Abducens Motoneurons Research Articles

Role for calbindin-D28K in in vitro classical conditioning of abducens nerve responses in turtles.

Intracellular calcium has a pivotal role in synaptic modifications that may underlie learning and memory. The present study examined whether there were changes in immunoreactivity levels of the AMPA receptor subunits GluR2/3 and calcium binding proteins during classical conditioning recorded in the abducens nerve of in vitro brain stem preparations from turtles. The results showed that abducens motor neurons in unconditioned turtle brain stems were immunopositive for GluR2/3, calbindin-D28K, and calmodulin, but were immunonegative for parvalbumin. After classical conditioning, immunoreactivity for calbindin-D28K in the abducens motor nuclei was significantly reduced, whereas there were no significant changes in GluR2/3, calmodulin, or parvalbumin. This reduction in calbindin-D28K immunoreactivity was not observed following conditioning in the NMDA receptor antagonist AP-5, which blocked conditioned responses, suggesting that these changes are NMDA receptor-dependent. Moreover, the degree of the decrease in calbindin-D28K immunoreactivity was negatively correlated with the level of conditioning. Consistent with the immunocytochemical findings, Western blot analysis showed that calbindin-D28K protein levels were reduced after classical conditioning. The results support the hypothesis that in vitro classical conditioning of abducens nerve responses utilizes intracellular calcium-dependent signaling pathways that require NMDA receptor function and suggest a specific role for the calcium binding protein calbindin-D28K.

A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.

During postnatal development, profound changes take place in the excitability of nerve cells, including modification in the distribution and properties of receptor-operated channels and changes in the density and nature of voltage-gated channels. We studied here the firing properties of abducens motoneurons (aMns) in transverse brainstem slices from postnatal day (P) 1-13 rats. Recordings were made from aMNs in the whole-cell configuration of the patch-clamp technique. Two main types of aMn could be distinguished according to their firing profile during prolonged depolarizations. Both types were identified as aMns by their fluorescence following retrograde labelling with the lipophilic carbocyanine DiI in the rectus lateralis muscle. The first type (BaMns) exhibited a burst of action potentials (APs) followed by an adaptation of discharge and were encountered in approximately 70 % of aMns. Their discharge profile resembled that of adult aMns and was encountered in all aMns after P9. BaMns exhibited a hyperpolarization-induced rebound potential that was blocked by low concentrations of Ni2+ or by Ca2+-free external solution. This current had the properties of the T-type current. Action potentials of BaMns showed a complex afterhyperpolarization (AHP). An inward rectification was evidenced following hyperpolarization and was blocked by external application of caesium or ZD7288, indicating the presence of the hyperpolarization-activated cationic current (IH). Blocking the IH current almost doubled the input resistance of BaMns. The second class of aMns (DaMns) displayed a delayed excitation that was mediated by A-type K+ currents and was observed only between P4 and P9. DaMns exhibited immature characteristics: an action potential with a simple AHP, a linear current-voltage relation and a large input resistance. The number of aMns remained unchanged when both types were present (P5-P6) and later in development when only BaMns were encountered (P19), suggesting that DaMns mature into BaMns during postnatal development. We conclude that aMns display profound reorganization in their intrinsic excitability during postnatal development.

Duane Radial Ray Syndrome (Okihiro Syndrome) Maps to 20q13 and Results from Mutations in SALL4, a New Member of the SAL Family

Duane syndrome is a congenital eye movement disorder characterized most typically by absence of abduction, restricted adduction, and retraction of the globe on attempted adduction. Duane syndrome can be coinherited with radial ray anomalies as an autosomal dominant trait, referred to as "Okihiro syndrome" or "Duane radial ray syndrome" (DRRS). We ascertained three pedigrees with DRRS and mapped their disease gene to a 21.6-cM region of chromosome 20 flanked by markers D20S888 and D20S102. A new member of the SAL family of proposed C(2)H(2) zinc finger transcription factors, SALL4, falls within the region. Mutation analysis of SALL4 in the three pedigrees revealed one nonsense and two frameshift heterozygous mutations. SALL4 represents the first identified Duane syndrome gene and the second malformation syndrome resulting from mutations in SAL genes and likely plays a critical role in abducens motoneuron development.

