Analysis of co-isogenic prion protein deficient mice reveals behavioral deficits, learning impairment, and enhanced hippocampal excitability.

BACKGROUND: Cellular prion protein (PrPC) is a cell surface GPI-anchored protein, usually known for its role in the pathogenesis of human and animal prionopathies. However, increasing knowledge about the participation of PrPC in prion pathogenesis contrasts with puzzling data regarding its natural physiological role. PrPC is expressed in a number of tissues, including at high levels in the nervous system, especially in neurons and glial cells, and while previous studies have established a neuroprotective role, conflicting evidence for a synaptic function has revealed both reduced and enhanced long-term potentiation, and variable observations on memory, learning, and behavior. Such evidence has been confounded by the absence of an appropriate knock-out mouse model to dissect the biological relevance of PrPC, with some functions recently shown to be misattributed to PrPC due to the presence of genetic artifacts in mouse models. Here we elucidate the role of PrPC in the hippocampal circuitry and its related functions, such as learning and memory, using a recently available strictly co-isogenic Prnp0/0 mouse model (PrnpZH3/ZH3). RESULTS: We performed behavioral and operant conditioning tests to evaluate memory and learning capabilities, with results showing decreased motility, impaired operant conditioning learning, and anxiety-related behavior in PrnpZH3/ZH3 animals. We also carried in vivo electrophysiological recordings on CA3-CA1 synapses in living behaving mice and monitored spontaneous neuronal firing and network formation in primary neuronal cultures of PrnpZH3/ZH3 vs wildtype mice. PrPC absence enhanced susceptibility to high-intensity stimulations and kainate-induced seizures. However, long-term potentiation (LTP) was not enhanced in the PrnpZH3/ZH3 hippocampus. In addition, we observed a delay in neuronal maturation and network formation in PrnpZH3/ZH3 cultures. CONCLUSION: Our results demonstrate that PrPC promotes neuronal network formation and connectivity. PrPC mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion may underlie an epileptogenic-susceptible brain that fails to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.

Nitric oxide production by brain stem neurons is required for normal performance of eye movements in alert animals.

Although nitric oxide (NO) is produced by discrete groups of neurons in the brain, participation of NO in premotor structures directly involved in reflexively evoked, sensory-motor functions has not been demonstrated so far. We now show that NO is a physiological mediator in the generation of a specific motor response in alert behaving animals. In the oculomotor system, numerous neurons expressing nitric oxide synthase (NOS) are located in the prepositus hypoglossi, a nucleus involved in the control of horizontal eye movements. Unilateral inhibition of NOS within this nucleus results in severe ocular nystagmus with slow phases directed to the contralateral side. Accordingly, local increases of NO or cyclic GMP produced a nystagmus in the opposite direction. It is concluded that a balanced production of NO by prepositus hypoglossi neurons is a necessary condition for the normal performance of eye movements in alert animals.

Aged wild-type and APP, PS1, and APP + PS1 mice present similar deficits in associative learning and synaptic plasticity independent of amyloid load.

Wild-type and single-transgenic (APP, PS1) and double-transgenic (APP+PS1) mice were studied at three different (3-, 12-, and 18-month-old) age periods. Transgenic mice had reflex eyelid responses like those of controls, but only 3-month-old mice were able to fully acquire conditioned eyeblinks, using a trace paradigm, whilst 12-month-old wild-type and transgenic mice presented intermediate values, and 18-month-old wild-type and transgenic mice were unable to acquire this type of associative learning. 18-month-old wild-type and transgenic mice presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals, but they did not show any activity-dependent potentiation of the CA3-CA1 synapse across conditioning sessions, as was shown by 3-month-old wild-type mice. Moreover, 18-month-old wild-type and transgenic mice presented a noticeable deficit in long-term potentiation evoked in vivo at the hippocampal CA3-CA1 synapse. The 18-month-old wild-type and transgenic mice also presented a significant deficit in prepulse inhibition as compared with 3-month-old controls. Except for results collected by prepulse inhibition, the above-mentioned deficits were not related with the presence of amyloid beta deposits. Thus, learning and memory deficits observed in aged wild-type and transgenic mice are not directly related to the genetic manipulations or to the presence of amyloid plaques.

