Anthony N. van den Pol

Hypothalamic Hypocretin (Orexin): Robust Innervation of the Spinal Cord

Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.

The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy.

The internal structure of the suprachiasmatic nucleus (SCN) was studied qualitatively and quantitatively in several hundred rat brains with a variety of methods including several types of Golgi impregnations, Nissl stains, horseradish peroxidase application, and electron microscopy. One suprachiasmatic nucleus has a volume of about 0.068 mm3 and contains close to 8,000 neurons. Dorsomedially, cells tend to be smaller and more tightly packed than ventrolaterally; significantly more somatic appositions occur in the dorsomedial SCN than in other parts of the nucleus. Two neurons with an extended region of somatic apposition may have no intercellular specializations, or they may be held together with various attachment plaques. Chains of neurons with long regions of somatosomatic apposition are found in the dorsomedial SCN with Nissl and silver stains, and with EM. The length of these chains is generally oriented in an antero‐posterior direction. Interspersed with the neurons are astroglia. Astroglia studied with Golgi impregnations, Cajals gold sublimate stain, and EM in SCN may have a rich cytoplasm falling in between the organelle‐rich cytoplasm of some large neurons and the organelle‐poor cytoplasm of some of the smaller SCN neurons. Nuclei of SCN glia and neurons are often invaginated and multiple nucleoli are a prominent feature of a large number of SCN neurons. With Golgi impregnations a number of relatively simple dendritic arbors exist. These include the simple bipolar cell, curly bipolar cell, radial neuron, monopolar neuron, and spinous cell. At the borders of the nucleus some dendrites may travel into the adjacent anterior hypothalamus; similarly, dendrites from cells outside SCN may enter into the nucleus boundaries. Compared with the rest of the hypothalamus, axons in SCN stain very poorly with conventional histological methods including Luxol blue, Bodian, Sevier‐Munger, Loyez, and Golgi, in part because of the fine caliber of the fibers. Golgi impregnated and silver‐stained axon fascicles composed primarily of unmyelinated axons may divide up within the nucleus or may pass through without maintaining local collaterals. Several different types of Golgi‐impregnated axonal arborizations terminate within SCN. Some have an extensive number of boutons ending on SCN somata, dendrites, and dendritic appendages. Other single axons pass through SCN without leaving any collaterals, or terminals in the nucleus. The majority of Golgi‐impregnated axons arising from SCN neurons maintain locally terminating collaterals, with boutons en passant and termineaux; these axons originate with equal frequency from the perikaryon or from a proximal dendrite. Golgi, horseradish peroxidase, and silver staining methods reveal axons connecting left and right SCN.

Hypocretin/Orexin Excites Hypocretin Neurons via a Local Glutamate Neuron—A Potential Mechanism for Orchestrating the Hypothalamic Arousal System

Neurons that release hypocretin/orexin modulate sleep, arousal, and energy homeostasis; the absence of hypocretin results in narcolepsy. Here we present data on the physiological characteristics of these cells, identified with GFP in transgenic mouse brain slices. Hypocretin-1 and -2 depolarized hypocretin neurons by 15mV and evoked an increase in spike frequency (+366% from a 1-3 Hz baseline). The mechanism for this appears to be hypocretin-mediated excitation of local glutamatergic neurons that regulate hypocretin neuron activity, in part by presynaptic facilitation of glutamate release. This represents a possible mechanism for orchestrating the output of the diffuse hypothalamic arousal system. No direct effect of hypocretin on membrane properties of hypocretin cells was detected. Norepinephrine and serotonin, transmitters of other arousal systems, decreased spike frequency and evoked outward currents, whereas acetylcholine and histamine had little effect.

