Refik Kanjhan

Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat

The aim of this study was to examine medullary and spinal axonal projections of inspiratory bulbospinal neurons of the rostral ventral respiratory group (VRG) in the rat. A direct visualization of long (9.8-33 mm) axonal branches, including those projecting to the contralateral side of the medulla oblongata and the spinal cord, was possible due to intracellular labeling with neurobiotin and long survival times (up to 22 h) after injections. Seven of the nine labeled neurons had bilateral descending axons, which were located in discrete regions of the spinal white matter; ipsilateral axons in the lateral and dorsolateral funiculus, contralateral in the ventral and ventromedial funiculus. The collaterals issued by these axons at the mid-cervical level formed close appositions with dendrites of phrenic motoneurons, which had also been labeled with neurobiotin. None of these collaterals crossed the midline. The significance of this finding is discussed in relation to the crossed-phrenic phenomenon. Additional spinal collaterals were identified in the C1 and T1 segments. Within the medulla, collaterals with multiple varicosities were identified in the lateral tegmental field and in the dorsomedial medulla (in the hypoglossal nucleus and in the nucleus of the solitary tract). These results demonstrate that inspiratory VRG neurons in the rat have some features which have not been previously described in the cat, including frequent bilateral spinal projection and projection to the nucleus of the solitary tract. In addition, this study shows that intracellular labeling with neurobiotin offers an effective way of tracing long axonal projections, supplementing results previously obtainable only with antidromic mapping, and providing morphological details which could not be observed in previous studies using labeling with horseradish peroxidase.

Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study "in vivo'.

1. Intracellular recordings were made in pentobarbitone‐anaesthetized rats from sixty‐eight neurones located in the rostral ventrolateral medulla (RVLM), which responded with inhibition (latency, 33.6 +/‐ 9.3 ms) after stimulation of the aortic depressor nerve with short bursts of pulses. This inhibition was due to chloride‐ and voltage‐dependent IPSPs. 2. Seventeen neurones could be excited antidromically after stimulation in the T2 spinal segment (conduction velocity 1.9‐8.5 m.s‐1) and were classified as RVLM presympathetic vasomotor neurones. 3. ‘Spontaneously’ active neurones (n = 29) displayed a largely irregular pattern of firing, with no clear relationship between the level of the membrane potential and cycles of phrenic nerve activity at end‐tidal CO2 < 5.0%. Cardiac cycle‐related shifts of the membrane potential were not considered indicative of baroreceptor input as they could be due to movement artifacts. 4. All neurones displayed large synaptic activity (EPSPs and IPSPs, peak‐to‐peak amplitude > 5.0 mV). The depolarizing IPSPs observed during injection of chloride and/or negative current consisted of a phasic and a tonic component. 5. The on‐going activity of these neurones resulted from synaptic inputs, with individual action potentials usually preceded by identifiable fast EPSPs. 6. No evidence was found for the presence of gradual depolarizations (autodepolarizations) between individual action potentials, and therefore under these experimental conditions the activity of RVLM presympathetic neurones did not depend on intrinsic pacemaker properties. 7. These results are consistent with the ‘network’ hypothesis for the generation of sympathetic vasomotor tone.

Barosensitive neurons in the rostral ventrolateral medulla of the rat in vivo: Morphological properties and relationship to C1 adrenergic neurons

The aim of this study, conducted in anaesthetized rats, was to examine the morphology of barosensitive neurons in the rostral ventrolateral medulla and their immunoreactivity for a catecholamine synthesizing enzyme, tyrosine hydroxylase. Thirty neurons displaying inhibitory postsynaptic potentials following stimulation of the aortic depressor nerve were intracellularly labelled with Lucifer Yellow or Neurobiotin. Some of these neurons could be excited antidromically from the second thoracic segment of the spinal cord, with conduction velocities of spinal axons ranging from 1.9 to 7.2 m/s. The filled somas were found immediately caudal to the facial nucleus and ventral or ventromedial to compact formation of the nucleus ambiguus. Some dendrites reached the ventral medullary surface. Axons usually projected dorsomedially and then made a sharp rostral and/or caudal turn. The caudally projecting axon could, in some cases, be followed to the first cervical segment of the spinal cord. Seven cells issued fine axon collaterals on the ipsilateral side. These were identified mainly in two areas: in the rostral ventrolateral medulla (or immediately dorsomedial to that region), and within the dorsal vagal complex. Seven of 27 examined cells (26%) were tyrosine hydroxylase-immunoreactive and were classified as C1 adrenergic neurons. No clear relationship was found between the presence or absence of adrenergic phenotype and the morphology of filled cells. However, the amplitude of aortic nerve-evoked inhibitory postsynaptic potentials was significantly larger in tyrosine hydroxylase-positive neurons. Possible reasons for the low percentage of barosensitive cells with tyrosine hydroxylase immunoreactivity found in this study, in comparison with previously published estimates, are discussed. This is the first study describing the morphology of neurons in this part of the medulla identified as barosensitive in vivo, and directly demonstrating adrenergic phenotype in a subset of these neurons.

