Ruben Guzman-Marin

Sleep-waking discharge patterns of median preoptic nucleus neurons in rats

Several lines of evidence show that the preoptic area (POA) of the hypothalamus is critically implicated in the regulation of sleep. Functionally heterogeneous cell groups with sleep‐related discharge patterns are located both in the medial and lateral POA. Recently a cluster of neurons showing sleep‐related c‐Fos immunoreactivity was found in the median preoptic nucleus (MnPN). To determine the specificity of the state‐related behaviour of MnPN neurons we have undertaken the first study of their discharge patterns across the sleep‐waking cycle. Nearly 76% of recorded cells exhibited elevated discharge rates during sleep. Sleep‐related units showed several distinct types of activity changes across sleep stages. Two populations included cells displaying selective activation during either non‐rapid eye movement (NREM) sleep (10%) or REM sleep (8%). Neurons belonging to the predominant population (58%) exhibited activation during both phases of sleep compared to wakefulness. Most of these cells showed a gradual increase in their firing rates prior to sleep onset, elevated discharge during NREM sleep and a further increase during REM sleep. This specific sleep‐waking discharge profile is opposite to that demonstrated by wake‐promoting monoaminergic cell groups and was previously found in cells localized in the ventrolateral preoptic area (vlPOA). We hypothesize that these vlPOA and MnPN neuronal populations act as parts of a GABAergic/galaninergic sleep‐promoting (‘anti‐waking’) network which exercises inhibitory control over waking‐promoting systems. MnPN neurons that progressively increase activity during sustained waking and decrease activity during sustained sleep states may be involved in homeostatic regulation of sleep.

Suppression of hippocampal plasticity-related gene expression by sleep deprivation in rats.

Previous work shows that sleep deprivation impairs hippocampal‐dependent learning and long‐term potentiation (LTP). Brain‐derived neurotrophic factor (BDNF), cAMP response‐element‐binding (CREB) and calcium–calmodulin‐dependent protein kinase II (CAMKII) are critical modulators of hippocampal‐dependent learning and LTP. In the present study we compared the effects of short‐ (8 h) and intermediate‐term (48 h) sleep deprivation (SD) on the expression of BDNF and its downstream targets, Synapsin I, CREB and CAMKII in the neocortex and the hippocampus. Rats were sleep deprived using an intermittent treadmill system which equated total movement in the SD and control treadmill animals (CT), but permitted sustained periods of rest in CT animals. Animals were divided into SD (treadmill schedule: 3 s on/12 s off) and two treadmill control groups, CT1 (15 min on/60 min off) and CT2 (30 min on/120 min off – permitting more sustained sleep). Real‐time Taqman RT‐PCR was used to measure changes in mRNA; BDNF protein levels were determined using ELISA. In the hippocampus, 8 h treatments reduced BDNF, Synapsin I, CREB and CAMKII gene expression in both SD and control groups. Following 48 h of experimental procedures, the expression of all these four molecular markers of plasticity was reduced in SD and CT1 groups compared to the CT2 and cage control groups. In the hippocampus, BDNF protein levels after 8 h and 48 h treatments paralleled the changes in mRNA. In neocortex, neither 8 h nor 48 h SD or control treatments had significant effects on BDNF, Synapsin I and CAMKII mRNA levels. Stepwise regression analysis suggested that loss of REM sleep underlies the effects of SD on hippocampal BDNF, Synapsin I and CREB mRNA levels, whereas loss of NREM sleep underlies the effects on CAMKII mRNA.

Activation of c-fos in GABAergic neurones in the preoptic area during sleep and in response to sleep deprivation

Neurones in the median preoptic nucleus (MnPN) and the ventrolateral preoptic area (vlPOA) express immunoreactivity for c‐Fos protein following sustained sleep, and display elevated discharge rates during both non‐REM and REM sleep compared to waking. We evaluated the hypothesis that MnPN and vlPOA sleep‐active neurones are GABAergic by combining staining for c‐Fos protein with staining for glutamic acid decarboxylase (GAD). In a group of six rats exhibiting spontaneous total sleep times averaging 82.2 ± 5.1% of the 2 h immediately prior to death, >75% of MnPN neurones that were Fos‐immunoreactive (IR) were also GAD‐IR. Similar results were obtained in the vlPOA. In a group of 11 rats exhibiting spontaneous sleep times ranging from 20 to 92%, the number of Fos + GAD‐IR neurones in MnPN and vlPOA was positively correlated with total sleep time. Compared to control animals, Fos + GAD‐IR cell counts in the MnPN were significantly elevated in rats that were sleep deprived for 24 h and permitted 2 h of recovery sleep. These findings demonstrate that a majority of MnPN and vlPOA neurones that express Fos‐IR during sustained spontaneous sleep are GABAergic. They also demonstrate that sleep deprivation is associated with increased activation of GABAergic neurones in the MnPN and vlPOA.

The Median Preoptic Nucleus Reciprocally Modulates Activity of Arousal-Related and Sleep-Related Neurons in the Perifornical Lateral Hypothalamus

The perifornical–lateral hypothalamic area (PF/LH) contains neuronal groups playing an important role in control of waking and sleep. Among the brain regions that regulate behavioral states, one of the strongest sources of projections to the PF/LH is the median preoptic nucleus (MnPN) containing a sleep-active neuronal population. To evaluate the role of MnPN afferents in the control of PF/LH neuronal activity, we studied the responses of PF/LH cells to electrical stimulation or local chemical manipulation of the MnPN in freely moving rats. Single-pulse electrical stimulation evoked responses in 79% of recorded PF/LH neurons. No cells were activated antidromically. Direct and indirect transynaptic effects depended on sleep–wake discharge pattern of PF/LH cells. The majority of arousal-related neurons, that is, cells discharging at maximal rates during active waking (AW) or during AW and rapid eye movement (REM) sleep, exhibited exclusively or initially inhibitory responses to stimulation. Sleep-related neurons, the cells with elevated discharge during non-REM and REM sleep or selectively active in REM sleep, exhibited exclusively or initially excitatory responses. Activation of the MnPN via microdialytic application of l-glutamate or bicuculline resulted in reduced discharge of arousal-related and in excitation of sleep-related PF/LH neurons. Deactivation of the MnPN with muscimol caused opposite effects. The results indicate that the MnPN contains subset(s) of neurons, which exert inhibitory control over arousal-related and excitatory control over sleep-related PF/LH neurons. We hypothesize that MnPN sleep-active neuronal group has both inhibitory and excitatory outputs that participate in the inhibitory control of arousal-promoting PF/LH mechanisms.

Mechanisms of nicotine actions on dorsal raphe serotoninergic neurons

Nicotine, locally administered into the dorsal raphe nucleus (DRN) of rat midbrain slices, increased the discharge rate of 70% of serotoninergic neurons, decreased it in 30% and induced reciprocal oscillatory increases in serotonin (5-hydroxytryptamine, 5-HT) and gamma-aminobutyric acid (GABA) release. All of nicotines stimulatory effects were maximal at 2.15 microM. Bicuculline, a GABA(A) receptor antagonist, increased the firing rate in 64% of serotoninergic neurons, decreased it in 36% and augmented serotonin and GABA release. Bicuculline increased nicotines stimulatory effects on firing rate but did not reverse the inhibitory ones. N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinil-cyclohexanecarboxamide (WAY-100635), a 5-HT(1A) receptor antagonist, increased the firing rate of 88% of serotoninergic neurons, as well as serotonin and GABA release and reversed nicotines inhibitory action on serotoninergic neurons. These data suggest that nicotine decreases the firing rate of one third of serotoninergic neurons through serotonin release and increases the firing rate of the remaining two thirds, due to stronger stimulatory than indirect inhibitory effects.

Transdermal nicotine on sleep and PGO spikes

There is conflicting evidence for the role of nicotine in sleep regulation. This study was undertaken to determine the effects of transdermal nicotine at doses of 17.5, 35 and 52.5 mg on sleep and PGO spike activity. Minor effects were observed on sleep with a general increase in waking. PGO spike activity was abolished by all patches. The results are discussed in terms of the mechanisms involved in the disappearance of PGO spikes as a result of nicotine.

Nicotine stimulation of dorsal raphe neurons: effects on laterodorsal and pedunculopontine neurons

Previous studies showed that nicotine suppresses the ponto-geniculo-occipital (PGO) spikes of rapid eye movement (REM) sleep in cats. This effect may depend on stimulation of dorsal raphe nucleus (DRN) serotoninergic neurons that inhibit the pedunculopontine (PPT) and laterodorsal tegmental (LDT) cholinergic neurons, generators of PGO spikes. For testing this hypothesis 37 experiments were performed in rat midbrain slices. Nicotine (2 mM), administered locally into DRN, significantly increased the firing rate of 81.1% DRN neurons and serotonin release while simultaneously and significantly decreasing the firing rate of 80.8% LDT neurons and of 81.8% PPT neurons. The inhibition of LDT neurons by nicotine administered into DRN was blocked by the 5-HT1A receptor antagonist WAY-100635 (140 nM) administered into LDT. These results indicate that nicotine inhibits the activity of LDT and PPT neurons and consequently the generation of PGO spikes through stimulation of DRN serotoninergic neurons.

Subcutaneous administration of nicotine changes dorsal raphe serotonergic neurons discharge rate during REM sleep

In the present study nicotine (0.1 mg/kg, s.c.) increased discharge rate of putative dorsal raphe (DRN) serotonergic neurons of behaving rats during REM sleep (362.61%), without any significant change during waking and non-REM sleep. Since serotonergic DRN neurons gate PGO onset, these results suggest that nicotine-induced suppression of PGO spikes during REM sleep previously reported is achieved through stimulation of dorsal raphe serotonergic cells.