Correlation between CGRP immunoreactivity and firing activity in cat abducens motoneurons.

A relationship between motoneuron activity and calcitonin gene-related peptide (CGRP) expression was previously suggested based on indirect inferences. We show here a positive correlation between CGRP immunoreactivity and firing activity in an experimental model that used tetanus neurotoxin (TeNT) to alter basal firing levels. A low dose (0.5 ng/kg) of TeNT injected in the lateral rectus muscle raised the basal firing rate of ipsilateral abducens motoneurons, estimated as the firing rate at straight-ahead gaze (F(0)); the firing rate returned to control values after 2 weeks. In contrast, a high dose (5 ng/kg) of TeNT decreased basal firing, which recovered slowly over a 7-week period. Expression of CGRP immunoreactivity by abducens motoneurons, preferentially related to betaCGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to approximately 120% by 7 days after low-dose TeNT, to include all available motoneurons in the nucleus. In addition, the average CGRP immunofluorescence optical density inside motoneurons almost doubled after 4 days and returned toward control values in the following 2 weeks. In contrast, a high-dose injection of TeNT reduced the number of CGRP-immunofluorescent motoneurons to 5.4% of control 7 days post injection, and the number returned to 77.6% after 42 days. CGRP immunofluorescence intensity inside motoneurons was also reduced. Regression analysis of F(0) values with either the number of CGRP-immunolabeled motoneurons, their average immunofluorescence intensity, or both factors combined resulted in positive correlations with regression coefficients of 0.87 or higher. Therefore, CGRP expression and firing activity in abducens motoneurons are positively correlated.

Estimation of premotor synaptic drives to simulated abducens motoneurons for control of eye position.

The firing rate of an abducens motoneuron (AbMN) is linearly related to eye position with slope K, above recruitment threshold theta. Within the AbMN population K increases as theta increases. It is possible that these properties depend on the synaptic drives generated by the major premotor inputs to AbMNs, namely position-vestibular-pause (PVP) cells and eye and head velocity (EHV) cells in the medial vestibular nucleus, and eye-position and burst-position cells in the nucleus prepositus hypoglossi (NPH). Premotor inputs to AbMNs were therefore modelled by a two-layer net, in which the output nodes represented the AbMNs (with fixed intrinsic properties) and the input nodes the three classes of premotor units ( n=20/class). Conjugate eye-position commands were used to generate the firing rates in premotor units found experimentally. The output of the net was compared with observed AbMN firing rates, and the resultant error used to adjust the magnitude and sign of the connection weights between premotor units and AbMNs. To provide additional constraints on permitted weights, the net was also trained under simulated smooth pursuit, cancellation of the vestibular-ocular reflex, and the vestibulo-ocular reflex itself (all at 0.5 Hz). Since the projections of EHV cells have not been clearly characterized, two versions of the model were trained, corresponding to different assumptions about these projections. In both versions, position-related AbMN firing rates were derived mainly from an excitatory drive from PVP cells and an inhibitory drive from NPH cells with the opposite ON direction. Variation in AbMN threshold theta and position sensitivity K depended on the strength of the drive from the NPH: the stronger the drive, the higher both K and theta. This arrangement was observed in six variants of the basic model with different parameter values, and in a simplified form (constant PVP drive and varying NPH drive) was able to generate qualitatively the observed relationship between K and theta even in the absence of input from EHV cells. It appears to be a robust mechanism for producing the experimentally observed variation in position-related firing of AbMNs, even without a contribution from their intrinsic properties, and predicts that local blocking of the inhibitory drive from cells in the NPH should lower both the position threshold and sensitivity of an individual AbMN. The model also indicates that if EHV cells have ipsilateral inhibitory projections, as has been proposed on the basis of their similarity with cells receiving input from the flocculus, then their role in eye-position control would reinforce that of cells in the NPH.

Influence of afferent synaptic innervation on the discharge variability of cat abducens motoneurones.

The discharge variability of abducens motoneurones was studied after blocking inhibitory synaptic inputs or both excitatory and inhibitory inputs by means of an intramuscular (lateral rectus) injection of either a low (0.5 ng kg(-1)) or a high dose (5 ng kg(-1)) of tetanus neurotoxin (TeNT), respectively. Motoneuronal firing increased after low-dose TeNT. High-dose treatment, however, produced a firing depression, and in some cells, a total lack of modulation in relation to eye movements. Firing became increasingly more regular with larger TeNT doses as shown by significant reductions in the coefficient of variation after low- and high-dose treatments. Similarly, autocorrelation histograms of interspike intervals increased the number of resolvable peaks twofold in low-dose-treated motoneurones and sevenfold in high-dose-treated motoneurones. The plots of standard deviation versus the mean instantaneous firing frequency showed an upward deflexion with low firing frequencies. The upward deflexion occurred in controls at 39.9 +/- 4.9 ms, an interval similar to the mean afterhyperpolarisation (AHP) duration (48.4 +/- 8.8 ms). Low-dose TeNT treatment shifted the deflexion point to 20.9 +/- 3.9 ms, whereas the high dose increased it to 60.7 +/- 6.1 ms, in spite of the fact that no differences in AHP parameters between groups were found. The density of synaptophysin-immunoreactive boutons decreased by 14 % after the low-dose treatment and 40.5 % after the high-dose treatment, indicating that protracted synaptic blockade produces elimination of synaptic boutons. It is concluded that abducens motoneurone spike variability during spontaneous ocular fixations depends largely on the balance between inhibitory and excitatory synaptic innervation.

GABA and glycine co-release optimizes functional inhibition in rat brainstem motoneurons in vitro.

Whole-cell patch clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) were obtained in identified abducens motoneurons (aMns) from young rats (P5-P13). Three types of mIPSC were distinguished according to their kinetics and their sensitivity to receptor antagonists: faster decaying events mediated by glycine receptors (glyRs), slower decaying events mediated by GABA(A) receptors (GABA(A)Rs), and mIPSCs displaying two components corresponding to GABA and glycine co-release. Dual component events accounted for approximately 30 % of mIPSCs, independently of the rat's age and were also identified during evoked transmitter release. In contrast, the kinetics of glyR- and GABA(A)R-mediated mIPSCs became faster during development. Monosynaptic inhibitory postsynaptic potentials (IPSPs) were able to fully inhibit motoneuron discharge elicited by current pulses. When the GABA(A)R-mediated component or the glyR-mediated component of the IPSP was blocked, the inhibition of motoneuron firing was reduced. The 20-80 % rise time and duration of GABA(A)R-mediated IPSPs were significantly longer than those mediated by glyRs. The time window of inhibition for each component was determined using single postsynaptic action potentials elicited with various delays from the onset of the IPSP. GlyR-mediated IPSPs induced fast transient inhibition whereas GABA(A)R-mediated IPSPs induced slow sustained suppression of firing. Using a modelling approach, we found that the two components summated non-linearly. We conclude that in developing aMns, co-release of GABA and glycine determines the strength and timing of inhibition through non-linear interactions between the two components, thus optimizing inhibition of motoneuron function.

Reversible deafferentation of abducens motoneurons and internuclear neurons with tetanus neurotoxin.

Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential affinity for inhibitory neurotransmission. Following its intramuscular injection, TeNT is retrogradely and trans-synaptically transported towards the premotor terminals. Therefore, we have used TeNT as a tool to study the consequences of functional deafferentation on motoneurons following its peripheral administration. For this, we injected the toxin into the lateral rectus muscle at doses of 5 or 0.5 ng/kg and recorded the discharge activity of abducens motoneurons and internuclear neurons in the alert cat. Our results showed that: (i) TeNT blocked selectively the afferent inhibitory signals on abducens neurons only when used at a low dose, whereas both excitatory and inhibitory synaptic drive was lost after the high dose treatment; (ii) all effects were reversible within one month; and (iii) strikingly, the internuclear neurons of the abducens nucleus showed similar discharge alterations to the motoneurons, suggesting a TeNT action on shared common afferences.

Glial Cell Line-Derived Neurotrophic Factor and Developing Mammalian Motoneurons: Regulation of Programmed Cell Death Among Motoneuron Subtypes

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF after in utero treatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability. In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRalpha1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRalpha2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRalpha1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRalpha1/GFRalpha2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRalpha2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Blocking the trigeminal EPSP in rat abducens motoneurons in vivo with the AMPA antagonists NBQX and GYKI 53655

In pentobarbitone-anaesthetized rats, the effects of two AMPA receptor antagonists, the competitive antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline (NBQX) and the non-competitive 2,3-benzodiazepine GYKI 53655, were compared on excitatory synaptic transmission of trigeminal origin in intracellularly-recorded abducens motoneurons. The effects of both antagonists were also investigated on the α-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA)-, kainate-, and N-methyl-D-aspartate (NMDA)-induced depression of extracellular antidromic field potentials in the abducens motor nucleus. Microiontophoretic application (≤100 nA) or intravenous injection of NBQX (≤5 mg/kg) affected both AMPA- and kainate-induced depressions whereas GYKI 53655 (≤100 nA; ≤4 mg/kg) blocked only the AMPA-induced depression. Neither NBQX or GYKI 53655 affected NMDA-induced depressions of antidromic field potentials. Using low intravenous (i.v.) doses of the antagonists NBQX or GYKI 53655 (2–2.5 mg/kg), a complete blockade of the composite disynaptic trigeminal excitatory post-synaptic potential (EPSP) was obtained without any changes in membrane potential, input resistance and antidromic action potentials in abducens motoneurons. GYKI 53655 was more potent at low i.v. doses (0.5–1.8 mg/kg) but NBQX had longer-lasting effects. The results show the existence of differences between the blocking action of NBQX and GYKI 53655 on AMPA-mediated receptor EPSP in abducens motoneurons.

Discharge characteristics of axotomized abducens internuclear neurons in the adult cat.

The aim of the present work was to characterize the axotomy-induced changes in the discharge properties of central nervous system neurons recorded in the alert behaving animal. The abducens internuclear neurons of the adult cat were the chosen model. The axons of these neurons course through the contralateral medial longitudinal fascicle and contact the medial rectus motoneurons of the oculomotor nucleus. Axotomy was carried out by the unilateral transection of this fascicle (right side) and produced immediate oculomotor deficits, mainly the incapacity of the right eye to adduct across the midline. Extracellular single-unit recording of abducens neurons was carried out simultaneously with eye movements. The main alteration observed in the firing of these axotomized neurons was the overall decrease in firing rate. During eye fixations, the tonic signal was reduced, and, on occasion, a progressive decay in firing rate was observed. On-directed saccades were not accompanied by the high-frequency spike burst typical of controls; instead, there was a moderate increase in firing. Similarly, during the vestibular nystagmus, neurons hardly modulated during both the slow and the fast phases. Linear regression analysis between firing rate and eye movement parameters showed a significant reduction in eye position and velocity sensitivities with respect to controls, during both spontaneous and vestibularly induced eye movements. These firing alterations were observed during the 3 month period of study after lesion, with no sign of recovery. Conversely, abducens motoneurons showed no significant alteration in their firing pattern. Therefore, axotomy produced long-lasting changes in the discharge characteristics of abducens internuclear neurons that presumably reflected the loss of afferent oculomotor signals. These alterations might be due to the absence of trophic influences derived from the target.

Plastic changes underlying vestibular compensation in the guinea-pig persist in isolated, in vitro whole brain preparations

Vestibular compensation for the postural and oculomotor deficits induced by unilateral labyrinthectomy is a model of post-lesional plasticity in the central nervous system. Just after the removal of one labyrinth, the deafferented, ipsilateral vestibular nucleus neurons are almost silent, and the discharge of the contralateral vestibular nucleus neurons is increased. The associated static disorders disappear in a few days, as normal activity is restored in both vestibular nuclei. In this study, we searched for traces of vestibular compensation in isolated whole brains taken from adult guinea-pigs. The electrophysiological responses evoked in control brains were compared to those evoked in brains taken from animals that had previously been labyrinthectomized. Guinea-pigs compensated for an initial labyrinthectomy within three days. In vivo, subsequent deafferentation of vestibular nucleus neurons on the intact side triggered “Bechterew's phenomenon”: a new postural and oculomotor syndrome appeared, similar to the one induced by the first lesion, but directed to the newly deafferented side. These disturbances would be caused by the new imbalance between the discharges of neurons in the two vestibular nuclei triggered by the second deafferentation. Experiments were designed to search for a similar imbalance in vitro in brains taken from labyrinthectomized animals, where the intact vestibular nerve is cut during the dissection. Isolated whole brains were obtained from young guinea-pigs at various times (one to seven days) following an initial labyrinthectomy. An imbalance between the resting activities of medial vestibular nucleus neurons on both sides of the brainstem was revealed in brains taken more than three days after the lesion: their discharge was higher on the compensated, initially lesioned side than on the newly deafferented side. In some cases, an oscillatory pattern of discharge, reminiscent of the spontaneous nystagmus associated in vivo with Bechterew's syndrome, appeared in both abducens nerves. These data demonstrate that most of the changes underlying vestibular compensation persist, and can thus be investigated in the isolated whole brain preparation. Brains removed only one day after the lesion displayed normal commissural responses and symmetric spinal inputs to vestibular nucleus neurons. However, an unusually large proportion of the neurons recorded on both sides of the preparation had very irregular spontaneous discharge rates. These data suggest that the first stages of vestibular compensation might be associated with transient changes in the membrane properties of vestibular nucleus neurons. Brains taken from compensated animals displayed a significant, bilateral decrease of the inhibitory commissural responses evoked in the medial vestibular nucleus by single-shock stimulation of the contralateral vestibular nerve. The sensitivity of abducens motoneurons on the initially lesioned, compensated side to synaptic activation from the contralesional vestibular nucleus neurons was also decreased. Both changes may explain the long-term, bilateral decrease of vestibular-related reflexes observed following unilateral labyrinthectomy. Spinal inputs to vestibular nucleus neurons became progressively asymmetric: their efficacy was increased on the lesioned side and decreased on the intact one. This last modification may support a functional substitution of the deficient, vestibular-related synergies involved in gaze and posture stabilization by neck-related reflexes.

Discharge profiles of abducens, accessory abducens, and orbicularis oculi motoneurons during reflex and conditioned blinks in alert cats.

