Quantitative analysis of synaptic contacts made between functionally identified oralis neurons and trigeminal motoneurons in cats.

A previous study revealed that rostrodorsomedial oralis (Vo.r) neurons synapsing on trigeminal motoneurons use GABA and/or glycine as neurotransmitters. To determine the number and spatial distribution of contacts, injections of biotinamide and horseradish peroxidase were made into a Vo.r neuron and an alpha-motoneuron in the jaw-closing (JC) and jaw-opening (JO) motor nucleus, respectively, in 39 cats. All Vo.r neurons responded to low-threshold mechanical stimulation of the oral tissues. Single Vo.r neurons terminating in the JC nucleus (Vo.r-dl neurons; n = 5) issued, on average, 10 times more boutons than Vo.r neurons terminating in the JO nucleus (Vo.r-vm neurons; n = 5; 4437 vs 445). The Vo.r-dl neuron-JC alpha-motoneuron pairs (n = 4) made contacts on either the soma-dendritic compartment or dendrites, and the Vo.r-vm neuron-JO motoneuron pairs (n = 2) made contacts on dendrites, with a range of two to seven contacts. In five of the six pairs, individual or groups of two to three terminals contacted different dendritic branches of a postsynaptic cell. The Vo.r-dl neurons innervated a greater number of counter-stained motoneuronal somata than did the Vo.r-vm neurons (216 vs 26). Total number of contacts per Vo.r neuron was higher for the Vo.r-dl than Vo.r-vm neurons (786 vs 72). The present study demonstrates that axonal branches of Vo.r neurons are divided into two types with different innervation domains on the postsynaptic neuron and that they are highly divergent. The overall effect exerted by these neurons is predicted to be much greater within the JC than JO motoneuron pool.

Transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase into trigeminal interneurones of the rat.

Intramuscular injections of either horseradish peroxidase (HRP) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) were made into the masseter muscle of rats. Both tracers labeled primary sensory neurones in the V mesencephalic nucleus, motoneurones in the V motor nucleus, and some motoneurones in the facial motor nucleus. WGA-HRP labeled additional neurones in the V main sensory nucleus and the rostral pole of the V nucleus oralis. These were classed as interneurones because they lay in areas outside those known to contain either first-order afferent or motoneurone somata. We argue that these were labeled by retrograde transport of tracer because they lay close to the V motor nucleus, and from some of them processes could be followed into the region of the V motor nucleus.

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex.

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst-firing activity that is phase-locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow-firing, fast-spiking, regular-spiking, and burst-firing neurons; previous work has suggested that the slow-firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst-firing neuron. Type I burst-firing neurons had significantly longer duration after hyperpolarisation potentials when held at -60 mV, and at -75 mV, type I neurons exhibited a low-threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1-12 local primary axon collaterals. All electrophysiological types, except for the type I burst-firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow-firing neurons. Branching of the primary axon collaterals of the fast-spiking and type I burst-firing neurons was more extensive than that of the other cell types, with those of the slow-firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast-spiking and burst-firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20-30-microm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB.

Differences in monosynaptic EPSPs elicited by masseter and temporalis spindle afferents in anaesthetised rats.

We have used the intracellular variant of the spike triggered averaging method to examine the monosynaptic connexions of masseter and temporalis spindle afferents on jaw-elevator motoneurones. Temporalis spindle afferents elicited larger averaged EPSPs in motoneurones than masseter spindle afferents, in part because transmission at synapses of temporalis afferents was associated with lower incidences of failures. We conclude that EPSP amplitude in this motor system in governed, at least in part, by the presynaptic neurone rather than the identity of the postsynaptic neurone.

Effects of afferent firing frequency on the amplitude of the monosynaptic EPSP elicited by trigeminal spindle afferents on trigeminal motoneurones.

We have examined the effect of changes in afferent firing frequency on the monosynaptic EPSP elicited by trigeminal muscle spindle afferents in elevator motoneurones, using both paired pulse and repetitive activation of the masseter nerve. Both modes of stimulation resulted in significant facilitation of EPSP amplitude over intervals of 5-19 ms, but no significant change in EPSP amplitude at either longer or shorter intervals. The facilitation obtained stands in contrast to the predominant depression of EPSP amplitude reported in hindlimb motoneurones following stimulation at decreasing intervals.

Contrasting effects of urethane and pentobarbitone anaesthesia on the electrical properties of rat jaw-elevator motoneurones.

Our main finding is that elevator motoneurones do not show sustained firing to intracellular injections of depolarising current pulses in rats anaesthetised with urethane. In contrast, virtually all elevator motoneurones show sustained firing in pentobarbitone-anaesthetised rats. The differences in firing are not associated with significant differences in membrane potential, spike amplitudes, AHP amplitude or duration, input resistance, time constant or rheobase (P greater than 0.06 in all cases). However, there are clear differences in the extent of sag seen under the two anaesthetics and so we tentatively suggest that the anaesthetics may differ in their effects on the inward rectifier.