Neuropeptide Transmission in Brain Circuits

Neuropeptides are found in many mammalian CNS neurons where they play key roles in modulating neuronal activity. In contrast to amino acid transmitter release at the synapse, neuropeptide release is not restricted to the synaptic specialization, and after release, a neuropeptide may diffuse some distance to exert its action through a G protein-coupled receptor. Some neuropeptides such as hypocretin/orexin are synthesized only in single regions of the brain, and the neurons releasing these peptides probably have similar functional roles. Other peptides such as neuropeptide Y (NPY) are synthesized throughout the brain, and neurons that synthesize the peptide in one region have no anatomical or functional connection with NPY neurons in other brain regions. Here, I review converging data revealing a complex interaction between slow-acting neuromodulator peptides and fast-acting amino acid transmitters in the control of energy homeostasis, drug addiction, mood and motivation, sleep-wake states, and neuroendocrine regulation.Neuropeptides are found in many mammalian CNS neurons where they play key roles in modulating neuronal activity. In contrast to amino acid transmitter release at the synapse, neuropeptide release is not restricted to the synaptic specialization, and after release, a neuropeptide may diffuse some distance to exert its action through a G protein-coupled receptor. Some neuropeptides such as hypocretin/orexin are synthesized only in single regions of the brain, and the neurons releasing these peptides probably have similar functional roles. Other peptides such as neuropeptide Y (NPY) are synthesized throughout the brain, and neurons that synthesize the peptide in one region have no anatomical or functional connection with NPY neurons in other brain regions. Here, I review converging data revealing a complex interaction between slow-acting neuromodulator peptides and fast-acting amino acid transmitters in the control of energy homeostasis, drug addiction, mood and motivation, sleep-wake states, and neuroendocrine regulation.

A fine-grained anatomical analysis of the role of the rat suprachiasmatic nucleus in circadian rhythms of feeding and drinking

Abstract Recent experiments have suggested that the suprachiasmatic nuclei (SCN) represent a neural timekeeper responsible for the maintenance of circadian rhythms of sleeping, drinking, general activity, plasma corticosterone, and pinealN-acetyltransferase. A rigorous evaluation of this proposal has been hampered, however, by the fact that past experiments have employed lesions many times larger than the volume of the SCN. To test the hypothesis that the SCN, as such, are necessary for certain circadian rhythms, the present study examined periodic variations in feeding and drinking before and after small (i.e. median size: 75% of total SCN volume) radiofrequency lesions and discrete knife cuts in the area of the SCN. The two behaviors were monitored in 120 male rats over a 7-week period. The animal room, kept at a constant temperature, was on a 12:12 light-dark cycle. Eating and drinking data were grouped in 6 four-hour time bins per day and analyzed using both autocorrelation coefficients and light-dark ratios. Brain-damaged animals were compared with sham-operated controls and blinded rats. Total damage to the suprachiasmatic nuclei, with little insult to adjacent areas (e.g. tractus infundibularis, supraoptic commissures. optic chiasm, area anterior to the SCN), was sufficient to eliminate circadian rhythms of both feeding and drinking. Conversely, lesions or knife cuts anterior, ventral or lateral to the SCN had little effect on the circadian rhythms. Damage directly posterior to the SCN abolished the circadian rhythms, probably by destroying efferents of the SCN. Loss of rhythms was sometimes seen with less than total destruction of the SCN. Rats with partial damage of the SCN showed a decrease in the autocorrelation coefficient describing their circadian rhythm. A computer-assisted procedure compared the three-dimensional reconstruction of each lesion with the circadian rhythm indicators, autocorrelation coefficients, and confirmed that the SCN and their immediate surround were necessary for the manifestation of circadian rhythms. Destruction of at least 50% of the SCN was sometimes associated with the appearance of a significant 8-h ultradian rhythm. This ultradian rhythm was not seen in any sham-operated control or blinded rat. The findings of the present study lend strong support to earlier suggestions that the SCN per se represent a critical area necessary for manifestation of certain circadian rhythms.

