Neuroprotective Role of Dietary Supplementation with Omega-3 Fatty Acids in the Presence of Basal Forebrain Cholinergic Neurons Degeneration in Aged Mice.

As major components of neuronal membranes, omega-3 polyunsaturated fatty acids (n-3 PUFA) exhibit a wide range of regulatory functions. Recent human and animal studies indicate that n-3 PUFA may exert beneficial effects on aging processes. Here we analyzed the neuroprotective influence of n-3 PUFA supplementation on behavioral deficits, hippocampal neurogenesis, volume loss, and astrogliosis in aged mice that underwent a selective depletion of basal forebrain cholinergic neurons. Such a lesion represents a valid model to mimic a key component of the cognitive deficits associated with dementia. Aged mice were supplemented with n-3 PUFA or olive oil (as isocaloric control) for 8 weeks and then cholinergically depleted with mu-p75-saporin immunotoxin. Two weeks after lesioning, mice were behaviorally tested to assess anxious, motivational, social, mnesic, and depressive-like behaviors. Subsequently, morphological and biochemical analyses were performed. In lesioned aged mice the n-3 PUFA pre-treatment preserved explorative skills and associative retention memory, enhanced neurogenesis in the dentate gyrus, and reduced volume and VAChT levels loss as well as astrogliosis in hippocampus. The present findings demonstrating that n-3 PUFA supplementation before cholinergic depletion can counteract behavioral deficits and hippocampal neurodegeneration in aged mice advance a low-cost, non-invasive preventive tool to enhance life quality during aging.

A whole-brain map of long-range inputs to GABAergic interneurons in the mouse medial prefrontal cortex.

The medial prefrontal cortex (mPFC) contains populations of GABAergic interneurons that play different roles in cognition and emotion. Their local and long-range inputs are incompletely understood. We used monosynaptic rabies viral tracers in combination with fluorescence micro-optical sectioning tomography to generate a whole-brain atlas of direct long-range inputs to GABAergic interneurons in the mPFC of male mice. We discovered that three subtypes of GABAergic interneurons in two areas of the mPFC are innervated by same upstream areas. Input from subcortical upstream areas includes cholinergic neurons from the basal forebrain and serotonergic neurons (which co-release glutamate) from the raphe nuclei. Reconstruction of single-neuron morphology revealed novel substantia innominata-anteromedial thalamic nucleus-mPFC and striatum-anteromedial thalamic nucleus-mPFC circuits. Based on the projection logic of individual neurons, we classified cortical and hippocampal input neurons into several types. This atlas provides the anatomical foundation for understanding the functional organization of the mPFC.

Stable Sequential Activity Underlying the Maintenance of a Precisely Executed Skilled Behavior.

A vast array of motor skills can be maintained throughout life. Do these behaviors require stability of individual neuron tuning or can the output of a given circuit remain constant despite fluctuations in single cells? This question is difficult to address due to the variability inherent in most motor actions studied in the laboratory. A notable exception, however, is the courtship song of the adult zebra finch, which is a learned, highly precise motor act mediated by orderly dynamics within premotor neurons of the forebrain. By longitudinally tracking the activity of excitatory projection neurons during singing using two-photon calcium imaging, we find that both the number and the precise timing of song-related spiking events remain nearly identical over the span of several weeks to months. These findings demonstrate that learned, complex behaviors can be stabilized by maintaining precise and invariant tuning at the level of single neurons.

Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling ß chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

Principles of auditory processing differ between sensory and premotor structures of the songbird forebrain.

Sensory and motor brain structures work in collaboration during perception. To evaluate their respective contributions, the present study recorded neural responses to auditory stimulation at multiple sites simultaneously in both the higher-order auditory area NCM and the premotor area HVC of the songbird brain in awake zebra finches (Taeniopygia guttata). Bird's own song (BOS) and various conspecific songs (CON) were presented in both blocked and shuffled sequences. Neural responses showed plasticity in the form of stimulus-specific adaptation, with markedly different dynamics between the two structures. In NCM, the response decrease with repetition of each stimulus was gradual and long-lasting and did not differ between the stimuli or the stimulus presentation sequences. In contrast, HVC responses to CON stimuli decreased much more rapidly in the blocked than in the shuffled sequence. Furthermore, this decrease was more transient in HVC than in NCM, as shown by differential dynamics in the shuffled sequence. Responses to BOS in HVC decreased more gradually than to CON stimuli. The quality of neural representations, computed as the mutual information between stimuli and neural activity, was higher in NCM than in HVC. Conversely, internal functional correlations, estimated as the coherence between recording sites, were greater in HVC than in NCM. The cross-coherence between the two structures was weak and limited to low frequencies. These findings suggest that auditory communication signals are processed according to very different but complementary principles in NCM and HVC, a contrast that may inform study of the auditory and motor pathways for human speech processing.NEW & NOTEWORTHY Neural responses to auditory stimulation in sensory area NCM and premotor area HVC of the songbird forebrain show plasticity in the form of stimulus-specific adaptation with markedly different dynamics. These two structures also differ in stimulus representations and internal functional correlations. Accordingly, NCM seems to process the individually specific complex vocalizations of others based on prior familiarity, while HVC responses appear to be modulated by transitions and/or timing in the ongoing sequence of sounds.

