Sharon Sampogna

Hypocretin (orexin) input to trigeminal and hypoglossal motoneurons in the cat: a double-labeling immunohistochemical study

In trigeminal and hypoglossal motor nuclei of adult cats, hypocretin immunoreactive fiber varicosities were observed in apposition to retrogradely labeled motoneuron somata and dendrites. Among those lateral hypothalamus neurons that project to the hypoglossal nucleus some were determined to be hypocretin immunoreactive and were located amongst the single-labeled hypocretinergic neurons. These data suggest that hypocretin may play a role in the synaptic control of these motoneurons.

Colocalization of γ-aminobutyric acid and acetylcholine in neurons in the laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and electron microscopic study

Cholinergic and gamma-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6-8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.

MCH-containing neurons in the hypothalamus of the cat: Searching for a role in the control of sleep and wakefulness

Neurons that utilize melanin-concentrating hormone (MCH) and others that employ hypocretin as neurotransmitter are located in the hypothalamus and project diffusely throughout the CNS, including areas that participate in the generation and maintenance of the states of sleep and wakefulness. In the present report, immunohistochemical methods were employed to examine the distribution of MCHergic and hypocretinergic neurons. In order to test the hypothesis that the MCHergic system is capable of influencing specific behavioral states, we studied Fos immunoreactivity in MCH-containing neurons during (1) quiet wakefulness, (2) active wakefulness with motor activity, (3) active wakefulness without motor activity, (4) quiet sleep and (5) active sleep induced by carbachol (AS-carbachol). We determined that MCHergic neuronal somata in the cat are intermingled with hypocretinergic neurons in the dorsal and lateral hypothalamus, principally in the tuberal and tuberomammillary regions; however, hypocretinergic neurons extended more in the anterior-posterior axis than MCHergic neurons. Axosomatic and axodendritic contacts were common between these neurons. In contrast to hypocretinergic neurons, which are known to be active during motor activity and AS-carbachol, Fos immunoreactivity was not observed in MCH-containing neurons in conjunction with any of the preceding behavioral conditions. Non-MCHergic, non-hypocretinergic neurons that expressed c-fos during active wakefulness with motor activity were intermingled with MCH and hypocretin-containing neurons, suggesting that these neurons are related to some aspect of motor function. Further studies are required to elucidate the functional sequela of the interactions between MCHergic and hypocretinergic neurons and the phenotype of the other neurons that were active during motor activity.

Distribution of hypocretin (orexin) immunoreactivity in the feline pons and medulla

The distribution of hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) immunoreactivities in the cat brainstem was examined using immunohistochemical techniques. Hcrt-1- and hcrt-2-positive fibers with varicosities were detected in almost all brainstem regions. However, no hcrt-1- or hcrt-2-immunoreactive neuronal somata were observed in the cat brainstem. Both hcrt-1- and hcrt-2-labeled fibers exhibited different densities in distinct regions of the brainstem. In most brainstem regions, the intensity of hcrt-1 immunoreactivity was higher than that of hcrt-2 immunoreactivity. The highest densities of hcrt-1- and hcrt-2-positive fibers were found in the nucleus raphe dorsalis (RD), the laterodorsal tegmental nucleus (LDT) and the locus coeruleus (LC), suggesting an important role for these peptides in functions related to sleep-wake behavior.

GABAergic neurons of the laterodorsal and pedunculopontine tegmental nuclei of the cat express c-fos during carbachol-induced active sleep

The laterodorsal and pedunculopontine tegmental nuclei (LDT-PPT) are involved in the generation of active sleep (AS; also called REM or rapid eye movement sleep). Although the LDT-PPT are composed principally of cholinergic neurons that participate in the control of sleep and waking states, the function of the large number of GABAergic neurons that are also located in the LDT-PPT is unknown. Consequently, we sought to determine if these neurons are activated (as indicated by their c-fos expression) during active sleep induced by the microinjection of carbachol into the rostro-dorsal pons (AS-carbachol). Accordingly, immunocytochemical double-labeling techniques were used to identify GABA and Fos protein, as well as choline acetyltransferase (ChAT), in histological sections of the LDT-PPT. Compared to control awake cats, there was a larger number of GABAergic neurons that expressed c-fos during AS-carbachol (31.5+/-6.1 vs. 112+/-15.2, P<0.005). This increase in the number of GABA+Fos+ neurons occurred on the ipsilateral side relative to the injection site; there was a small decrease in GABA+Fos+ cells in the contralateral LDT-PPT. However, the LDT-PPT neurons that exhibited the largest increase in c-fos expression during AS-carbachol were neither GABA+ nor ChAT+ (47+/-22.5 vs. 228.7+/-14.0, P<0.0005). The number of cholinergic neurons that expressed c-fos during AS-carbachol was not significantly different compared to wakefulness. These data demonstrate that, during AS-carbachol, GABAergic as well as an unidentified population of neurons are activated in the LDT-PPT. We propose that these non-cholinergic LDT-PPT neurons may participate in the regulation of active sleep.

