Hypothalamic hypocretinergic/orexinergic neurons projecting to the oral pontine rapid eye movement sleep inducing site in the cat.

The cat ventral oral pontine reticular nucleus (vRPO) is responsible for the generation and maintenance of rapid eye movement (REM) sleep. Hypothalamic neurons containing the peptide hypocretin-1 (also called orexin-A) which will be herewith defined as orexinergic (Orx) neurons, occupy a pre-eminent place in the integration and stabilization of arousal networks as well as in the physiopathology of narcolepsy/cataplexy. In the previous investigations, low-volume and dose microinjections of hypocretin-1 in cat vRPO produced a specific and significant suppression of REM sleep. The aim of this study is to map the hypothalamic Orx neurons that project to the vRPO and suppress REM sleep generation in the cat. Five adult cats received microinjections of the retrograde tracer cholera toxin (CTb) into the vRPO. Brains were processed employing both CTb staining and antiorexin-A immunocytochemistry techniques. A large number of double-labeled neurons (Orx-CTb) intermingled with the single CTb-positive and single Orx neurons were detected in the ipsilateral lateral, perifornical, dorsal, anterior, perimammillothalamic, and posterior hypothalamic areas but were very scarce in the paraventricular, dorsomedial, ventromedial, and periventricular hypothalamic nuclei. A considerable number of double-labeled neurons were also observed in both the dorsal and the lateral hypothalamic areas in the contralateral hypothalamus. Our results suggest that the widely distributed Orx neuronal hypothalamic groups could physiologically inhibit REM sleep generation in vRPO.

Functional Anatomy of Non-REM Sleep.

The state of non-REM sleep (NREM), or slow wave sleep, is associated with a synchronized EEG pattern in which sleep spindles and/or K complexes and high-voltage slow wave activity (SWA) can be recorded over the entire cortical surface. In humans, NREM is subdivided into stages 2 and 3-4 (presently named N3) depending on the proportions of each of these polygraphic events. NREM is necessary for normal physical and intellectual performance and behavior. An overview of the brain structures involved in NREM generation shows that the thalamus and the cerebral cortex are absolutely necessary for the most significant bioelectric and behavioral events of NREM to be expressed; other structures like the basal forebrain, anterior hypothalamus, cerebellum, caudal brain stem, spinal cord and peripheral nerves contribute to NREM regulation and modulation. In NREM stage 2, sustained hyperpolarized membrane potential levels resulting from interaction between thalamic reticular and projection neurons gives rise to spindle oscillations in the membrane potential; the initiation and termination of individual spindle sequences depends on corticothalamic activities. Cortical and thalamic mechanisms are also involved in the generation of EEG delta SWA that appears in deep stage 3-4 (N3) NREM; the cortex has classically been considered to be the structure that generates this activity, but delta oscillations can also be generated in thalamocortical neurons. NREM is probably necessary to normalize synapses to a sustainable basal condition that can ensure cellular homeostasis. Sleep homeostasis depends not only on the duration of prior wakefulness but also on its intensity, and sleep need increases when wakefulness is associated with learning. NREM seems to ensure cell homeostasis by reducing the number of synaptic connections to a basic level; based on simple energy demands, cerebral energy economizing during NREM sleep is one of the prevalent hypotheses to explain NREM homeostasis.

Functional anatomy of the sleep-wakefulness cycle: wakefulness.

Sleep is a necessary, diverse, periodic, and an active condition circadian and homeostatically regulated and precisely meshed with waking time into the sleep-wakefulness cycle (SWC). Photic retinal stimulation modulates the suprachiasmatic nucleus, which acts as the pacemaker for SWC rhythmicity. Both the light period and social cues adjust the internal clock, making the SWC a circadian, 24-h period in the adult human. Bioelectrical and behavioral parameters characterize the different phases of the SWC. For a long time, lesions and electrical stimulation of brain structures, as well as connection studies, were the main methods used to decipher the foundations of the functional anatomy of the SWC. That is why the first section of this review presents these early historical studies to then discuss the current state of our knowledge based on our understanding of the functional anatomy of the structures underlying the SWC. Supported by this description, we then present a detailed review and update of the structures involved in the phase of wakefulness (W), including their morphological, functional, and chemical characteristics, as well as their anatomical connections. The structures for W generation are known as the "ascending reticular activating system", and they keep and maintain the "thalamo-cerebral cortex unit" awake. This system originates from the neuronal groups located within the brainstem, hypothalamus, and basal forebrain, which use known neurotransmitters and whose neurons are more active during W than during the other SWC states. Thus, synergies among several of these neurotransmitters are necessary to generate the cortical and thalamic activation that is characteristic of the W state, with all the plastic qualities and nuances present in its different behavioral circumstances. Each one of the neurotransmitters exerts powerful influences on the information and cognitive processes as well as attentional, emotional, motivational, behavioral, and arousal states. The awake "thalamo-cerebral cortex unit" controls and adjusts the activation pattern through a top-down action on the subcortical cellular groups that are the origin of the "ascending reticular activating system".