Circuit-based interrogation of sleep control.

Sleep is a fundamental biological process observed widely in the animal kingdom, but the neural circuits generating sleep remain poorly understood. Understanding the brain mechanisms controlling sleep requires the identification of key neurons in the control circuits and mapping of their synaptic connections. Technical innovations over the past decade have greatly facilitated dissection of the sleep circuits. This has set the stage for understanding how a variety of environmental and physiological factors influence sleep. The ability to initiate and terminate sleep on command will also help us to elucidate its functions within and beyond the brain.

Cell migration in the developing rodent olfactory system.

The components of the nervous system are assembled in development by the process of cell migration. Although the principles of cell migration are conserved throughout the brain, different subsystems may predominantly utilize specific migratory mechanisms, or may display unusual features during migration. Examining these subsystems offers not only the potential for insights into the development of the system, but may also help in understanding disorders arising from aberrant cell migration. The olfactory system is an ancient sensory circuit that is essential for the survival and reproduction of a species. The organization of this circuit displays many evolutionarily conserved features in vertebrates, including molecular mechanisms and complex migratory pathways. In this review, we describe the elaborate migrations that populate each component of the olfactory system in rodents and compare them with those described in the well-studied neocortex. Understanding how the components of the olfactory system are assembled will not only shed light on the etiology of olfactory and sexual disorders, but will also offer insights into how conserved migratory mechanisms may have shaped the evolution of the brain.

Regulation of the orexigenic neuropeptide, enkephalin, by PPARδ and fatty acids in neurons of the hypothalamus and forebrain.

Ingestion of a high-fat diet composed mainly of the saturated fatty acid, palmitic (PA), and the unsaturated fatty acid, oleic (OA), stimulates transcription in the brain of the opioid neuropeptide, enkephalin (ENK), which promotes intake of substances of abuse. To understand possible underlying mechanisms, this study examined the nuclear receptors, peroxisome proliferator-activated receptors (PPARs), and tested in hypothalamic and forebrain neurons from rat embryos whether PPARs regulate endogenous ENK and the fatty acids themselves affect these PPARs and ENK. The first set of experiments demonstrated that knocking down PPARδ, but not PPARα or PPARγ, increased ENK transcription, activation of PPARδ by an agonist decreased ENK levels, and PPARδ neurons coexpressed ENK, suggesting that PPARδ negatively regulates ENK. In the second set of experiments, PA treatment of hypothalamic and forebrain neurons had no effect on PPARδ protein while stimulating ENK mRNA and protein, whereas OA increased both mRNA and protein levels of PPARδ in forebrain neurons while having no effect on ENK mRNA and increasing ENK levels. These findings show that PA has a strong, stimulatory effect on ENK and weak effect on PPARδ protein, whereas OA has a strong stimulatory effect on PPARδ and weak effect on ENK, consistent with the inhibitory effect of PPARδ on ENK. They suggest a function for PPARδ, perhaps protective in nature, in embryonic neurons exposed to fatty acids from a fat-rich diet and provide evidence for a mechanism contributing to differential effects of saturated and monounsaturated fatty acids on neurochemical systems involved in consummatory behavior. Our findings show that PPARδ in forebrain and hypothalamic neurons negatively regulates enkephalin (ENK), a peptide known to promote ingestive behavior. This inverse relationship is consistent with our additional findings, that a saturated (palmitic; PA) compared to a monounsaturated fatty acid (oleic; OA) has a strong stimulatory effect on ENK and weak effect on PPARδ. These results suggest that PPARδ protects against the neuronal effects of fatty acids, which differentially affect neurochemical systems involved in ingestive behavior.

A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

Male song quality modulates c-Fos expression in the auditory forebrain of the female canary.