MCHergic projections to the nucleus pontis oralis participate in the control of active (REM) sleep.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are located in the lateral hypothalamus and incerto-hypothalamic area and project diffusely throughout the central nervous system, including areas that participate in the generation and maintenance of sleep and wakefulness. Recent studies have shown that hypothalamic MCHergic neurons are active during active sleep (AS), and that intraventricular microinjections of MCH induce AS sleep; however, there are no data available regarding the manner in which MCHergic neurons participate in the control of this behavioral state. Utilizing immunohistochemical and retrograde tracing techniques, we examined, in the cat, projections from MCHergic neurons to the nucleus pontis oralis (NPO), which is considered to be the executive area that is responsible for the generation and maintenance of AS. In addition, we explored the effects on sleep and waking states produced by the microinjection of MCH into the NPO. We first determined that MCHergic fibers and terminals are present in the NPO. We also found that when a retrograde tracer (cholera toxin subunit B) was placed in the NPO MCHergic neurons of the hypothalamus were labeled. When MCH was microinjected into the NPO, there was a significant increase in the amount of AS (19.8+/-1.4% versus 11.9+/-0.2%, P<0.05) and a significant decrease in the latency to AS (10.4+/-4.2 versus 26.6+/-2.3 min, P<0.05). The preceding anatomical and functional data support our hypothesis that the MCHergic system participates in the regulation of AS by modulating neuronal activity in the NPO.

Fos and serotonin immunoreactivity in the raphe nuclei of the cat during carbachol-induced active sleep: A double-labeling study

The microinjection of carbachol into the nucleus pontis oralis produces a state which is polygraphically and behaviorally similar to active sleep (rapid eye movement sleep). In the present study, using double-labeling techniques for serotonin and the protein product of c-fos (Fos), we sought to examine whether immunocytochemically identified serotonergic neurons of the raphe nuclei of the cat were activated, as indicated by their expression of c-fos, during this pharmacologically-induced behavioral state (active sleep-carbachol). Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited a significantly greater number of c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus. Whereas most of the c-fos-expressing neurons in the raphe dorsalis were small, those in the raphe magnus were medium-sized and in the raphe pallidus they were small and medium-sized. The mean number of serotonergic neurons that expressed c-fos (i.e. double-labeled cells) was similar in control and active sleep-carbachol cats. These data indicate that there is an increased number of non-serotonergic, c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus during the carbachol-induced state.(ABSTRACT TRUNCATED AT 250 WORDS)

Brainstem glycinergic neurons and their activation during active (rapid eye movement) sleep in the cat.

It is well established that, during rapid eye movement (REM) sleep, somatic motoneurons are subjected to a barrage of inhibitory synaptic potentials that are mediated by glycine. However, the source of this inhibition, which is crucial for the maintenance and preservation of REM sleep, has not been identified. Consequently, the present study was undertaken to determine in cats the location of the glycinergic neurons, that are activated during active sleep, and are responsible for the postsynaptic inhibition of motoneurons that occurs during this state. For this purpose, a pharmacologically-induced state of active sleep (AS-carbachol) was employed. Antibodies against glycine-conjugated proteins were used to identify glycinergic neurons and immunocytochemical techniques to label the Fos protein were employed to identify activated neurons. Two distinct populations of glycinergic neurons that expressed c-fos were distinguished. One population was situated within the nucleus reticularis gigantocellularis (NRGc) and nucleus magnocellularis (Mc) in the rostro-ventral medulla; this group of neurons extended caudally to the ventral portion of the nucleus paramedianus reticularis (nPR). Forty percent of the glycinergic neurons in the NRGc and Mc and 25% in the nPR expressed c-fos during AS-carbachol. A second population was located in the caudal medulla adjacent to the nucleus ambiguus (nAmb), wherein 40% of the glycinergic cells expressed c-fos during AS-carbachol. Neither population of glycinergic cells expressed c-fos during quiet wakefulness or quiet (non-rapid eye movement) sleep. We suggest that the population of glycinergic neurons in the NRGc, Mc, and nPR participates in the inhibition of somatic brainstem motoneurons during active sleep. These neurons may also be responsible for the inhibition of sensory and other processes during this state. It is likely that the group of glycinergic neurons adjacent to the nucleus ambiguus (nAmb) is responsible for the active sleep-selective inhibition of motoneurons that innervate the muscles of the larynx and pharynx.

Apnea promotes glutamate-induced excitotoxicity in hippocampal neurons.

Patients with obstructive sleep apnea (OSA) exhibit hippocampal damage and cognitive deficits. To determine the effect of apnea on the synaptic transmission in the hippocampus, we performed electrophysiological studies in an in vivo guinea pig model of OSA. Specifically, we determined the cornu ammonis region 1 (CA1) field excitatory postsynaptic potential (fEPSP) response to cornu ammonis region 3 (CA3) stimulation and examined the presynaptic mechanisms underlying the changes in the fEPSP. Single episodes of apnea resulted in a maximal potentiation of the fEPSPs at 1 to 3 min after the termination of each episode of apnea. The mean amplitude and slope of the post-apneic fEPSP was significantly larger compared with the pre-apneic control. These changes were accompanied by a significant decrease in the paired-pulse facilitation ratio during the post-apneic period compared with the pre-apneic control. The N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801, when applied locally to the CA1 recording site by pressure ejection, blocked the apnea-induced potentiation of the fEPSP. In the experimental animals that were subjected to extended periods of recurrent apnea, CA1 neurons exhibited positive immunoreactivity for fragmented DNA strands, which indicates apoptotic cell death. The present results demonstrate that apnea-induced potentiation of the hippocampal CA1 fEPSP is mediated by an NMDA receptor mechanism. We therefore conclude that recurrent apnea produces abnormally high levels of glutamate that results in the apoptosis of CA1 neurons. We hypothesize that this damage is reflected by the cognitive deficits that are commonly observed in patients with breathing disorders such as OSA.

Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated.