Double- and triple-labeling of functionally characterized central neurons projecting to peripheral targets studied in vitro

doi:10.1016/0306-4522(90)90075-F

Neuroscience

Volume 38, Issue 3, 1990, Pages 829-841

https://doi.org/10.1016/0306-4522(90)90075-F Get rights and content

Abstract

The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysioiogical properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties.

The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.

Reference (26)

BorkeR.C. et al.
Brain stem afferents of hypoglossal neurons in the rat
Brain Res.
(1983)
GibbA.J. et al.
A preparation for patch clamp studies of labelled, identified neurones from guinea pig spinal cord
J. Neurosci. Meth.
(1987)
GimlichR.L. et al.
Improved fluorescent compounds for tracing cell lineage
Devl Biol.
(1985)
GloverJ.C. et al.
Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo
J. Neurosci. Meth.
(1986)
HorikawaK. et al.
A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates
J. Neurosci. Meth.
(1988)
JankowskaE. et al.
Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells
Brain Res.
(1976)
PurvesD. et al.
Imaging mammalian nerve cells and their connections over time in living animals
Trends Neurosci.
(1987)
SmithsonK.G. et al.
A reliable method for immunocytochemical identification of Lucifer Yellow injected, peptide-containing mammalian central neurons
J. Neurosci. Meth.
(1984)
StewartW.W.
Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer
Cell
(1978)
SunM.K. et al.
Reticulospinal pacemaker neurons of the rat rostral ventrolateral medulla with putative sympathoexcitatory function: an intracellular study in vitro
Brain Res
(1988)

BooneT.B. et al.

The ultrastructure of two distinct neuron populations in the hypoglossal nucleus of the rat

Expl Brain Res.

(1984)

BrownA.G. et al.

Intracellular Staining of Mammalian Neurons

BucklandR.M.

Strong signals from streptavidin-biotin

Nature

(1986)

Cited by (59)

CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus
2016, Brain Research
Citation Excerpt :
The hypoglossal nucleus, where the overwhelming majority of neurons are cholinergic hypoglossal motoneurons (HMs) (Davidoff and Schulze, 1988; Viana et al., 1990), is suggested to provide a fundamental function in respiration, swallowing, suckling, and mastication (Sawczuk and Mosier, 2001).
6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) is a most commonly used antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the central nervous system. During the past two decades, studies had demonstrated that CNQX could partially activate AMPA receptors that are located on the hippocampal and cerebellar interneurons, thus subsequently leading to the facilitation of inhibitory transmission. However, whether CNQX could enhance inhibitory synaptic transmission in the hypoglossal nucleus remains elusive. Here, using whole-cell patch-clamp recording in the brainstem slice, we showed that CNQX greatly increased both frequency and amplitude of spontaneous inhibitory postsynaptic currents in the hypoglossal motoneurons, whereas D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), N-methyl-D-aspartate (NMDA) receptor antagonist, had no effect on inhibitory synaptic transmission. Application of bicuculline and strychnine further identified that CNQX not only increased GABAergic sIPSCs but also glycinergic one in these motoneurons. Similar enhancement of inhibitory transmission was observed with application of 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), a quinoxaline derivative of CNQX, but not with application of GYKI 53655, a non-competitive antagonist of AMPA receptor. In the presence of tetradotoxin, the effect of CNQX on sIPSCs was abolished, suggesting that an increase in presynaptic interneuron spike firing rate induced by CNQX was responsible for the facilitation of sIPSCs. Taken together, these results demonstrated that the excitatory effect of CNQX on presynaptic interneurons triggered enhancement of both GABAergic and glycinergic synaptic transmission within the rat hypoglossal nucleus.
Motor control during sleep and wakefulness: Clarifying controversies and resolving paradoxes
2013, Sleep Medicine Reviews
Citation Excerpt :
Therefore, even if there were to be an effect of a glycine blocker on the dendritic field of other cells, it would be insignificant both on a temporal and anatomical basis with respect to the control of the motoneuron membrane potential and the blockade of the somatic REM-specific IPSPs. However, no technique is perfect: with intracellular recording one can only sample the activity of one motoneuron at a time, although this is not a significant drawback since motor pools, and especially the hypoglossal,46 are relatively homogenous. In addition, there have not been any reports of motoneurons, in any of the motor pools that have been studied from an intracellular perspective (i.e., spinal cord, masseter or hypoglossal), that exhibit a pattern of control during REM sleep that is different from that described above.
Data accumulated during the last 40 years, since the discovery that there is a loss of muscle tone during REM sleep, have delineated many of the neurotransmitter systems, synaptic mechanisms and neuronal circuitries involved in the control of somatic motoneurons during sleep and waking states. Nevertheless, there are still a number of extant controversies as well as paradoxical and conflicting data. For example, the paradoxical modulation of motor activity that occurs in individuals with cataplexy during wakefulness compared to REM sleep is unresolved as are the mechanisms that are responsible for the control of hypoglossal motoneurons during normal states and those that are operative during sleep disorders such as obstructive sleep apnea. In addition, the circuitry whereby the hypocretinergic system promotes motor activation during wakefulness, and motor inhibition during REM sleep, has yet to be clarified. The use of new techniques, such those involving optogenetics and nanoparticles, will help to clarify the preceding issues and provide as a foundation for addressing a number of current critical unanswered questions such as those dealing with the differential control of motor activity in newborns and the aged. The resulting data will strengthen the foundation for the development of efficacious therapeutics to treat disorders of motor control that occur during sleep as well as wakefulness.
Inspiratory-phase short time scale synchrony in the brainstem slice is generated downstream of the pre-Bötzinger complex
2008, Neuroscience
Citation Excerpt :
In these models, the peaks and troughs of synchronous oscillations are generated by alternating excitatory and inhibitory synaptic inputs, respectively, that are connected via recurrent axon collaterals. The XII nucleus is ill-equipped to generate short time scale synchronous oscillations because HMs lack recurrent axon collaterals and interneurons within the XII nucleus that could provide inhibitory synaptic inputs are scarce (Viana et al., 1990). Therefore, oscillations are more likely generated in the premotor area, which contains both excitatory and inhibitory neurons (Li et al., 1997), these oscillations would be transmitted to the XII nucleus.
Respiratory neurons are synchronized on a long time scale to generate inspiratory and expiratory-phase activities that are critical for respiration. Long time scale synchrony within the respiratory network occurs on a time scale of more than hundreds of milliseconds to seconds. During inspiration, neurons are synchronized on a short time scale to produce synchronous oscillations, which shape the pattern of inspiratory motor output. This latter form of synchrony within the respiratory network spans a shorter time range of tens of milliseconds. In the neonatal mouse rhythmically active medullary slice preparation, we recorded bilateral inspiratory activity from hypoglossal (XII) rootlets to study where in the slice synchronous oscillations are generated. Based on previous work that proposed the origin of these oscillations, we tested the pre-Bötzinger complex (PreBötC) and the XII motor nucleus. Unilateral excitation of the PreBötC, via local application of a perfusate containing high K⁺, increased mean inspiratory burst frequency bilaterally (296±66%; n=10, P<0.01), but had no effect on the relative power of oscillations. In contrast, unilateral excitation of the XII nucleus increased both mean peak integrated activity bilaterally (ipsilateral: 41±10%, P<0.01; contralateral: 17±7%; P<0.05, n=10) and oscillation power in the ipsilateral (50±17%, n=7, P<0.05), but not in the contralateral rootlet. Cross-correlation analysis of control inspiratory activity recorded from the left and right XII rootlets produced cross-correlation histograms with significant peaks centered around a time lag of zero and showed no subsidiary harmonic peaks. Coherence analysis of left and right XII rootlet recordings demonstrated that oscillations are only weakly coherent. Together, the findings from local application experiments and cross-correlation and coherence analyses indicate that short time scale synchronous oscillations recorded in the slice are likely generated in or immediately upstream of the XII motor nucleus.
Respiratory pre-motor control of hypoglossal motoneurons in the rat
2002, Neuroscience
The goal of this study was to determine the origin and transmission pathway of respiratory drive to hypoglossal motoneurons. First we recorded intracellularly from 28 antidromically activated inspiratory hypoglossal motoneurons (resting membrane potential, −50±3 mV), and found that injection of chloride ions had no discernible effect on the shape of their membrane potential trajectories. We concluded that the membrane potential trajectories of these hypoglossal motoneurons were determined primarily by inspiratory excitation. To determine the origin of this excitation we cross-correlated the extracellular discharge of medullary inspiratory neurons, including those in the hypoglossal motor nucleus, with the hypoglossal nerve discharge. We found 27 inspiratory neurons within the hypoglossal motor nucleus that were not antidromically activated from the ipsilateral hypoglossal nerve; their cross-correlograms featured either central peaks (1.7±0.2 ms) alone (n=14; 39%), or central peaks (1.3±0.2 ms) followed by troughs (1.3±0.1 ms) at short latencies (1.1±0.4 ms) (n=13; 36%), and suggest that these neurons are hypoglossal interneurons. We recorded from 238 inspiratory neurons throughout the rest of the medulla; the cross-correlograms of 19 neurons (8%), located mostly in the lateral tegmental field, displayed narrow half-amplitude peaks (1.0±0.1 ms) at short latencies (0.9±0.1 ms), which we interpreted as evidence for monosynaptic excitation of hypoglossal motoneurons.
We conclude that the respiratory control of hypoglossal motoneurons originates from inspiratory premotor neurons scattered throughout the lateral tegmental field and interneurons within the hypoglossal motor nucleus.
Diversity of neuron-specific K<sup>+</sup>-Cl<sup>-</sup> cotransporter expression and inhibitory postsynaptic potential depression in rat motoneurons
2002, Journal of Biological Chemistry
Citation Excerpt :
To assure that the tissue was taken from the XII or DMV nucleus, the micropunches were taken from within the boundaries of the respective nuclei. The resulting isolated neurons retained several original morphological features resembling those described previously for XII and DMV neurons (16-19). For intracellular recording, the electrolyte filling the pipette (50–80 megaohms) was connected via Ag-AgCl electrode to the input stage of an intracellular recording amplifier (IR283; Neuro Data).
Motoneurons receive a robust recurrent synaptic inhibition by γ-aminobutyric acid and glycine, which activate Cl⁻ channels. Thus, Cl⁻ homeostasis determines the efficacy of synaptic inhibition in the motoneurons.In situ hybridization reveals that the neuronal K⁺-Cl⁻ cotransporter isoform 2 (KCC2), a major mechanism in maintaining a low Cl⁻ concentration in neurons, is abundantly expressed in the facial, hypoglossal (XII), and spinal motoneurons innervating striated muscle, whereas the dorsal vagal motoneurons (DMVs) controlling smooth muscle exhibited little expression of KCC2. This raises a general interest in the correlation between KCC2 expression and inhibitory postsynaptic potential (IPSP) performance in the native circuits. Intracellular and whole-cell patch recordings revealed that an activity-dependent depression of IPSPs and positive shift of IPSP reversal potentials were more prominent in the DMV than in the XII. Cl⁻ influx through Cl⁻ channels was extruded more potently in the XII than in the DMV, suggesting that differences in Cl⁻extrusion account for these dynamic differences of IPSP. Cl⁻ extrusion was inhibited by either furosemide or an increase in extracellular potassium concentrations. Thus, the rigid maintenance of IPSP and rapid Cl⁻ extrusion in the XII reflects an intense expression of KCC2. KCC2 expression may strongly influence the IPSP depression and functional properties of the motoneurons innervating striated muscles.
The effects of membrane potential oscillations on the excitability of rat hypoglossal motoneurons
2022, Frontiers in Physiology