In canaries, specific phrases of male song (sexy songs, SS) that are difficult to produce are especially attractive for females. Females exposed to SS produce more copulation displays and deposit more testosterone into their eggs than females exposed to non-sexy songs (NS). Increased expression of the immediate early genes c-Fos or zenk (a.k.a. egr-1) has been observed in the auditory forebrain of female songbirds hearing attractive songs. C-Fos immunoreactive (Fos-ir) cell numbers were quantified here in the brain of female canaries that had been collected 30min after they had been exposed for 60min to the playback of SS or NS or control white noise. Fos-ir cell numbers increased in the caudomedial mesopallium (CMM) and caudomedial nidopallium (NCM) of SS birds as compared to controls. Song playback (pooled SS and NS) also tended to increase average Fos-ir cell numbers in the mediobasal hypothalamus (MBH) but this effect did not reach full statistical significance. At the individual level, Fos expression in CMM was correlated with its expression in NCM and in MBH but also with the frequency of calls that females produced in response to the playbacks. These data thus indicate that male songs of different qualities induce a differential metabolic activation of NCM and CMM. The correlation between activation of auditory regions and of the MBH might reflect the link between auditory stimulation and changes in behavior and reproductive physiology.

Somatostatin and corticotrophin releasing hormone cell types are a major source of descending input from the forebrain to the parabrachial nucleus in mice.

The pontine parabrachial nucleus (PBN) receives substantial descending input from higher order forebrain regions that exerts inhibitory and excitatory influences on taste-evoked responses. Somatostatin (Sst) and corticotrophin releasing hormone (Crh) reporter mice were used in conjunction with injection of the retrograde tracer CTb-488 into the caudal PBN to determine the extent to which Sst and Crh cell types contribute to the descending pathways originating in the lateral hypothalamus (LH), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and insular cortex (IC). Five to 7 days following injections, the animals were euthanized and tissue sections prepared for confocal microscopy. Crh cell types in each forebrain site except IC project to the PBN with the greatest percentage originating in the BNST. For Sst cell types, the largest percentage of double-labeled cells was found in the CeA followed by the BNST. Few retrogradely labeled cells in the LH coexpressed Sst, whereas no double-labeled cells were observed in IC. The present results suggest that Sst and Crh cell types are a substantial component of the descending pathways from the amygdala and/or BNST to the PBN and are positioned to exert neuromodulatory effects on central taste processing.

Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: effects of age and sex.

Naturally occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described for the ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase-3 to identify dying cells in the brains of male and female mice from postnatal day (P) 1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain (the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and ventromedial nucleus of the hypothalamus; the basolateral, central, and medial amygdala; the lateral and principal nuclei of the bed nuclei of the stria terminalis; the caudate-putamen; the globus pallidus; the lateral septum; and the islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7, and the average rate of cell death varied tenfold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region-specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain.

Mutation of the BiP/GRP78 gene causes axon outgrowth and fasciculation defects in the thalamocortical connections of the mammalian forebrain.

Proper development of axonal connections is essential for brain function. A forward genetic screen for mice with defects in thalamocortical development previously isolated a mutant called baffled. Here we describe the axonal defects of baffled in further detail and identify a point mutation in the Hspa5 gene, encoding the endoplasmic reticulum chaperone BiP/GRP78. This hypomorphic mutation of BiP disrupts proper development of the thalamocortical axon projection and other forebrain axon tracts, as well as cortical lamination. In baffled mutant brains, a reduced number of thalamic axons innervate the cortex by the time of birth. Thalamocortical and corticothalamic axons are delayed, overfasciculated, and disorganized along their pathway through the ventral telencephalon. Furthermore, dissociated mutant neurons show reduced axon extension in vitro. Together, these findings demonstrate a sensitive requirement for the endoplasmic reticulum chaperone BiP/GRP78 during axon outgrowth and pathfinding in the developing mammalian brain.

Large-scale synchronized activity during vocal deviance detection in the zebra finch auditory forebrain.