The morphology of the axons and axon collaterals of rat jaw-elevator motoneurones.

We have made intracellular injections of horseradish peroxidase into the somata of jaw-elevator motoneurones and subsequently reconstructed the axonal morphology of 4 cells. In each case the axons gave off collaterals which were essentially restricted to the ventral portion of the V motor nucleus. This observation provides the first evidence that these motoneurones may exert recurrent synaptic effects.

Quantitative analysis and postsynaptic targets of GABA-immunoreactive boutons within the rat trigeminal motor nucleus.

We have used the post-embedding immunogold labelling method using antibodies to gamma-aminobutyric acid (GABA) to obtain quantitative data on the distribution, frequency, postsynaptic targets and ultrastructural characteristics of GABA-immunoreactive (GABA-IR) boutons in the trigeminal motor nucleus of rats. We have also combined this method with horseradish peroxidase tracing to obtain specific evidence for termination of some GABA-IR boutons onto identified jaw-elevator motoneurones. Twenty-eight percent of all synapses in the motor nucleus involved GABA-IR boutons. Seventy-three percent of the GABA-IR boutons formed axo-dendritic synapses, 13% axo-somatic synapses and 14% axo-axonic synapses. Ninety-three percent of GABA-IR boutons formed symmetrical synapses. Overall, 58% of all boutons contained only flattened vesicles, while 26% contained round vesicles and 16% a mixture of vesicle types. Measurements of bouton cross sectional area, apposition length, and active zone length were obtained from serial reconstructions of 15 GABA-IR boutons and 30 unlabelled boutons. In each case mean values for GABA-IR boutons were significantly smaller than those for nonlabelled boutons.

Monosynaptic connexions of single V interneurones to the contralateral V motor nucleus in anaesthetised rats.

We have used the extracellular spike triggered averaging method to obtain evidence for a monosynaptic connexion of single V (trigeminal) interneurones, located in the region immediately caudal to the V motor nucleus, onto neurones within the contralateral V motor nucleus. The extracellular fields recorded in the contralateral nucleus are of smaller amplitude than those detected within the ipsilateral nucleus and the implications of this are discussed.

Light and electron microscopical localisation of 5-HT-immunoreactive boutons in the rat trigeminal motor nucleus.

We have used pre-embedding EM immunohistochemical methods to obtain quantitative data on the frequency and post-synaptic targets of 5-hydroxytryptamine-immunoreactive (5-HT-IR) boutons within the rat V motor nucleus. Thirteen percent (69/531) of all synaptic contacts in the motor nucleus involved 5-HT-IR boutons. Seventy-four percent of 5-HT-IR boutons made axo-dendritic contacts, 20% axo-somatic contacts, and 6% axo-axonic contacts. We conclude that a significant fraction of boutons in the motor nucleus are 5-HT-IR and most contribute to postsynaptic rather than presynaptic effects on trigeminal motoneurones.

Visualisation of CGRP and ChAT-like immunoreactivity in identified trigeminal neurones by combined peroxidase and alkaline phosphatase enzymatic reactions.

We report here a method that allows simultaneous visualisation of two antigens within single neurones. In essence this involves the combined use of horseradish peroxidase and alkaline phosphatase reactions to visualise two markers. Using this method we show that ChAT-and CGRP-like immunoreactivity can be co-localised within single neurones of the V to VII motor nuclei. In the case of the V motor nucleus, we show that each marker can be localised in motoneurones labelled with horseradish peroxidase.

'Fusimotor set': new evidence for alpha-independent control of gamma-motoneurones during movement in the awake cat.

The discharge activity of single muscle spindle receptors was recorded in freely moving cats. Large changes in responsiveness to length variations was observed in different types of movement. In separate stimulations on anaesthetised cats, the activity of the gamma-fusimotor neurones responsible for these changes was reconstructed. The results suggested that fusimotor action on a given spindle afferent during the movements studied was not rigidly alpha-linked, but 'set' by the CNS to steady levels, and that it could switch from largely static (gamma s) to largely dynamic (gamma d) according to the motor tasks performed.

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals.

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and gamma-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.

The membrane properties and firing characteristics of rat jaw-elevator motoneurones.