[The "thalamus-cerebral cortex" anatomofunctinal unit in the waking state]. / La unidad anatomofuncional "tálamo-corteza cerebral" en el estado de vigilia.

The "thalamus-cerebral cortex unit" is awakened by the "ascending reticular activating system", which originates from neuronal groups located within the brainstem, hypothalamus and basal forebrain, all of which use known neurotransmitters and whose neurons are more active during wakefulness than during the other behavioral states. Synergies among several of these neurotransmitters are necessary to generate the cortical and thalamic activation that is characteristic of wakefulness. During the wakefulness the complex interrelated structures of the "thalamus-cerebral cortex unit" provide the pathway that interconnects cortex and thalamus and allow the modulation, and organization of the mechanisms producing the adequate activity of the different thalamic and cortical formations and the organization of cognitive processes and performance of appropriate behavioral responses.

[REM sleep modulation by non-GABAergic neurons of the hypothalamus and basal forebrain]. / Modulación del sueño REM por neuronas no-GABAérgicas del hipotálamo y prosencéfalo basal.

The ventral part of the oral pontine reticular nucleus (vRPO) is a demonstrated site of brainstem REM-sleep generation and maintenance. The vRPO has reciprocal connections with structures that control other states of the sleep-wakefulness cycle, many situated in the basal forebrain and the diencephalon. The aim of the present revision is to map, using the results described in previous publications of our group, the local origin of the basal forebrain and hypothalamus non-GABAergic projections to the vRPO, and specially the contribution of the hypothalamic neurons positive to hypocretin/orexin (H/O) peptides. I summarize non-GABAergic projections to the vRPO from the: ipsilateral central amygdaline nucleus and the stria terminalis bed nuclei, bilateral projections, but most abundant in the ipsilateral side, from the median preoptic nucleus, medial and lateral preoptic areas, abundant from the zona incerta and dorsal, lateral, posterior and perifornical hypothalamic areas. Very abundant bilateral projections of H/O neurons to the vRPO are described, expressive of the important modulation exerted by these neurons on the vRPO nucleus. I discuss the functional significance of the above results and the corresponding mechanisms, supported by physiological and ultrastructural results of our group. Based on the connections and action mechanisms of H/O neurons on the vRPO, which produce the decreased activity of neurons in this nucleus and, therefore, inhibition of REM sleep, I reflect briefly on narcolepsy pathophysiology.

Kainic acid intraocular injections during the postnatal critical period induce plastic changes in the visual system.

Changes in the retino-collicular projection and in the number of optic nerve (ON) axons in adult rats were analyzed after partial loss of retinal ganglion cells (RGCs), induced by intravitreal injections of kainic acid (KA) on postnatal days 2-3 (P2-P3) or 10-12 (P10-P12). KA injected at P2-P3 decreased the volume of the adult contralateral superior colliculus (SC) and the density of the retino-collicular contralateral projection, but maintained the neonatal pattern in the ipsilateral projection from the un-injected eye. ON axon number was significantly increased in the un-injected eye but decreased in the KA-injected eye. Thus, restriction of the ipsilateral retino-collicular projection and RGC death in the un-injected eye are modified by KA at P2-P3, during the postnatal critical period, but not at P10-P12, after it is over. We suggest that, in the SC contralateral to the KA-injected eye, the disappearance of axon terminals belonging to RGC killed by KA would decrease competition between ipsilateral and contralateral terminals, thus contributing to maintaining the neonatal pattern in the ipsilateral retino-collicular projection. The reduction in RGC death in the un-injected eye could also be related to the disappearance of RGC terminals in the contralateral SC, which would have increased neurotrophic factor availability.

[Vegetative condition (state) and conscious thought. Reflections scientific neuro and ethics]. / Estado vegetativo y pensamiento consciente. Reflexiones neurocientíficas y éticas.

Thirty years ago, the study of the brain lesions of a patient who had remained in a coma for eight years, together with my experience on the degree of wakefulness of animals with similar lesions, compelled me to reflecting on the level of consciousness that our patient had. Recent findings from functional magnetic resonance image, that have shown aspects of speech perception, emotional processing, language comprehension and even conscious awareness might be retained in patients who behaviourally meet all of the criteria that define the vegetative state, have allowed me to make new neuroscientists and ethical reflections.

[Origins and first steps of the Spanish Society for Neuroscience]. / Orígenes y primeros pasos en la Sociedad Española de Neurociencia (SENC).

I recall the background, the environment, the people and the events that led to the birth of the Spanish Society for Neuroscience (SENC) and remember how and why the multidisciplinary Neurobiology teachers at the Medical School of the Universidad Autónoma de Madrid decided to organize the First Meeting of Spanish Neurobiologists in 1979. Our principal aim was to promote Neuroscience research in Spain. For this was necessary: to know each other, support each other and organize and set up a modern and solid framework for training young researchers in Neuroscience. After reporting the results and circumstances of the first two Meetings, in 1980 and 1981, I discuss the impact of the Sixth European Neuroscience Congress held in Torremolinos in 1982 on Neuroscience in our country. The 1983 Meeting of the Spanish Neurobiologists decided to create the Spanish Society for Neuroscience. The effort of the heterogeneous Management Commission, the preparation of the Bylaws, the selection of the first members and the birth of the Society in 1985 are outlined. I continue in describing the components and work of the three first Boards of Directors and events of the corresponding Congresses until the consolidation of SENC in national and international scientific fields. My talk runs through the development of our Society, its growth in membership and quality and our hopes for the future.

Sleep-wakefulness effects after microinjections of hypocretin 1 (orexin A) in cholinoceptive areas of the cat oral pontine tegmentum.

Hypocretinergic/orexinergic neurons, which are known to be implicated in narcolepsy, project to the pontine tegmentum areas involved in the control of rapid eye movement (REM) sleep. Here, we report the effects on sleep-wakefulness produced by low-volume microinjections of hypocretin (Hcrt)1 (20-30 nL, 100, 500 and 1000 microm) and carbachol (20-30 nL, 0.1 m) delivered in two areas of the oral pontine tegmentum of free-moving cats with electrodes for chronic sleep recordings: in the dorsal oral pontine tegmentum (DOPT) and in the ventral part of the oral pontine reticular nucleus (vRPO). Carbachol in the DOPT produced dissociate polygraphic states, with some but not all REM sleep signs. In contrast, carbachol in the vRPO produced a shift with short latency from wakefulness (W) to REM sleep with all of its polygraphic and behavioral signs. Hcrt-1 in the DOPT increased W and decreased both slow-wave sleep (SWS) and REM sleep during the first 3 h post-drug. The same doses of Hcr-1 in the vRPO produced a significant suppression of REM sleep without a definitive trend for changes in the other states. Both groups showed significant decreases in the number of transitions from SWS to REM sleep. Thus, Hcrt-1 produced distinct effects in cholinoceptive areas of the oral pontine tegmentum; in the DOPT it promoted W, suppressed SWS and probably defacilitated REM sleep, and in the vRPO it directly inhibited REM sleep. Hypocretinergic/orexinergic signaling is lost in narcoleptics and this absence would mean that pontine defacilitation/inhibition of REM sleep would also be absent, explaining why these patients can fall directly into REM sleep from W.

GABAergic and non-GABAergic thalamic, hypothalamic and basal forebrain projections to the ventral oral pontine reticular nucleus: their implication in REM sleep modulation.