The discharge profiles of identified abducens, accessory abducens, and orbicularis oculi motoneurons have been recorded extra- and intracellularly in alert behaving cats during spontaneous, reflexively evoked, and classically conditioned eyelid responses. The movement of the upper lid and the electromyographic activity of the orbicularis oculi muscle also were recorded. Animals were conditioned by short, weak air puffs or 350-ms tones as conditioned stimuli (CS) and long, strong air puffs as unconditioned stimulus (US) using both trace and delayed conditioning paradigms. Motoneurons were identified by antidromic activation from their respective cranial nerves. Orbicularis oculi and accessory abducens motoneurons fired an early, double burst of action potentials (at 4-6 and 10-16 ms) in response to air puffs or to the electrical stimulation of the supraorbital nerve. Orbicularis oculi, but not accessory abducens, motoneurons fired in response to flash and tone presentations. Only 10-15% of recorded abducens motoneurons fired a late, weak burst after air puff, supraorbital nerve, and flash stimulations. Spontaneous fasciculations of the orbicularis oculi muscle and the activity of single orbicularis oculi motoneurons that generated them also were recorded. The activation of orbicularis oculi motoneurons during the acquisition of classically conditioned eyelid responses happened in a gradual, sequential manner. Initially, some putative excitatory synaptic potentials were observed in the time window corresponding to the CS-US interval; by the second to the fourth conditioning session, some isolated action potentials appeared that increased in number until some small movements were noticed in eyelid position traces. No accessory abducens motoneuron fired and no abducens motoneuron modified their discharge rate for conditioned eyelid responses. The firing of orbicularis oculi motoneurons was related linearly to lid velocity during reflex blinks but to lid position during conditioned responses, a fact indicating the different neural origin and coding of both types of motor commands. The power spectra of both reflex and conditioned lid responses showed a dominant peak at approximately 20 Hz. The wavy appearance of both reflex and conditioned eyelid responses was clearly the result of the high phasic activity of orbicularis oculi motor units. Orbicularis oculi motoneuron membrane potentials oscillated at approximately 20 Hz after supraorbital nerve stimulation and during other reflex and conditioned eyelid movements. The oscillation seemed to be the result of both intrinsic (spike afterhyperpolarization lasting approximately 50 ms, and late depolarizations) and extrinsic properties of the motoneuronal pool and of the circuits involved in eye blinks.

Modulation of kainate-induced responses by pentobarbitone and GYKI-53784 in rat abducens motoneurons in vivo

The modulation of kainate-induced responses by pentobarbitone and the 2,3-benzodiazepine GYKI-53784 (LY303070), a potent non-competitive AMPA antagonist, was studied in vivo using both extracellular recordings of antidromic field potentials and intracellular recordings from abducens motoneurons in ketamine/diazepam-anesthetized rats. In previous studies on pentobarbitone-anesthetized rats [M. Ouardouz, J. Durand, GYKI-52466 antagonizes glutamate responses but not NMDA and kainate responses in rat abducens motoneurons, Neurosci. Lett. 125 (1991) 5–8; M. Ouardouz, J. Durand, Involvement of AMPA receptors in trigeminal postsynaptic potentials recorded in rat abducens motoneurons in vivo, Eur. J. Neurosci. 6 (1994) 1662–1668; A. Ruiz, J. Durand, Blocking the trigeminal EPSPs in rat abducens motoneurons in vivo with the AMPA antagonists, NBQX and GYKI-53655, J. Neurophysiol. (1998) submitted], we showed that 2,3-benzodiazepines do not affect kainate-induced depolarizations in abducens motoneurons. Here, we tested whether pentobarbitone is involved in the pharmacological discrimination by 2,3-benzodiazepines between AMPA- and kainate-induced responses. Kainate-induced depolarizations were reversibly depressed after application of either GYKI-53784 and pentobarbitone. However, kainate-induced depolarizations were not inhibited by GYKI-53784 with pentobarbitone; they were even potentiated sometimes. Using extracellular recordings, we confirmed that in the presence of pentobarbitone, GYKI-53784 counteracts the effects of AMPA but not of kainate on antidromic field potentials in the abducens nucleus. Blockade of kainate-induced responses by GYKI-53784 was reversed with pentobarbitone, which appears relevant to the discrimination between AMPA- and kainate receptor-mediated responses in vivo. In the presence of pentobarbitone, kainate would depolarize motoneurons mainly via kainate receptors since kainate-induced responses were not depressed by 2,3-benzodiazepines. This finding strongly favors the existence of kainate receptors in adult motoneurons but their role is still unknown.

