem also increased higher-frequency EEG energies during REM and NREM sleep and transiently produced a behavioral state that appeared intermediate between wake and sleep. Our findings demonstrate that hypnotics that improve sleep quality in adults may produce profoundly different behavioral changes in neonates.

A preliminary study of sleep ontogenesis in the ferret (Mustela putorius furo)

We investigated sleep ontogenesis in the ferret-a placental mammal that is highly altricial compared to other mammalian species. Because altriciality is linked with elevated rapid-eye-movement (REM) sleep amounts during infancy, it was expected that ferret kits would display very high levels of this state. Longitudinal polysomnographic measurements were made from 8 ferret kits from approximately eye-opening (postnatal day [P]30)-P50 using an experimental routine that minimized the effects of maternal separation. These data were compared to values from 8 adult ferrets (>3 months of age) and 6 neonatal cats (mean age: P31.7). We find that the polygraphic features of REM and non-REM (NREM) sleep are present by at least P30. Over the next 2 weeks, REM sleep amounts slightly declined while wakefulness and NREM sleep amounts increased. However, a comparison to published values from developing cats and rats showed that the ferret did not exhibit a disproportionate amount of REM sleep at similar postnatal ages or relative to a common developmental milestone (eye-opening).