The ventral part of the oral pontine reticular nucleus (vRPO) is a demonstrated site of brainstem REM-sleep generation and maintenance. The vRPO has reciprocal connections with structures that control other states of the sleep-wakefulness cycle, many situated in the basal forebrain and the diencephalon. Some of these connections utilize the inhibitory neurotransmitter GABA. The aim of the present work is to map the local origin of the basal forebrain and diencephalon projections to the vRPO whether GABAergic or non-GABAergic. A double-labelling technique combining vRPO injections of the neuronal tracer, cholera-toxin (CTB), with GAD-immunohistochemistry, was used for this purpose in adult cats. All of the numerous CTB-positive neurons in the reticular thalamic and dorsocaudal hypothalamic nuclei were double-labelled (CTB/GAD-positive) neurons. Approximately 15%, 14% and 16% of the CTB-positive neurons in the zona incerta and the dorsal and lateral hypothalamic areas are, respectively, CTB/GAD-positive neurons. However, only some double-labelled neurons were found in other hypothalamic nuclei with abundant CTB-positive neurons, such as the paraventricular nucleus, perifornical area and H1 Forel field. In addition, CTB-positive neurons were abundant in the central amygdaline nucleus, terminal stria bed nuclei, median preoptic nucleus, medial and lateral preoptic areas, dorsomedial and ventromedial hypothalamic nuclei, posterior hypothalamic area and periventricular thalamic nucleus. The GABAergic and non-GABAergic connections described here may be the morphological pillar through which these prosencephalic structures modulate, either by inhibiting or by exciting, the vRPO REM-sleep inducing neurons during the different sleep-wakefulness cycle states.

[Modulation by the GABA of the ventro-oral-pontine reticular REM sleep-inducing neurons]. / Modulación por el acido gamma-aminobutírico (GABA) del sueño de movimientos oculares rápidos (REM).

From a multidisciplinary study in our laboratory we have compiled numerous findings on the role played by the inhibitory neurotransmitter GABA in the ventral part of the oral pontine reticular nucleus (vRPO), REM sleep induction and maintenance brainstem structure. Functional GABA in the vRPO is located in a few small and scattered neuronal bodies, and in an abundant number of synaptic terminals: 30% of all synaptic terminals in vRPO are GABAergic. These terminals form inhibitory, symmetric synapses on the soma and different segments of the dendritic tree of the vRPO neurons, mainly in those of large diameter. In unitary intracellular studies, in vitro, we have demonstrated that GABA produces hyperpolarization of the vRPO neurons. In vivo experiments in freely moving cats, local microinjections of the GABA(A) receptor agonist muscimol decreased REM sleep. The different densities of GABA-immunoreactions and the diverse and complex morphological ultrastructure of the vRPO GABAergic terminals suggest that they have different origins and physiologic functions. There are GABAergic projections to the vRPO from diencephalic structures related with the other phases of the sleep-wakefulness cycle: wakefulness and non-REM sleep, which may be anatomical substrata for the GABAergic inhibition of the vRPO REM sleep-inducing neurons during these other phases.

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat.

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase-wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus alpha and peri-alpha, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.

Projections from the cat posterior lateral hypothalamus to the ventral part of the oral pontine reticular nucleus contain a GABAergic component.

The posterior lateral hypothalamus (PLH) has long been considered crucial to normal wakefulness while the ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. However, to date, there is no information on the ultrastructure or neurotransmitter content of the hypothalamo-reticular projection. In the present study, we examined the morphology and synaptic organization of PLH terminals in the vRPO using PLH injections of biotinylated dextran amine (BDA) as well as of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Since some PLH neurons are GABAergic, we used a post-embedding immunogold technique to determine whether any anterogradely labeled terminals were GABA-immunopositive. Electron microscope analyses revealed a variety of ultrastructural features in the vRPO anterogradely labeled terminals. Although most labeled terminals (over 63%) formed symmetric synapses on vRPO somata and dendrites, others made asymmetric synapses on vRPO dendrites. The relative percentages of labeled terminals observed on large, medium and small diameter dendrites were 44.3 +/- 5.5%, 35.3 +/- 3.0% and 20.4 +/- 3.1%, respectively. Finally, post-embedding immunogold technique revealed that there are GABA-immunopositive and immunonegative components to this projection, indicating that GABA is one of the transmitters used by the PLH cells that project to the vRPO. Furthermore, most, if not all, of the GABA-labeled axon terminals formed symmetric synapsis. In conclusion, our results suggest that the PLH could modulate the physiological responses of vRPO neurons through a GABAergic pathway as well as by other inhibitory and/or excitatory pathways. Activation of the descending PLH GABAergic projection may inhibit the REM sleep-inducing neurons within the vRPO and thus contribute to the suppression of REM sleep activation during wakefulness.

[The orbitofrotal cortex I: anatomy and memory procesing]. / La corteza orbitofrontal I: anatomía y procesamiento de la memoria.

The orbitofrontal cortex has extensive tight connections with the medial temporal tobe and medial thalamic structures, which are responsible for memory processing and consolidation. What is more, this cortex is constantly activated in the memory encoding processes. This makes the orbitofrontal cortex a critical region for memory formation. This cortex is also connected with the motor and hetero- and uni- modal association sensory cortices, the limbic cortices, and subcortical structures responsible for functions related with these systems. All these facts convert the orbitofrontal cortex into a nodal region within the neural networks responsible of selecting, assembling and analyzing, based on our memory, present and pass experiences, so that we can organize and decide the most appropriate behaviour in a given situation.