View all citing articles on Scopus

View full text

Article preview

Neuroscience

Abstract

Reference (26)

Brain stem afferents of hypoglossal neurons in the rat

Brain Res.

A preparation for patch clamp studies of labelled, identified neurones from guinea pig spinal cord

J. Neurosci. Meth.

Improved fluorescent compounds for tracing cell lineage

Devl Biol.

Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo

J. Neurosci. Meth.

A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates

J. Neurosci. Meth.

Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells

Brain Res.

Imaging mammalian nerve cells and their connections over time in living animals

Trends Neurosci.

A reliable method for immunocytochemical identification of Lucifer Yellow injected, peptide-containing mammalian central neurons

J. Neurosci. Meth.

Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer

Cell

Reticulospinal pacemaker neurons of the rat rostral ventrolateral medulla with putative sympathoexcitatory function: an intracellular study in vitro

Brain Res

The ultrastructure of two distinct neuron populations in the hypoglossal nucleus of the rat

Expl Brain Res.

Intracellular Staining of Mammalian Neurons

Strong signals from streptavidin-biotin

Nature

Cited by (59)

CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus

Motor control during sleep and wakefulness: Clarifying controversies and resolving paradoxes

Inspiratory-phase short time scale synchrony in the brainstem slice is generated downstream of the pre-Bötzinger complex

Respiratory pre-motor control of hypoglossal motoneurons in the rat

Diversity of neuron-specific K<sup>+</sup>-Cl<sup>-</sup> cotransporter expression and inhibitory postsynaptic potential depression in rat motoneurons

The effects of membrane potential oscillations on the excitability of rat hypoglossal motoneurons