Physiological Properties of Hypothalamic MCH Neurons Identified with Selective Expression of Reporter Gene after Recombinant Virus Infection

Neurons that synthesize melanin-concentrating hormone (MCH) may modulate arousal and energy homeostasis. The scattered MCH neurons have been difficult to study, as they have no defining morphological characteristics. We have developed a viral approach with AAV for selective long-term reporter gene (GFP) expression in MCH neurons, allowing the study of their cellular physiology in hypothalamic slices. MCH neurons showed distinct membrane properties compared to other neurons infected with the same virus with a cytomegalovirus promoter. Transmitters of extrahypothalamic arousal systems, including norepinephrine, serotonin, and the acetylcholine agonist muscarine, evoked direct inhibitory actions. Orexigenic neuropeptide Y was inhibitory by pre- and postsynaptic mechanisms; an anorexigenic melanocortin agonist had no effect. In contrast, the hypothalamic arousal peptide hypocretin/orexin evoked a direct inward current and increased excitatory synaptic activity and spike frequency in the normally silent MCH neurons. Together, these data support the view that MCH neurons may integrate information within the arousal system in favor of energy conservation.

Enhanced early developmental expression of the metabotropic glutamate receptor mGluR5 in rat brain: Protein, mRNA splice variants, and regional distribution

Glutamate stimulates phosphatidyl inositol hydrolysis and mobilizes intracellular calcium through the mediation of metabotropic glutamate receptors (mGluRs), in particular the “Group I” receptors mGluRs, mGluR1, and mGluR5. This activity is markedly enhanced in developing brain relative to the adult. To determine whether this may be due to an increased amount of mGluR5 present in the developing brain, we examined mGluR5 expression using western blotting to measure mGluR5 protein, reverse transcription polymerase chain reaction (RT‐PCR) to measure mGluR5 mRNA, and immunocytochemistry to assess the regional distribution of mGluR5 morphologically. Western blotting revealed that in all brain regions examined there is more mGluR5 protein present in developing brain than in the adult. In most regions, the developmental decrease was over two‐fold. Total mGluR5 mRNA also decreased with development in most regions, but to a much lesser extent than the protein, suggesting that there is considerable post‐transcriptional regulation of the expression of this receptor. RT‐PCR analysis also demonstrated that in most regions the mGluR5a splice variant is most abundant in the young animals but mGluR5b predominates in the adult. Light microscopic immunocytochemistry indicated that expression is widespread in developing brain, and that the developmental decrease in receptor concentration is due to both an increased growth of receptor‐poor tissue regions and decreased expression within receptor‐rich regions.

Melanin concentrating hormone depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus

The neuropeptide melanin concentrating hormone (MCH) is synthesised only by neurons of the lateral hypothalamic (LH) area in the CNS. MCH cells project widely throughout the brain. Despite the growing interest in this peptide, in part related to its role in feeding, little has been done to characterise its physiological effects in neurons. Using whole‐cell recording with current and voltage clamp, we examined the cellular actions in neurons from the LH. MCH induced a consistent decrease in the frequency of action potentials and reduced synaptic activity. Most fast synaptic activity in the hypothalamus is mediated by GABA or glutamate. MCH inhibited the synaptic activity of both glutamatergic and GABAergic LH neurons, each tested independently. MCH reduced the amplitude of glutamate‐evoked currents and reduced the amplitude of miniature excitatory currents, indicating an inhibitory modulation of postsynaptic glutamate receptors. In the presence of tetrodotoxin to block action potentials, MCH caused a depression in the frequency of miniature glutamate‐mediated postsynaptic currents, suggesting a presynaptic site of receptor expression. In voltage clamp experiments, MCH depressed the amplitude of calcium currents, suggesting that a mechanism of inhibition may involve a reduced calcium‐dependent release of amino acid transmitter. Previous reports have suggested that MCH activated potassium channels in non‐neuronal cells transfected with the MCH receptor gene. We found no effect of MCH on voltage‐dependent potassium channels in LH neurons. Baclofen, a GABAB receptor agonist, activated G‐protein gated inwardly rectifying potassium (GIRK)‐type channels; in the same neurons, MCH had no effect on GIRK channels. MCH showed no modulation of sodium currents. Blockade of the Gi/Go protein with pertussis toxin eliminated the actions of MCH. The inhibitory actions of MCH on both excitatory and inhibitory synaptic events, coupled with opposing excitatory actions of hypocretin, another LH peptide that projects to many of the same loci, suggest a substantial level of complexity in neuropeptide modulation of LH actions.

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

Optogenetic Stimulation of MCH Neurons Increases Sleep

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.