[The biography of a neuron]. / Biografía de una neurona.

A summary review of current concepts regarding the major events in the long life cycle (the "biography") of a typical human brain neuron, and the main cellular and molecular mechanisms involved, is provided. Once born in the embryo, neurons never undergo cell division: Their differentiated phenotype, which includes the multiple neuronal networks that each cell establishes, is the cumulative result, over years of development, if many cell-autonomous (intrinsic) and intercellular (extrinsic) signaling events that regulate gene expression. The effect of such signals is not just qualitative, but dependent on their precise timing and dosage. Moreover, most intercelullar signals are powerfully regulated by tissue spatial constraints, or by the patterned bioelectrical activity (spontaneous and/or experience-related) of the developing neuronal networks. Thus, a major part of the biological information required to build each adult neuron is coded by a myriad of temporally-, spatially- or activity-dependent signaling events that occur within the developing neuronal networks themselves over a very protracted period. These facts cast serious doubt on the biological soundness of cell-replacement strategies as substitutes for damaged adult brain neurons.

[Sleep, learning and memory]. / Sueño, aprendizaje y memoria.

As a subject, "Sleep, Learning and Memory" is quite lively. Many papers have been published on this subject in the last 20 years. However, these papers present major contradictions. This is logic because it is a complex subject and, consquently, without simple solutions. It is important to be precise, as much on a basic as on a clinical experimental level, in a large number of questions. For a correct interpretation of the data it is necessary to sustain a unified vision of human nervous system function. Today we can affirm that, for correct learning and memory processing, a normal and harmonic sleep-wakefulness cycle, including the interphase transition states, is essential. Many findings demonstrate the special and determining role of REM sleep in the mechanisms of memory consolidation.

Cajal hoy: la permanencia de un genio / No disponible

No disponible

[Adult neurogenesis and stem cells. Functional capacity]. / Neurogénesis en el adulto y células madre. Su capacidad funcional.

The ability of stem cells to give rise to new neurons in the adult central nervous system is a phenomenon that has raised many hopes. The possible doors opened by the potentiality of these cells in benefit of neurological patients are numerous. But we need more research and facts to avoid raising false expectations. There is no sound scientific proof to sustain the excitement created by embryonic stem cell therapy. In general, because this therapy is still a distant goal; in particular because the widespread degenerative processes underlying Parkinson's and Alzheimer's diseases make complete repair through transplant practically impossible. It is also hard to understand why research in human embryonic stem cells is being promoted when there are no previous sound results using animal embryonic stem cells. In addition, there are existing alternatives to embryonic stem cells, without ethical problems, that can obtain cells to restore damaged adult tissues, using more promising techniques with already contrasted results.

Brain structures and mechanisms involved in the generation of REM sleep.

This article reviews the central nervous mechanisms involved in the broad network that generates and maintains REM sleep. Experimental investigations have identified the pontine tegmentum as the critical substrate for REM sleep mechanisms. Several pontine structures are involved in the generation of each particular polygraphic event that characterizes REM sleep: desynchronization in the electroencephalogram, theta rhythm in the hippocampus, muscle atonia, pontogeniculooccipital waves and rapid eye movements. The pontine tegmentum also holds the region where cholinergic stimulation can trigger all the behavioural and bioelectric signs of REM sleep. The exact location has been investigated and amply discussed over the last few years. Studies in the authors>> laboratory, mapping the pontine tegmentum with small volume carbachol (a cholinergic agonist) microinjections, have demonstrated that the executive neurons for REM sleep generation are neither located in the dorsal part of the pontine tegmentum, nor diffusely spread through the medial pontine reticular formation: they are concentrated in a discrete area in the ventral part of the oral pontine reticular nucleus (vRPO). In turn, the vRPO has connections with structures involved in the generation of the other states of the sleep-wake cycle as well as with structures responsible for the generation of each of the different events characterizing REM sleep. This allows us to propose the vRPO as the crucial region for REM sleep generation. Related research, with invivo and invitro experiments, into the actions of different neurotransmitters on vRPO neurones indicates that not only acetylcholine but other neurotransmitters have an active key role in vRPO REM sleep generation mechanisms.