1. We have determined the membrane and firing properties of fifty-six jaw-elevator motoneurones in rats that were anaesthetized with pentobarbitone, paralysed and artificially ventilated. 2. Forty-two neurones were identified as masseter motoneurones and fourteen as masseter synergist motoneurones. The membrane potentials for the sample ranged from -60 to -86 (mean = -68; S.D. = 7.3; n = 56), and spike amplitudes from 50 to 95 mV. The duration of the after-hyperpolarization following antidromic spikes in masseter motoneurones ranged from 15 to 50 ms (mean = 30; S.D. = 12.8) and their amplitudes from 1.0 to 4.5 mV (mean = 2.7; S.D. = 2.2; n = 42). 3. The mean input resistance for the total sample was 2.3 M omega (S.D. = 0.9; n = 56), membrane time constant 3.9 ms (S.D. = 0.9; n = 48) and rheobase 4.2 nA (S.D. = 2.6; n = 56). The distribution of these parameters was independent of membrane potential. We found no significant interrelationships between the membrane properties and one interpretation of this is that our sample may be drawn from a homogenous population of motoneurones. We also suggest that elevator motoneurones may have a lower Rm (specific membrane resistivity) value than cat hindlimb motoneurones because they have a similar range of input resistance values but only half the total surface area. 4. Forty-six out of forty-nine neurones fired repetitively to a depolarizing current pulse at a mean threshold of 1.6 x rheobase. Current-frequency plots were constructed for thirteen neurones and all but one showed a primary and secondary range in the firing of the first interspike interval. The mean slope in the primary range was 31 impulses s-1 nA-1 and 77 impulses s-1 nA-1 for the secondary range. The mean minimal firing frequency for steady firing was 26 impulses s-1 and, in response to an increase of stimulation, the rate increased monotonically with a slope of 11 impulses s-1 nA-1. 5. The dynamic sensitivity of twelve neurones was assessed from their response to ramp waveforms of current of constant amplitude but varying frequencies (0.2-2 Hz). Firing initially increased along a steep slope up to a frequency of between 40 and 60 impulses s-1 and then increased along a much shallower slope. Both the threshold for eliciting firing and the firing at the transition point of the two slopes remained constant with changes in ramp frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Tendon organ firing during active muscle lengthening in awake, normally behaving cats.

Recordings were obtained of the discharge of single tendon organ (Ib) and muscle spindle (Ia) afferents of the ankle extensor muscles during movement in normal cats. During very slow, smooth increases and decreases in muscle force, Ib afferents showed from one to five stepwise changes in firing rate, attributable to the recruitment of motor units inserting into the receptor capsule. These 'recruitment steps' in Ib firing rate became smoothed and tended to merge during faster variations in muscle force, and were rarely discernible in normal movements such as slow stepping. Rapid imposed stretches resulted in Ib firing patterns which fitted well a dynamic function of whole muscle force. Comparisons were made between the responses of Ib and Ia afferents during rapid, imposed muscle stretch. The segmentation of discharge typical of Ia afferents was not present in Ib afferents, despite segmentation of the e.m.g. of the receptor-bearing muscles. This would imply that Ib afferents exert a rapidly fluctuating reflex action against a relatively steady background of Ib input. Ankle extensor Ib firing during stepping was characterized by feeble firing during the swing phase and substantial, smoothly modulated firing during the stance phase. Taken together with previous chronic recordings, the data support the view that the ensemble of Ib afferents from a muscle signals a dynamic, non-linear function of whole muscle force over a wide range of normal movement.

The electrical geometry, electrical properties and synaptic connections onto rat V motoneurones in vitro.

1. We have developed a tissue slice preparation which allows the study of the actions of single presynaptic neurones onto single trigeminal motoneurones in the immature rat. Our aim in this first stage of the work has been to assess the validity of this preparation as a model for responses obtained in vivo from trigeminal motoneurones in adult rats. We have quantified the integrative properties of the motoneurones and also the variability in transmission at synapses of single presynaptic neurones onto the motoneurones. This data has then been compared to similar published data obtained from adult (rat) trigeminal motoneurones in vivo. 2. Quantitative reconstructions were made of the morphology of three motoneurones which had been labelled with biocytin by intracellular injection. The neurones gave off six to nine dendrites, of mean length 522 microns (S.D. = 160; n = 22), which branched on average 10.5 times to produce 11.45 end-terminations per dendrite (S.D. = 8.57; n = 22). The mean surface area of the dendrites was 0.92 x 10(4) microns2 (S.D. = 0.67; n = 22), and, for individual cells, the ratio of the combined dendritic surface area to the total neuronal surface area ranged from 98.3 to 99.2% (n = 3). At dendritic branch points the ratio of the summed diameters of the daughter dendrites to the 3/2 power against the parent dendrite to the 3/2 power was 1.09 (S.D. = 0.21; n = 217), allowing branch points to be collapsed into a single cylinder. The equivalent cylinder diameter of the combined dendritic tree remained approximately constant over the proximal 25-40% of the equivalent electrical length of the dendritic tree and then showed tapering. The tapering could be ascribed to termination of dendrites at different electrical distances from the soma. 3. Electrical properties were determined for a total of eighty-seven motoneurones, all with membrane potentials more negative than 60 mV (mean = 66.0 mV; S.D. = 5.2) and spikes which overshot zero (mean spike amplitude = 77 mV; S.D. = 10.5; n = 87). The spikes were followed by after-hyperpolarizations (AHPs) of mean amplitude 2.2 mV (S.D. = 1.7; n = 47), and mean duration 54.1 ms (S.D. = 9.5; n = 47). The mean input resistance of the neurones was 7.5 M omega (S.D. = 2.5; n = 69), the mean membrane time constant was 3.5 ms (S.D. = 2.2; n = 35), and the mean rheobase was 1.6 nA (S.D. = 1.1; n = 56).(ABSTRACT TRUNCATED AT 400 WORDS)