Trigeminal disynaptic circuit mediating corneal afferent input to M. depressor palpebrae inferioris motoneurons in the pigeon (Columba livia).

Corneal afferent projections to the trigeminal brainstem nuclear complex (TBNC) and associated structures, as determined by transganglionic transport of various tracers, were found to be predominantly concentrated in two distinct patches in the dorsolateral medulla at periobex levels. One was in the external cuneate nucleus, and the other was in the ventralmost part of the ophthalmic division of the TBNC. The projections of putative second-order neurons in these regions, as determined by injections of wheat germ agglutinin conjugated to horseradish peroxidase into the dorsolateral medulla, were found to include the dorsal trigeminal motor nucleus (Vd), which innervates the M. depressor palpebrae inferioris. Electrical stimulation of Vd, which elicited lower eyelid movements, was then used to guide injections of tracer into Vd, which retrogradely labeled clusters of neurons in the corneal afferent recipient regions of the dorsolateral medulla. The lower eyelid of pigeons, unlike the nictitating membrane and upper lid, does not appear to be appreciably involved in either reflex blinking in response to relatively mild stimulation of the cornea (e.g., air puff), or in eye closure during the saccade-like head movements associated with walking, or in eye closure during pecking; but in response to a stimulus that makes corneal contact, an upward movement of the lower lid follows descent of the nictitating membrane and upper lid as part of a defensive eye-closing mechanism. The anatomical results thus appear to define a dedicated disynaptic trigeminal sensorimotor circuit for the control of lower eyelid motility in response to mechanical or noxious stimuli of the cornea. Injections of tracers into the lower and upper eyelids labeled palpebral sensory afferents that terminated predominantly in maxillary and ophthalmic portions, respectively, of the dorsal horn of upper cervical spinal segments. These terminal fields were therefore largely separate from those of corneal afferents. There were no specific corneal afferent projections upon accessory abducens motoneurons that innervate the two muscles controlling the nictitating membrane.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague–Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35 S -labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Extraocular Motor Unit and Whole-Muscle Responses in the Lateral Rectus Muscle of the Squirrel Monkey

Because primate studies provide data for the current experimental models of the human oculomotor system, we investigated the relationship of lateral rectus muscle motoneuron firing to muscle unit contractile characteristics in the squirrel monkey. Also examined was the correlation of whole-muscle contractile force with the degree of evoked eye displacement. A force transducer was used to record lateral rectus whole-muscle or muscle unit contraction in response to abducens whole-nerve stimulation or stimulation of single abducens motoneurons or axons. Horizontal eye displacement was recorded using a magnetic search coil. (1) Motor units could be categorized based on contraction speed (fusion frequency) and fatigue. (2) The kt value (change in motoneuronal firing necessary to increase motor unit force by 1.0 mg) of the units correlated with maximum tetanic tension. (3) There was some tendency for maximum tetanic tension of this unit population to separate into three groups. (4) At a constant frequency of 100 Hz, 95% of the motor units demonstrated significantly different force levels dependent on immediately previous stimulation history (hysteresis). (5) A mean force change of 0.32 gm/ degrees and a mean frequency change of 4.7 Hz/ degrees of eye displacement were observed in response to whole-nerve stimulation. These quantitative data provide the first contractile measures of primate extraocular motor units. Models of eye movement dynamics may need to consider the nonlinear transformations observed between stimulation rate and muscle tension as well as the probability that as few as two to three motor units can deviate the eye 1 degrees.