Auditory systems bias responses to sounds that are unexpected on the basis of recent stimulus history, a phenomenon that has been widely studied using sequences of unmodulated tones (mismatch negativity; stimulus-specific adaptation). Such a paradigm, however, does not directly reflect problems that neural systems normally solve for adaptive behavior. We recorded multiunit responses in the caudomedial auditory forebrain of anesthetized zebra finches (Taeniopygia guttata) at 32 sites simultaneously, to contact calls that recur probabilistically at a rate that is used in communication. Neurons in secondary, but not primary, auditory areas respond preferentially to calls when they are unexpected (deviant) compared with the same calls when they are expected (standard). This response bias is predominantly due to sites more often not responding to standard events than to deviant events. When two call stimuli alternate between standard and deviant roles, most sites exhibit a response bias to deviant events of both stimuli. This suggests that biases are not based on a use-dependent decrease in response strength but involve a more complex mechanism that is sensitive to auditory deviance per se. Furthermore, between many secondary sites, responses are tightly synchronized, a phenomenon that is driven by internal neuronal interactions rather than by the timing of stimulus acoustic features. We hypothesize that this deviance-sensitive, internally synchronized network of neurons is involved in the involuntary capturing of attention by unexpected and behaviorally potentially relevant events in natural auditory scenes.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

Expression and rapid experience-dependent regulation of type-A GABAergic receptors in the songbird auditory forebrain.

GABAergic transmission influences sensory processing and experience-dependent plasticity in the adult brain. Little is known about the functional organization of inhibitory circuits in the auditory forebrain of songbirds, a robust model extensively used in the study of central auditory processing of behaviorally relevant communication signals. In particular, no information is currently available on the expression and organization of GABAA receptor-expressing neurons. Here, we studied the distribution and regulation of GABAA receptors in the songbird auditory forebrain, with a specific focus on α5, a subunit implicated in tonic inhibition and sensory learning. We obtained a zebra finch cDNA that encodes the α5-subunit (GABRA5) and carried out a detailed analysis of its expression via in situ hybridization. GABRA5 was highly expressed in the caudomedial nidopallium (NCM), caudomedial mesopallium, and field L2. Using double fluorescence in situ hybridization, we demonstrate that a large fraction of GABRA5-expressing neurons is engaged by auditory experience, as revealed by the song-induced expression of the activity-dependent gene zenk. Remarkably, we also found that α5 expression is rapidly regulated by sensory stimulation: 30 min of conspecific song playbacks significantly increase the number of GABRA5-expressing neurons in NCM, but not in other auditory areas. This effect is selective for α5, but not γ2 transcripts. Our results suggest that α5-containing GABAA receptors likely play a key role in central auditory processing and may contribute to the experience-dependent plasticity underlying auditory learning.

Electrophysiological analysis of the inhibitory effects of FMRFamide-like peptides on the pacemaker activity of gonadotropin-releasing hormone neurons.

Gonadotropin-releasing hormone (GnRH) neurons in the terminal nerve (TN) show endogenous pacemaker activity, which is suggested to be dependent on the physiological conditions of the animal. The TN-GnRH neurons have been suggested to function as a neuromodulatory neuron that regulates long-lasting changes in the animal behavior. It has been reported that the TN-GnRH neurons are immunoreactive to FMRFamide. Here, we find that the pacemaker activity of TN-GnRH neuron is inhibited by FMRFamide: bath application of FMRFamide decreased the frequency of pacemaker activity of TN-GnRH neurons in a dose-dependent manner. This decrease was suppressed by a blockage of G protein-coupled receptor pathway by GDP-ß-S. In addition, FMRFamide induced an increase in the membrane conductance, and the reversal potential for the FMRFamide-induced current changed according to the changes in [K(+)](out) as predicted from the Nernst equation for K(+). We performed cloning and sequence analysis of the PQRFamide (NPFF/NPAF) gene in the dwarf gourami and found evidence to suggest that FMRFamide-like peptide in TN-GnRH neurons of the dwarf gourami is NPFF. NPFF actually inhibited the pacemaker activity of TN-GnRH neurons, and this inhibition was blocked by RF9, a potent and selective antagonist for mammalian NPFF receptors. These results suggest that the activation of K(+) conductance by FMRFamide-like peptide (≈NPFF) released from TN-GnRH neurons themselves causes the hyperpolarization and then inhibition of pacemaker activity in TN-GnRH neurons. Because TN-GnRH neurons make tight cell clusters in the brain, it is possible that FMRFamide-like peptides released from TN-GnRH neurons negatively regulates the activities of their own (autocrine) and/or neighboring neurons (paracrine).

Distribution of leptin-sensitive cells in the postnatal and adult mouse brain.