The morphology and electrical geometry of rat jaw-elevator motoneurones.

1. The aim of this work was to quantify both the morphology and electrical geometry of the dendritic trees of jaw-elevator motoneurones. To do this we have made intracellular recordings from identified motoneurones in anaesthetized rats, determined their membrane properties and then filled them with horseradish peroxidase by ionophoretic ejection. Four neurones were subsequently fully reconstructed and the lengths and diameters of all the dendritic segments measured. 2. The mean soma diameter was 25 microns and values of mean dendritic length for individual cells ranged from 514 to 773 microns. Dendrites branched on average 9.1 times to produce 10.2 end-terminations. Dendritic segments could be represented as constant diameter cylinders between branch points. Values of dendritic surface area ranged from 1.08 to 2.52 x 10(5) microns 2 and values of dendritic to total surface area from 98 to 99%. 3. At branch points the ratio of the summed diameters of the daughter dendrites to the 3/2 power against the parent dendrite to the 3/2 power was exactly 1.0. Therefore the individual branch points could be collapsed into a single cylinder. Furthermore for an individual dendrite the diameter of this cylinder remained constant with increasing electrical distance from the soma. Thus individual dendrites can be represented electrically as cylinders of constant diameter. 4. However dendrites of a given neurone terminated at different electrical distances from the soma. The equivalent-cylinder diameter of the combined dendritic tree remained constant over the proximal half and then showed a pronounced reduction over the distal half. The reduction in equivalent diameter could be ascribed to the termination of dendrites at differing electrical distances from the soma. Therefore the complete dendritic tree of these motoneurones is best represented as a cylinder over the proximal half of their electrical length but as a cone over the distal half.

Proprioceptive input from the jaw muscles and its influence on lapping, chewing, and posture.

Experiments are described which define criteria for identifying fusimotor axons discharging in filaments of the masseter nerve in lightly anaesthetized cats. During reflex movements of the jaw two patterns of discharge were observed in different fusimotor fibres. One type, called "sustained," fired at a fairly constant increased rate. The other, called "modulated," fired at high frequencies during the extrafusal muscle contraction. Evidence from spindle primary and secondary recordings in similar experiments strongly suggests that the "sustained" type were dynamic fusimotor fibres and the "modulated" type were static fusimotor fibres. New spindle recordings in normal unanaesthetized cats indicate that the modulated pattern of static fusimotor discharge also occurs in these conditions. Its effect is to reduce the tendency for spindle afferents to become silent during muscle shortening. A proposal is made that the static fusimotor discharge in cyclic movements may represent a temporal "template" for the intended movement as directed by the central pattern generator.

Fusimotor activity in masseter nerve of the cat during reflex jaw movements.

1. Unit recordings have been made from the central ends of filaments of the masseter nerve in lightly anaesthetized cats. Evidence is presented to show that fusimotor activity may be distinguished from alpha motor activity. 2. During reflex cyclic movements induced by intra-oral stimulation, two distinct patterns of fusimotor firing emerged. One type of unit increased firing at the beginning and sustained this with little modulation throughout the movements. The other type was strongly modulated approximately in parallel with the alpha motor activity. 3. By comparison with records of jaw elevator spindle afferents under similar conditions, it was deduced that the sustained type of action was due to dynamic fusimotor neurones while the modulated type was due to static fusimotor neurones. 4. The patterns of fusimotor activity seen in these rhythmic movements under light anaesthesia agree well with the patterns deduced from spindle recordings in the conscious cat during mastication. 5. The results emphasize the importance of looking beyond a simple hypothesis of 'alpha-gamma co-activation' to explain fusimotor function. It is proposed that tonic dynamic fusimotor activity is set at the beginning of a movement to determine the incremental sensitivity of primary endings to stretch. The static fusimotor fibres are activated principally during shortening to help keep both primary and secondary endings active.