Vincent A. Pieribone

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology

Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structures, the nucleus paragigantocellularis (PGi) and the perifascicular area of the nucleus prepositus hypoglossi (PrH), both located in the rostral medulla. Minor afferents to LC were found in the dorsal cap of the paraventricular hypothalamus and spinal lamina X. Recent studies have also revealed limited inputs from two areas nearby the LC, the caudal midbrain periaqueductal gray (PAG) and the ventromedial pericoerulear region. The pericoeruleus may provide a local circuit interface to LC neurons. Recent electron microscopic analyses have revealed that LC dendrites extend preferentially into the rostromedial and caudal juxtaependymal pericoerulear regions. These extracoerulear LC dendrites may receive afferents in addition to those projecting to LC proper. However, single-pulse stimulation of inputs to such dendritic regions reveals little or no effect on LC neurons. Double-labeling studies have revealed that a variety of neurotransmitters impinging on LC neurons originate in its two major afferents, PGi and PrH. The LC is innervated by PGi neurons that stain for markers of adrenalin, enkephalin or corticotropin-releasing factor. Within PrH, large proportions of LC-projecting neurons stained for GABA or met-enkephalin. Finally, in contrast to previous conclusions, the dorsal raphe does not provide the robust 5-HT innervation found in the LC. We conclude that 5-HT inputs may derive from local 5-HT neurons in the pericoerulear area. Neuropharmacology experiments revealed that the PGi provides a potent excitatory amino acid (EAA) input to the LC, acting primarily at non-NMDA receptors in the LC. Other studies indicated that this pathway mediates certain sensory responses of LC neurons. NMDA-mediated sensory responses were also revealed during local infusion of magnesium-free solutions. Finally, adrenergic inhibition of LC from PGi could also be detected in nearly every LC neuron tested when the EAA-mediated excitation is first eliminated. In contrast to PGi, the PrH potently and consistently inhibited LC neurons via a GABAergic projection acting at GABAA receptors within LC. Such PrH stimulation also potently attenuated LC sensory responses. Finally, afferents to PGi areas that also contain LC-projecting neurons were identified. Major inputs were primarily autonomic in nature, and included the caudal medullary reticular formation, the parabrachial and Kölliker-Fuse nuclei, the PAG, NTS and certain hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution and significance of CNS adrenoceptors examined with in situ hybridization.

Several of the established alpha 1-, alpha 2- and beta-adrenoceptors have now been isolated and cloned. The in situ hybridization method has been used to map the distribution of many of these adrenoceptors within cells of the CNS. These studies add complementary and new information to our knowledge of adrenoceptor localization provided previously by radioligand-mediated autoradiography. Neuronal cell groups containing one or more mRNAs for seven adrenoceptor subtypes throughout the rat CNS have been mapped. In the present review Anthony Nicholas, Tomas Hökfelt and Vincent Pieribone will examine these localizations and discuss the additional information these maps supply, as well as some implications for understanding central noradrenaline and adrenaline systems.

Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.

DISTRIBUTION OF ALPHA 1 ADRENOCEPTORS IN RAT BRAIN REVEALED BY IN SITU HYBRIDIZATION EXPERIMENTS UTILIZING SUBTYPE-SPECIFIC PROBES

The distribution of neurons in the rat CNS that synthesize mRNA for the alpha 1A/D and alpha 1B adrenoceptors was revealed by the in situ hybridization method. Forty-eight-mer DNA probes were synthesized to two different and unique regions of both the alpha 1A/D and alpha 1B mRNAs. Tissue sections from all levels of the CNS and some peripheral ganglia were incubated in a hybridization cocktail containing one of these four probes. The two mRNAs were expressed in a discrete and often complementary manner to each other, and identical hybridization patterns were seen for the probes directed against the same mRNA. The alpha 1A/D probes hybridized heavily with neurons in the internal granular and internal plexiform layers of the olfactory bulb, in layers II-V of most areas of the cerebral cortex, and in the lateral aspect of the lateral amygdaloid nucleus, with pyramidal neurons of CA1-CA4 regions, hilar and granular neurons of the dentate gyrus, and neurons in the reticular thalamic nucleus, cranial and spinal motor nuclei, and the inferior olivary nucleus. Light labeling was seen in a variety of other regions in the brain and spinal cord. The alpha 1B probes hybridized heavily with neurons in the mid layers of cerebral cortex and with virtually all neurons in the thalamus, except the reticular and habenular nuclei. In addition, labeling was seen in the lateral and central amygdaloid nuclei, in brainstem and spinal motor nuclei, over most neurons of the dorsal and medullary raphe nuclei and neurons of the intermediolateral cell column in the spinal cord. Light labeling was seen in the septal nucleus, the horizontal limb of the diagonal band, the paraventricular and lateral hypothalamic nuclei, the pontine and medullary reticular formation, and in most laminae in the spinal cord. The patterns of labeling obtained with the alpha 1B probes resemble the labeling seen in previous autoradiographic ligand binding studies utilizing “general” alpha 1 ligands, while the labeling patterns seen with the alpha 1A/D probes do not correspond to any published alpha 1 receptor distribution pattern, indicating that this mRNA likely encodes for a novel adrenoceptor. The present findings further expand the heterogeneity of adrenoceptor mRNAs presented in two accompanying studies (Nicholas et al., 1993a,b). This differential distribution of adrenoceptors subtypes provides a framework for the functional diversity to the apparently widespread, diffuse, and rather homogeneous noradrenergic innervation of the CNS.