Leptin plays a pivotal role in the regulation of energy homeostasis and neuroendocrine functions, and increasing evidence indicates that leptin acts on the brain to mediate many of these effects. Recent data have also suggested that leptin influences brain development during early postnatal life. Here we examined the distribution of cells that express mRNA encoding the long form of the leptin receptor (LepRb) in postnatal and adult mouse brains by using in situ hybridization. In both adults and neonates, LepRb mRNA was largely restricted to regions known to control energy balance. Labeled cells were found in the arcuate, ventromedial, and dorsomedial nuclei of the hypothalamus as well as in the lateral hypothalamic area. Heavily labeled cells were also found in the median preoptic and ventral premammillary nuclei, two hypothalamic nuclei that are known to control reproduction. Moreover, during postnatal and adult life, clearly labeled cells were found in extrahypothalamic autonomic control sites such as the nucleus of the tractus solitarius. Importantly, this receptor can induce intracellular signaling because peripheral injection of leptin caused STAT3 phosphorylation in most sites in which LepRb mRNA was expressed. LepRb mRNA was also transiently elevated in certain regions of the postnatal mouse brain, such as the cortex, hippocampus, and laterodorsal nucleus of the thalamus. Taken together, these observations are consistent with the proposed roles of leptin in feeding and neuroendocrine regulation. They also identify regions where LepRb mRNA is expressed during early postnatal life and suggest new roles for leptin in the nervous system during development.

Auditory spatial tuning at the crossroads of the midbrain and forebrain.

The barn owl's midbrain and forebrain contain neurons tuned to sound direction. The spatial receptive fields of these neurons result from sensitivity to combinations of interaural time (ITD) and level (ILD) differences over a broad frequency range. While a map of auditory space has been described in the midbrain, no similar topographic representation has been found in the forebrain. The first nuclei that belong exclusively to the forebrain and midbrain pathways are the thalamic nucleus ovoidalis (Ov) and the external nucleus of the inferior colliculus (ICx), respectively. The midbrain projects to the auditory thalamus before sharp spatial receptive fields emerge; although Ov and ICx receive projections from the same midbrain nuclei, they are not directly connected. We compared the spatial tuning in Ov and ICx. Thalamic neurons respond to a broader frequency range and their ITD and ILD tuning varied more across frequency. However, neurons in Ov showed spatial receptive fields as selective as neurons in ICx. Thalamic spatial receptive fields were tuned to frontal and contralateral space and correlated with their tuning to ITD and ILD. Our results indicate that spatial tuning emerges in both pathways by similar combination selectivity to ITD and ILD. However, the midbrain and the thalamus do not appear to repeat exactly the same processing, as indicated by the difference in frequency range and the broader tuning to binaural cues. The differences observed at the initial stages of these sound-localization pathways may reflect diverse functions and coding schemes of midbrain and forebrain.

Characterization and mapping of sleep-waking specific neurons in the basal forebrain and preoptic hypothalamus in mice.

We recorded 872 single units across the complete sleep-waking cycle in the mouse preoptic area (POA) and basal forebrain (BFB), which are deeply involved in the regulation of sleep and wakefulness (W). Of these, 552 were sleep-active, 96 were waking-active, 106 were active during both waking and paradoxical sleep (PS), and the remaining 118 were state-indifferent. Among the 872, we distinguished slow-wave sleep (SWS)-specific, SWS/PS-specific, PS-specific, W-specific, and W/PS-specific neurons, the last group being further divided into specific tonic type I slow (TI-Ss) and specific tonic type I rapid (TI-Rs) both discharging specifically in association with cortical activation during both W and PS. Both the SWS/PS-specific and PS-specific neurons were distributed throughout a wide region of the POA and BFB, whereas the SWS-specific neurons were mainly located in the middle and ventral half of the POA and adjacent BFB, as were the W-specific and W/PS-specific neurons. At the transition from waking to sleep, the majority of SWS-specific and all SWS/PS-specific neurons fired after the onset of cortical synchronization (deactivation), whereas all W-specific and W/PS-specific neurons showed a significant decrease in firing rate >0.5 s before the onset. At the transition from SWS to W, the sleep-specific neurons showed a significant decrease in firing rate 0.1 s before the onset of cortical activation, while the W-specific and W/PS-specific neurons fired >0.5 s before the onset. TI-Ss neurons were characterized by a triphasic broad action potential, slow single isolated firing, and an antidromic response to cortical stimulation, whereas TI-Rs neurons were characterized by a narrow action potential and high frequency burst discharge in association with theta waves in PS. These data suggest that the forebrain sleep/waking switch is regulated by opposing activities of sleep-promoting (SWS-specific and SWS/PS-specific) and waking-promoting (W-specific and W/PS-specific) neurons, that the initiation of sleep is caused by decreased activity of the waking-promoting neurons (disfacilitation), and that the W/PS-specific neurons are deeply involved in the processes of cortical activation/deactivation.