[Hemoperitoenum as presentation of hepatocellular carcinoma: experience in three cases with spontaneous tumoral rupture and review of the literature]. / Hemoperitoneo como forma de presentación del carcinoma hepatocelular: experiencia de tres casos con rotura tumoral espontánea y revisión de la literatura.

Hemoperitoneum due to spontaneous rupture of hepatocellular carcinoma (HCC) constitutes a life-threatening situation if no appropriate therapy is provided. This complication is a well-known form of HCC presentation in countries with high incidence of liver tumours, but is an unusual event in Western countries, where it has been described in 5% or less of cases with HCC. We report three patients admitted to our centre with acute hemoperitoneum secondary to non-traumatic rupture as a first manifestation of not previously diagnosed HCC. A review of the related literature is also performed.

Activity-dependent changes of the hippocampal CA3-CA1 synapse during the acquisition of associative learning in conscious mice.

Contemporary neuroscientists are paying increasing attention to subcellular, molecular and electrophysiological mechanisms underlying learning and memory processes. Recent efforts have addressed the development of transgenic mice affected at different stages of the learning process, or emulating pathological conditions involving cognition and motor-learning capabilities. However, a parallel effort is needed to develop stimulating and recording techniques suitable for use in behaving mice, in order to grasp activity-dependent neural changes taking place during the very moment of the process. These in vivo models should integrate the fragmentary information collected by different molecular and in vitro approaches. In this regard, long-term potentiation (LTP) has been proposed as the neural mechanism underlying synaptic plasticity. Moreover, N-methyl-d-aspartate (NMDA) receptors are accepted as the molecular substrate of LTP. It now seems necessary to study the relationship of both LTP and NMDA receptors with the plastic changes taking place, in selected neural structures, during actual learning. Here, we review data on the involvement of the hippocampal CA3-CA1 synapse in the acquisition of classically conditioned eyelid conditioned responses (CRs) in behaving mice. Available data show that LTP, evoked by high-frequency stimulation of Schaffer collaterals, disturbs both the acquisition of CRs and the physiological changes that occur at the CA3-CA1 synapse during learning. Moreover, the administration of NMDA-receptor antagonists is able not only to prevent LTP induction in vivo, but also to hinder the formation of both CRs and functional changes in strength of the CA3-CA1 synapse. Thus, there is experimental evidence relating activity-dependent synaptic changes taking place during actual learning with LTP mechanisms and with the role of NMDA receptors in both processes.

Classical eyeblink conditioning during acute hypobaric hypoxia is improved in acclimatized mice and involves Fos expression in selected brain areas.

This work attempts to evaluate the cognitive aspects of the acclimatization ability of mice submitted to simulated altitude. Critical altitudes were detected by evaluating open field activity, combined or not with object recognition tasks, at different acute simulated altitudes. Results showed impaired cognitive abilities at approximately 3,733 m and above. To evaluate acclimatization capabilities, mice submitted to hypobaric hypoxia at approximately 5,000 m for 1 wk were tested for learning and memory performances with classical eyeblink conditioning at the same altitude or at land altitude. Results showed total acclimatization in mice conditioned at approximately 5,000 m but no improved performance in those conditioned at land altitudes compared with controls. Selected brain sites of conditioned animals were analyzed by immunohistochemistry to detect expression of the protein product of the protooncogene c-fos (Fos) in relation to both motor learning processes and hypobaric conditions. In the nucleus of the solitary tract, a higher expression of Fos was found in the acute hypobaric conditioned animals than in control conditioned and nonconditioned animals. Similar patterns between groups were found in the other brain areas, mainly in the piriform cortex and area 1 of the cingulate cortex and in the hippocampus. Differences between hemispheres were detected only in acute hypobaric animals. The present results show that acclimatization to high altitude prevents the impairment of classical eyeblink conditioning evoked by hypobaric hypoxic conditions but does not improve this task when acquired under land conditions, although it could diminish the activation requirements for its performance.

Behaviour of Medial Rectus Motoneurons in the Alert Cat.

The activity of identified medial rectus motoneurons was recorded in alert cats during spontaneous and vestibular induced eye movements. Medial rectus motoneurons fired a burst of spikes slightly preceding adducting saccades and increased their discharge rate linearly with successive eye positions in the adducting direction. Conduction velocity (21.3 - 98.2 m/s), eye position sensitivity (ks, 7.1 +/- 1.5 spikes/s/deg), and eye velocity sensitivity (rs, 1 +/- 0.2 spikes/s/deg/s) during spontaneous eye movements, and time constants calculated from phase lead analysis (To, 135 +/- 36 ms) showed values similar to those described previously for cat abducens motoneurons. The firing rate during repeated fixation of the same eye position was affected significantly by the direction of the preceding saccade and by the animal's level of alertness. Eye velocity sensitivity was not significantly affected by changes in the animal's level of alertness. A weak negative relationship (coefficient of correlation=-0.56) was observed between eye velocity sensitivity (rv) and sinusoidal rotational frequency, with no change in eye position sensitivity (kv) with stimulus frequency. The subsequent changes in the time constant (Tv) calculated as Tv=rv/kv in relation to stimulus frequency suggests that the oculomotor system deviates from a (linear) first-order model.

Afferents to the oculomotor nucleus in the goldfish (Carassius auratus) as revealed by retrograde labeling with horseradish peroxidase.

The goal of this work was to compare the distribution and morphology of neurons projecting to the oculomotor nucleus in goldfish with those previously described in other vertebrate groups. Afferent neurons were revealed by retrograde labeling with horseradish peroxidase. The tracer was electrophoretically injected into the oculomotor nucleus. The location of the injection site was determined by the antidromic field potential elicited in the oculomotor nucleus by electrical stimulation of the oculomotor nerve. Labeled axons whose trajectories could be reconstructed were restricted to the medial longitudinal fasciculus. In order of quantitative importance, the afferent areas to the oculomotor nucleus were: (1) the ipsilateral anterior nucleus and the contralateral tangential and descending nuclei of the octaval column. Furthermore, a few labeled cells were found dorsomedially to the caudal pole of the unlabeled anterior octaval nucleus; (2) the contralateral abducens nucleus. The labeled internuclear neurons were arranged in two groups within and 500 microns behind the caudal subdivision of the abducens nucleus; (3) a few labeled cells were observed in the rhombencephalic reticular formation near the abducens nucleus, most of which were contralateral to the injection site. Specifically, stained cells were found in the caudal pole of the superior reticular nucleus, throughout the medial reticular nucleus and in the rostral area of the inferior reticular nucleus; (4) eurydendroid cells of the cerebellum, located close to the contralateral eminentia granularis pars lateralis, were also labeled; and (5) a small and primarily ipsilateral group of labeled cells was located at the mesencephalic nucleus of the medial longitudinal fasciculus. The similarity in the structures projecting to the oculomotor nucleus in goldfish to those in other vertebrates suggests that the neural network involved in the oculomotor system is quite conservative throughout phylogeny. Nevertheless, in goldfish these projections appeared with some specific peculiarities, such as the cerebellar and mesencephalic afferents to the oculomotor nucleus.

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.

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.

Response of abducens internuclear neurons to axotomy in the adult cat.

The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons of the oculomotor nucleus constitutes an optimal model for investigating the effects of axotomy in the central nervous system. We have analyzed the morphological changes induced by this lesion on both the cell bodies and the transected axons of abducens internuclear neurons in the adult cat. Axotomy was performed by the transection of the medial longitudinal fascicle. Cell counts of Nissl-stained material and calretinin-immunostained abducens internuclear neurons revealed no cell death by 3 months postaxotomy. Ultrastructural examination of these cells at 6, 14, 24, and 90 days postaxotomy showed normal cytological features. However, the surface membrane of axotomized neurons appeared contacted by very few synaptic boutons compared to controls. This change was quantified by measuring the percentage of synaptic coverage of the cell bodies and the linear density of boutons. Both parameters decreased significantly after axotomy, with the lowest values at 90 days postlesion ( approximately 70% reduction). We also explored axonal regrowth and the possibility of reinnervation of a new target by means of anterograde labeling with biocytin. At all time intervals analyzed, labeled axons were observed to be interrupted at the caudal limit of the lesion; in no case did they cross the scar tissue to reach the distal part of the tract. Nonetheless, a conspicuous axonal sprouting was present at the caudal aspect of the lesion site. Structures suggestive of axonal growth were found, such as large terminal clubs, from which short filopodium-like branches frequently emerged. Similar findings were obtained after parvalbumin and calretinin immunostaining. At the electron microscopy level, biocytin-labeled boutons originating from the sprouts appeared surrounded by either extracellular space, which was extremely dilated at the lesion site, or by glial processes. The great majority of labeled boutons examined were, thus, devoid of neuronal contact, indicating absence of reinnervation of a new target. Altogether, these data indicate that abducens internuclear neurons survive axotomy in the adult cat and show some form of axonal regrowth, even in the absence of target connection.

Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat.

Chewing, swallowing, breathing, and vocalization in mammals require precise coordination of tongue movements with concomitant activities of the mimetic muscles. The neuroanatomic basis for this oro-facial coordination is not yet fully understood. After the stereotaxic microinjection of retrograde and anterograde neuronal tracers (biotin-dextran, Fluoro-Ruby, Fluoro-Emerald, and Fluoro-Gold) into the facial and hypoglossal nuclei of the rat, we report here a direct bilateral projection of hypoglossal internuclear interneurons onto facial motoneurons. We also confirm the existence of a small pool of neurons in the dorsal part of the brainstem reticular formation that project ipsilaterally to both facial and hypoglossal nuclei. For precise tracer injections, both motor nuclei were located and identified by the electrical antidromic activation of their constituent motoneurons. Injections of retrograde tracers into the facial nucleus consistently labeled neurons in the hypoglossal nucleus. These neurons prevalently lay in the ipsilateral side, were small in size, and, like classic intrinsic hypoglossal local-circuit interneurons, had several thin dendrites. Reverse experiments - injections of anterograde tracers into the hypoglossal nucleus - labeled fine varicose nerve fiber terminals in the facial nucleus. These fiber terminals were concentrated in the intermediate subdivision of the facial nucleus, with a strong ipsilateral prevalence. Double injections of different tracers into the facial and the hypoglossal nuclei revealed a small, but constant, number of double-labeled neurons located predominantly ipsilateral in the caudal brainstem reticular formation. Hypoglossal internuclear interneurons projecting to the facial nucleus, as well as those neurons of the parvocellular reticular formation that project to both facial and hypoglossal nuclei, could be involved in oro-facial coordination.

Localization of parvalbumin, calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat.

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15-20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions.

Influence of the postsynaptic target on the functional properties of neurons in the adult mammalian central nervous system.

In this review we have attempted to summarize present knowledge concerning the regulatory role of target cells on the expression and maintenance of the neuronal phenotype during adulthood. It is well known that in early developmental stages the survival of neurons is maintained by specific neurotrophic factors derived from their target tissues. Neuronal survival is not the only phenotype that is regulated by target-derived neurotrophic factors since the expression of electrophysiological and cytochemical properties of neurons is also affected. However, a good deal of evidence indicates that the survival of neurons becomes less dependent on their targets in the adult stage. The question is to what extent are target cells still required for the maintenance of the pre-existing or programmed state of the neuron; i.e., what is the functional significance of target-derived factors during maturity? Studies addressing this question comprise a variety of neuronal systems and technical approaches and they indicate that trophic interactions, although less apparent, persist in maturity and are most easily revealed by experimental manipulation. In this respect, research has been directed to analyzing the consequences of disconnecting a group of neurons from their target-by either axotomy or selective target removal using different neurotoxins-and followed (or not) by the implant of a novel target, usually a piece of embryonic tissue. Numerous alterations have been described as taking place in neurons following axotomy, affecting their morphology, physiology and metabolism. All these neuronal properties return to normal values when regeneration is successful and reinnervation of the target is achieved. Nevertheless, most of the changes persist if reinnervation is prevented by any procedure. Although axotomy may represent, besides target disconnection, a cellular lesion, alternative approaches (e.g., blockade of either the axoplasmic transport or the conduction of action potentials) have been used yielding similar results. Moreover, in the adult mammalian central nervous system, neurotoxins have been used to eliminate a particular target selectively and to study the consequences on the intact but target-deprived presynaptic neurons. Target depletion performed by excitotoxic lesions is not followed by retrograde cell death, but targetless neurons exhibit several modifications such as reduction in soma size and in the staining intensity for neurotransmitter-synthesizing enzymes. Recently, the oculomotor system has been used as an experimental model for evaluating the functional effects of target removal on the premotor abducens internuclear neurons whose motoneuronal target is destroyed following the injection of toxic ricin into the extraocular medial rectus muscle. The functional characteristics of these abducens neurons recorded under alert conditions simultaneously with eye movements show noticeable changes after target loss, such as a general reduction in firing frequency and a loss of the discharge signals related to eye position and velocity. Nevertheless, the firing pattern of these targetless abducens internuclear neurons recovers in parallel with the establishment of synaptic contacts on a presumptive new target: the small oculomotor internuclear neurons located in proximity to the disappeared target motoneurons. The possibility that a new target may restore neuronal properties towards a normal state has been observed in other systems after axotomy and is also evident from experiments of transplantation of immature neurons into the lesioned central nervous system of adult mammals. It can be concluded that although target-derived factors may not control neuronal survival in the adult nervous system, they are required for the maintenance of the functional state of neurons, regulating numerous aspects of neuronal structure, chemistry and electro-physiology.(ABSTRUCT TRUNCATED)

Dose-dependent, central effects of botulinum neurotoxin type A: a pilot study in the alert behaving cat.

We investigated, in alert behaving cats, the long-term effects of botulinum neurotoxin (BoNT) type A injected into the lateral rectus muscle of the eye. We studied orthodromic field potentials recorded in the injected muscle, eye movements, and the discharge characteristics of the innervating abducens motoneurons. Single BoNT injections at doses from 0.01 to 0.3 ng/kg reduced, or even completely eliminated, eye movements in the abducting direction for up to 2 months without affecting the motoneuron discharge profile that remained related to actual eye movements of the contralateral unparalyzed eye. This result indicates that abducens motoneurons were still under the influence of the ocular motor central control system regardless of their ineffective action on lateral rectus muscle fibers. We also conclude that paralysis per se is not enough to initiate axotomy-like neural responses in ocular motoneurons. The injection of BoNT at a dose of 3 ng/kg produced significant changes in the discharge pattern of abducens motoneurons lasting up to 3 months-the maximum time checked. This finding was probably due to retrograde and, perhaps, transneuronal effects of BoNT when injected in a high dose. The results give some indications of the maximum allowable dose that can be used without the induction of unwanted side effects in the motoneuronal pool innervating the injected muscle.

Discharge of identified deep cerebellar nuclei neurons related to eye blinks in the alert cat.

The activity of identified cerebellar nuclear neurons was recorded in the alert cat during blinks induced by corneal air puffs, light flashes and tones. Eyelid response to air puffs consisted of an early (16.5 +/- 2.7 ms) downward movement followed by two to three late downward steps. Blinks induced by flashes or tones presented longer latencies (52.6 +/- 4.8 and 50.1 +/- 8.0 ms). Type A neurons (n = 86) increased their spike activity in coincidence with the beginning of the blink, regardless of the stimulus modality. The late eyelid downward responses were accompanied by corresponding increases in the firing rate of the neuron. Type A neurons were activated mostly from the red nucleus (48/86) or the restiform body (24/86). Type B neurons (n = 30) fired a brief burst of spikes slightly preceding the blink, followed by a noticeable decrease in their firing rate. As for type A, the discharge response of type B neurons was always the same regardless of the sensory modality. These neurons were activated from the red nucleus (18/30), oculomotor complex (6/30) and restiform body (6/30). Although no precise temporal coupling was found between the beginning of the neuronal response and the start of either the stimulus or the motor response, linear regression analysis demonstrated significant relationships between mean firing rate of type A and B neurons and eyelid position, velocity and/or acceleration. Deep cerebellar nuclei neurons presented here seem to be directly involved in the execution of reflexively induced blinks following the smaller details of eyelid motor performance. The opposite behavior of type A and B cells suggests an interplay of reciprocal actions to determine the ongoing displacements of the lid. Finally, the cerebellum seems to influence blinks through a spread action on many brainstem sites and not exclusively on the red nucleus.

Behavior of neurons in the abducens nucleus of the alert cat--I. Motoneurons.

The activity of 53 antidromically identified abducens motoneurons was analyzed in alert cats during spontaneous and vestibular induced eye movements. Conduction velocities ranged from 13 to 70 m/s and all motoneurons increased their discharge rates with successive eye positions in the abducting direction. Motoneurons were recruited from -19 degrees to +7 degrees. Within the oculomotor range frequency saturation was never observed for any cell. The slope of rate-position (k) relationships ranged from 2 to 17.7 spikes/s/deg (n = 40, mean 8.7 +/- 2.5). Regression analysis showed that the rate-position plots could be fit by straight lines but in most cases exponential curves produced slightly better statistical fits. Steeper slopes suggest that successively larger increases in k are required for the lateral rectus muscle to maintain more eccentric fixations in the on direction. Interspike intervals for a constant eye position exhibited low variability (less than 3.5%) for fixations shorter than 1 s. Over longer periods, variability increased in proportion to the duration of the fixation in exponential-like fashion up to 14%. Abducens motoneurons showed considerable variability in frequency during repeated fixations of the same eye position. Discharge rates were found to depend upon both the direction of the previous eye movement and, more importantly, the animal's level of alertness. The rate-position regression lines for fixation periods after saccades in the on direction significantly differed in slopes (100%) and thresholds (20%) from those in the off direction. The observed static hysteresis in abducens motoneuron behavior was in opposite direction to that previously described for the mechanical properties of the lateral rectus. This suggests both neural and mechanical factors are significantly involved in determining final eye position. The animal's level of alertness was evaluated in this study by counting the number of saccadic movements/s occurring in "alert" (1 +/- 0.2 saccades/s), and "drowsy" (0.5 +/- 0.2 saccades/s) circumstances. Comparison of the rate-position regression lines between the two conditions showed a significant decrease in slopes (100%) and elevation of thresholds (70%). Discharge rate of abducens motoneurons increased abruptly 8.9 +/- 2.8 ms prior to saccades in the horizontal on direction, and decreased 14.8 +/- 4.05 m before saccades in the off direction. During purely vertical saccades the firing frequency of abducens motoneurons did not change. Burst frequency did not saturate during saccades, but increased with saccadic velocity in a linear fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavior of neurons in the abducens nucleus of the alert cat--II. Internuclear neurons.

The activity of 43 antidromically identified abducens internuclear neurons with conduction velocities ranging from 14 to 54 m/s was analyzed in alert cats during spontaneous and vestibular induced eye movements. The discharge rate of internuclear neurons significantly increased with successive adducting positions of the contralateral eye. Slopes of rate-position (k) relationships ranged from 3.1 to 17.9 spikes/deg (mean 12.01 +/- 3.1). Threshold ranged from -19 degrees to +3 degrees. Frequency saturation was never observed for any internuclear neuron within the oculomotor range. Although straight lines were selected to illustrate the rate-position relationships, exponential curves always provided the best statistical fit demonstrating that an enhancement in frequency potentiation (k) must accompany more eccentric fixations in the on direction. Internuclear neurons showed a low variability in firing rate (less than 3.0%) for fixations less than 1 s. Variability increased with both longer and repeated fixations of the same eye position. Discharge rates were found to depend upon both the direction of the preceding eye movement and the animal's level of alertness. Separate regression lines of rate-position relations following saccades in the on and off directions differed significantly in slope (100%), but not threshold. The observed static hysteresis in an identified non-motoneuron shows this property to be in a central neural circuit prior to the extraocular motoneuron. The slopes (k) of rate-position plots for all internuclear neurons decreased significantly (100%) when level of alertness changed from "alert" (1 +/- 0.2 saccades/s) to "drowsy" (0.5 +/- 0.2 saccades/s). Thresholds, however, were not significantly altered. Discharge rate of abducens internuclear neurons increased abruptly 10.4 +/- 2.5 ms preceding saccades in the on direction, and decreased 20.5 +/- 7.8 ms before saccades in the off direction. Internuclear neuronal activity was not affected by pure vertical saccades. During on direction saccades, firing frequency did not saturate, but increased with velocity in a linear fashion. Exponential functions often fit the data better due to the difference in slopes of rate-velocity plots for on vs off direction saccades. Slopes (rs) of rate-velocity regression lines during spontaneous saccades ranged from 0.99 to 4.10 spikes/s/deg/s (mean 2.16 +/- 0.93). During saccades in the off direction activity always decreased, but it seldom ceased. Rate-velocity regression lines measured during the fast phase of vestibular nystagmus (rsv = 2.09 +/- 0.88) showed no significant differences from rs slopes in 82% of the cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of proprioception in the control of lid position during reflex and conditioned blink responses in the alert behaving cat.

The contribution of the orbicularis oculi muscle to the determination of lid position, and the putative role of eyelid proprioception in the control of reflex and conditioned eye blinks, were studied in alert behaving cats. Upper lid movements and the electromyographic activity of the orbicularis oculi muscle were recorded during reflexively evoked blinks and during the classical conditioning of the eyelid response. Blinks were evoked by air puffs, flashes and electrical stimulation of the supraorbitary branch of the trigeminal nerve. Eyelid responses were conditioned with a trace classical conditioning paradigm consisting of a short, weak air puff, followed 250 ms later by a long, strong air puff. Orbicularis oculi muscle activation during reflex blinks was independent of lid position and was not modified by the presence of weights acting in the upward or downward directions. Local anesthesia of the supraorbital nerve reduced blinks evoked by air puffs applied to the lower jaw, but did not affect flash-evoked blinks. No relationship was established between initial lid position and the first downward component of conditioned eyelid responses. In contrast, initial lid position was related to the first upward component of the same conditioned response. It is concluded that orbicularis oculi motor units receive no feedback proprioceptive signals from the eyelid, other than those coming from cutaneous receptors, and that lid position is determined by the activity of the levator palpebrae superioris muscle. The lack of sensory information about lid position in facial motoneurons probably has some functional implications on the central control of cognitive and emotional facial expressions.

A neurophysiological study of prepositus hypoglossi neurons projecting to oculomotor and preoculomotor nuclei in the alert cat.

The activity of 62 antidromically identified prepositus hypoglossi neurons was recorded in 10 alert cats during spontaneous, vestibular or visually induced eye movements. Neurons were antidromically activated from stimulating electrodes implanted in the ipsilateral medial longitudinal fasciculus (n = 24), the ipsilateral interstitial nucleus of Cajal (n = 6), the ipsilateral parabigeminal nucleus (n = 2), the contralateral superior colliculus (n = 6) and the contralateral cerebellar posterior peduncle (n = 24). Neurons were identified as eye-movement-related when their rate-position and/or rate-velocity plots showed correlation coefficients greater than or equal to 0.6. They were further classified as "position", "position-velocity" and "velocity-position" according to their relative eye position and velocity coefficients. However, they seemed to be distributed as a continuum in which a progressive decrease of eye velocity sensitivity was accompanied by a proportional increase in eye position sensitivity. "Position-velocity" neurons (n = 9) were mainly horizontal type II neurons projecting to the vicinity of the oculomotor complex; two of these neurons with vertical sensitivity were also activated from the interstitial nucleus of Cajal. Mean position and velocity sensitivity of these neurons were 5.2 spikes/s per degree and 0.62 spikes/s per degree per second, respectively. Pure "position" neurons (n = 7) also showed activation during ipsilateral eye fixations; their mean position gain was 7.3 spikes/s per degree and they projected to the ipsilateral oculomotor and Cajal nuclei, and to the contralateral superior colliculus. "Velocity-position" neurons (n = 18) were type I or II neurons with rather irregular tonic firing rates and a mean velocity gain of 0.75 spikes/s per degree per second. Type II "velocity-position" neurons projected mainly to the oculomotor area, while type I neurons projected preferentially to the cerebellum. A special type of "pause" neuron (n = 5), with very low firing rate and pausing mainly for contralateral saccades, was activated exclusively from the contralateral posterior peduncle. Many neurons with weak eye movement sensitivity (n = 22) were activated mainly (73%) from the cerebellum. It can be concluded that the prepositus hyperglossi nucleus distributes specific eye movement related signals to motor and premotor brainstem and cerebellar structures. The variability of interspike intervals of representative prepositus hypoglossi neurons of each class was compared to the discharge variability of identified abducens motoneurons.(ABSTRACT TRUNCATED AT 400 WORDS)