Effects of botulinum neurotoxin type A on the expression of gephyrin in cat abducens motoneurons

In this study, we investigated the effects of long-term synaptic blockade on postsynaptic receptor clustering at central inhibitory glycinergic synapses. High doses of botulinum neurotoxin type A injected in the lateral rectus muscle completely abolishes inhibitory postsynaptic potentials onto abducens motoneurons within 2 days postinjection, and transmission remains blocked for at least 2 months. Using this model, we analyzed the expression of gephyrin, a glycine receptor clustering protein, on the membrane of motoneuron somata after botulinum neurotoxin type A injection in their target muscle. Immunofluorescence or electron microscopy immunohistochemistry revealed gephyrin-immunoreactive clusters (most < 0.5 microm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructurally, presynaptic terminals containing flattened synaptic vesicles (F terminals) were found associated with multiple gephyrin-immunoreactive postsynaptic densities (average 1.24 gephyrin clusters/F+ profile). No significant changes in gephyrin-immunoreactive clusters were observed at 5 days postinjection, but we found significant reductions (25-40%) in the density of gephyrin clusters 19 and 35 days postinjection. Hence, the physiological alterations reported in this model precede structural changes on postsynaptic receptor cluster density. The decrease in gephyrin-immunoreactive clusters was paralleled by reductions in synaptic covering (F+ terminals per 100 microm of membrane). Presumed inactive F+ terminals that remained attached to the motoneuron surface displayed normal gephyrin-immunoreactive clusters; however, the pre- and postsynaptic membranes in between synaptic active zones frequently appeared separated by enlarged extracellular spaces. We concluded that postsynaptic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.

Intracellular Correlates of Acquisition and Long-Term Memory of Classical Conditioning in Purkinje Cell Dendrites in Slices of Rabbit Cerebellar Lobule HVI

Intradendritic recordings in Purkinje cells from a defined area in parasaggital slices of cerebellar lobule HVI, obtained after rabbits were given either paired (classical conditioning) or explicitly unpaired (control) presentations of tone and periorbital electrical stimulation, were used to assess the nature and duration of conditioning-specific changes in Purkinje cell dendritic membrane excitability. We found a strong relationship between the level of conditioning and Purkinje cell dendritic membrane excitability after initial acquisition of the conditioned response. Moreover, conditioning-specific increases in Purkinje cell excitability were still present 1 month after classical conditioning. Although dendritically recorded membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in cells from paired or control animals, the size of a potassium channel-mediated transient hyperpolarization was significantly smaller in cells from animals that received classical conditioning. In slices of lobule HVI obtained from naive rabbits, the conditioning-related increases in membrane excitability could be mimicked by application of potassium channel antagonist tetraethylammonium chloride, iberiotoxin, or 4-aminopyridine. However, only 4-aminopyridine was able to reduce the transient hyperpolarization. The pharmacological data suggest a role for potassium channels and, possibly, channels mediating an IA-like current, in learning-specific changes in membrane excitability. The conditioning-specific increase in Purkinje cell dendritic excitability produces an afterhyperpolarization, which is hypothesized to release the cerebellar deep nuclei from inhibition, allowing conditioned responses to be elicited via the red nucleus and accessory abducens motorneurons.

Conditions of dominant effectiveness of distal sites of active uniform dendrites with distributed tonic inputs

This simulation study aimed at assessing linkage between the membrane properties and the effectiveness of somatopetal current transfer from activated tonic excitatory inputs homogeneously distributed along uniform dendrites. It was shown that in the dendrites having anN-shaped steady current-voltage membrane characteristic due to the negative slope within a certain range of potentials, distal sites can be more effective than proximal sites in somatopetal current transfer from tonically activated excitatory synaptic inputs. Inhomogeneous dendritic depolarization produced by these inputs should be found everywhere within a range of the negative slope. In simulated dendrites receiving, as in rat abducens motoneurons, voltage-sensitive synaptic inputs of anN-methyl-D-aspartate (NMDA) type, such spatial effects occurred at low depolarization produced by subcritical excitation. At supercritical excitation, depolarization increased and left the range of the negative slope, and proximal sites became much more effective than distal ones. It is suggested that persistent inward currents (including other than of NMDA nature) can provide similar effects.