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.

Cellular localization of messenger RNA for beta-1 and beta-2 adrenergic receptors in rat brain: An in situ hybridization study

Selective, 35S-labeled, oligonucleotide probes were designed from sequences of the rat beta-1 and beta-2 adrenoceptor messenger RNAs for use in situ hybridization experiments on sections of unfixed rat brain and spinal cord. After hybridized sections were exposed to film or dipped in autoradiographic emulsion, specific and selective labeling patterns characteristic for each receptor messenger RNA and region of the central nervous system were observed. For example, labeling for beta-1 messenger RNA was found in the anterior olfactory nucleus, cerebral cortex, lateral intermediate septal nucleus, reticular thalamic nucleus, oculomotor complex, vestibular nuclei, deep cerebellar nuclei, trapezoid nucleus, abducens nucleus, ventrolateral pontine and medullary reticular formations, the intermediate gray matter of the spinal cord and in the pineal gland, while beta-2 messenger RNA labeling was strongest in the olfactory bulb, piriform cortex, hippocampal formation, thalamic intralaminar nuclei and cerebellar cortex. In some of these regions the beta-1 labeling seemed mainly confined to the cell nucleus. Whether or not this apparently nuclear labeling is specific, i.e. indicates synthesis of beta-1 receptor, remains to be established. However, all labeling patterns described disappeared when excess unlabeled probes were added to their respective radiolabeled probes or when sense probes were employed. Since the in situ method labels only cell bodies that produce the messenger RNA for these two beta receptor subtypes, a comparison between these maps and those of past autoradiographic studies mapping the location of central beta receptors using drugs as radioligands may produce further insights regarding the pre- and postsynaptic localization of these receptors in the various parts of the central nervous system circuitry.

Galanin induces a hyperpolarization of norepinephrine-containing locus coeruleus neurons in the brainstem slice

Galanin applied in the bath or by micropipette directly on to locus coeruleus neurons in an in vitro slice preparation caused a hyperpolarization accompanied by a small decrease in membrane resistance. Immunohistochemical staining of intracellularly filled neurons indicated that the effect of galanin was exerted on norepinephrine neurons of the locus coeruleus. The galanin effect was variable in amplitude and duration and often showed desensitization, with subsequent applications producing a smaller response. When cells were exposed to tetrodotoxin or tetrodotoxin/low calcium media, the galanin response was still present. Under voltage clamp galanin application caused a net outward current that did not reverse in normal potassium concentrations; however, by increasing extracellular potassium concentrations the net outward current was reversed and the reversal potential shifted to a less negative potential. The response to galanin was identical when either KCl or KAc was used as the intracellular electrode solution. Tetraethylammonium chloride significantly reduced or abolished the response to galanin in most cells, although in a few cells the galanin response was not affected. Glibenclamide, a blocker of ATP-sensitive potassium channels, did not affect the galanin hyperpolarization. In addition, diazoxide had no effect on the membrane properties of locus coeruleus neurons. These results demonstrate that galanin exerts its inhibitory effect in the locus coeruleus via an increase in K+ conductance; however, not via the pancreatic type of ATP-sensitive K+ channels. Cryostat sections of the locus coeruleus incubated in 125I-labeled galanin revealed binding sites in the locus coeruleus at all levels. Sections of the locus coeruleus processed for ultrastructural immunocytochemistry showed galanin immunoreactivity in many neuronal somata and dendritic processes within the nucleus, confirming earlier evidence for the coexistence of galanin and noradrenaline in locus coeruleus neurons. Galanin-immunoreactive soma and dendrites in the locus coeruleus less frequently received galanin-immunoreactive synapses of axonal origin. These findings suggest that endogenous galanin in the locus coeruleus is mainly released from noradrenaline galanin somata and/or dendrites to act on autoreceptors or on receptors on adjacent neurons.

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

A Genetically Targetable Fluorescent Probe of Channel Gating with Rapid Kinetics

We have developed a genetically targetable, optical channel-gating reporter that converts rapid membrane potential changes into changes in fluorescence intensity. We have named this construct SPARC (sodium channel protein-based activity reporting construct). Green fluorescent protein was inserted into an intracellular loop of a reversibly nonconducting form of the rat mu I skeletal muscle voltage-gated sodium channel. Rapid changes of the membrane potential modulate the fluorescence of the inserted green fluorescent protein. This change in fluorescence can faithfully report depolarizing pulses as short as 2 ms. The fluorescence signal does not inactivate during extended depolarizations. Several features of the probes response properties indicate that it likely reports gating charge movement of a single domain of rat mu I skeletal muscle. This probe provides a new approach for studying rapid channel movements and may possibly act as a fluorescent activity reporter in excitable cells.

Midbrain serotonergic neurons are central pH chemoreceptors

Serotonergic neurons in the medulla are central respiratory chemoreceptors. Here we show that serotonergic neurons in the midbrain of rats are also highly chemosensitive to small changes in CO2/pH and are closely associated with large penetrating arteries. We propose that midbrain raphé neurons are sensors of blood CO2 that maintain pH homeostasis by inducing arousal, anxiety and changes in cerebrovascular tone in response to respiratory acidosis.