A comparative study of pre-sympathetic and Bötzinger neurons in the rostral ventrolateral medulla (RVLM) of the rat

The aim of this study was to investigate the degree of functional and anatomical overlap between two major neuronal subpopulations in the rostral ventrolateral medulla: pre-sympathetic (sympathoexcitatory) neurons, and expiratory neurons of the Bötzinger complex. Extracellular recordings were made with dye-filled microelectrodes in pentobarbital anesthetized, paralyzed and artificially ventilated adult Wistar rats. Tests applied included stimulation of baroreceptor afferents, activation of peripheral chemoreceptors and lung stretch receptors, changes in central respiratory drive with hyper- or hypoventilation, nociceptive stimulation, and antidromic stimulation from the T2 segment of the spinal cord or medulla oblongata at obex level. The two groups of neurons showed different patterns of spontaneous activity and generally different responses to these stimuli. The recording positions showed some overlap, but the majority of Bötzinger neurons were dorsolateral to pre-sympathetic neurons. There was a large overlap between the location of pre-sympathetic neurons and the lateral part of the C1 adrenergic group, but only a small overlap between these adrenergic neurons and Bötzinger neurons. These results indicate that the anatomically adjacent pre-sympathetic and Bötzinger expiratory neurons form two functionally distinct neuronal subpopulations.

OPIOIDS AND PAIN

1. The central nervous system in mammals is able to react to painful stimuli at many levels that are involved in transmission, modulation and sensation of pain. Endogenous opioid peptides and their receptors are located at key points in pain pathways, and response to pain can be modulated by local application of opioids at many sites. Mechanisms of opioid analgesia at peripheral, spinal, medullary and midbrain levels are only incompletely understood; forebrain systems are even less appreciated. Local circuits in the spinal dorsal horn play a critical role in processing nociceptive afferent input and in mediating the actions of descending pain modulating systems.

Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat

There has been controversy whether the dorsal respiratory group (DRG), identified in the cat and several other species as a concentration of mainly inspiratory neurons located in the ventrolateral subnucleus of the solitary tract, also exists in the rat. The aim of this study was to re-examine this question by systematically exploring this region with extracellular microelectrodes, in anesthetized and artificially ventilated rats. One-hundred and forty-two units were recorded which fired in phase with central respiratory cycles (determined by recording from the phrenic nerve) and/or lung inflations. One-hundred and nineteen recordings were thought to be from neuronal cell bodies (confirmed in some cases by excitatory responses to microelectrophoretic administration of DL-homocysteic acid), while the remaining 23 were from lung vagal afferents. Most neurons in the former group (87/119) were inspiratory. Out of 96 neurons tested for spinal projections only 14 (12 inspiratory, 2 expiratory) responded antidromically following stimulation at C3 segment. These results confirm the existence of the DRG in the rat and demonstrate that neurons located in this region have firing patterns generally similar to those previously described in the cat. The main difference is the relative paucity in the rat of neurons projecting spinally below the C2 level, which indicates that most DRG neurons in this species do not project directly to phrenic and intercostal motoneurons, but to other, as yet unidentified, neuronal groups within the brainstem or upper cervical segments.

Purinergic Regulation of Sound Transduction and Auditory Neurotransmission

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X2 receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K+ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K+ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K+ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K+ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca2+ influx and gating of Ca2+ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Glycinergic and GABAergic Synaptic Activity Differentially Regulate Motoneuron Survival and Skeletal Muscle Innervation

GABAergic and glycinergic synaptic transmission is proposed to promote the maturation and refinement of the developing CNS. Here we provide morphological and functional evidence that glycinergic and GABAergic synapses control motoneuron development in a region-specific manner during programmed cell death. In gephyrin-deficient mice that lack all postsynaptic glycine receptor and some GABAA receptor clusters, there was increased spontaneous respiratory motor activity, reduced respiratory motoneuron survival, and decreased innervation of the diaphragm. In contrast, limb-innervating motoneurons showed decreased spontaneous activity, increased survival, and increased innervation of their target muscles. Both GABA and glycine increased limb-innervating motoneuron activity and decreased respiratory motoneuron activity in wild-type mice, but only glycine responses were abolished in gephyrin-deficient mice. Our results provide genetic evidence that the development of glycinergic and GABAergic synaptic inputs onto motoneurons plays an important role in the survival, axonal branching, and spontaneous activity of motoneurons in developing mammalian embryos.

A method for the three-dimensional reconstruction of Neurobiotin™-filled neurons and the location of their synaptic inputs

Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit α1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses.