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.

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.

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.

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.

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.

Involvement of cerebral cortical structures in the classical conditioning of eyelid responses in rabbits.

The classical conditioning of the eyelid motor system in alert behaving rabbits has been used to study the expression of Fos in the hippocampus, and in the occipital, parietal, piriform and temporal cortices. Animals were classically conditioned with both delay and trace conditioning paradigms. As conditioned stimulus, both short and long (20 and 100 ms) tones (600 Hz, 90 dB) or short, weak (20 ms, 1kg/cm(2)) air puffs were used. The unconditioned stimulus was always a long, strong (100 ms, 3 kg/cm(2)) air puff that started 250-270 ms after the onset of the conditioned stimulus. The expression of Fos was significantly increased after both delayed and trace conditioning in the hippocampus, and in the parietal and piriform cortices contralateral to the unconditioned stimulus presentation side, compared with equivalent ipsilateral structures in conditioned animals, or with Fos production in the same contralateral structures in pseudo-conditioned and control animals. Fos expression in some cortical sites was specific to tone versus air puff stimuli when used as conditioned stimulus. Thus, Fos expression was significantly increased in the contralateral temporal lobe when tones were used as conditioned stimulus, for both delayed and trace conditioning paradigms, but not when animals were conditioned to short, weak air puffs. The present results indicate a specific Fos activation in several cerebral cortical structures during associative eyelid conditioning.

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.

Changes in eye blink responses following hypoglossal-facial anastomosis in the cat: evidence of adult mammal motoneuron unadaptability to new motor tasks.

Hypoglossal-facial anastomosis is used in humans to restore the activity of the mimic musculature following irrecoverable facial nerve lesions. As eyelid movement kinetics is very well known, we have used this experimental model in cats to follow the evolution of blink responses and the adaptability of hypoglossal motor pools to new motor tasks. Although the electromyographic activity of the orbicularis oculi muscle in response to corneal air puffs, flashes of light or electrical stimulation of the supraorbital nerve was not recovered in the seven months following this crossed anastomosis, reflex blinks were got back by the increased activity of the retractor bulbi and extraocular recti muscles. The lid of the anastomosed side oscillated in perfect synchronization with tongue movements during licking, while it was severely affected in its motor function during optokinetic stimulation because of the spontaneous appearance of tongue-related hypoglossal activity. Present results suggest that adult mammal motoneurons are unable to readapt their motor programs to the kinetic needs of new motor targets and that most of the functional recovery observed in the cat was achieved by the compensatory hyperactivity of motor systems not directly affected by the surgery.

Involvement of cerebellar cortex and nuclei in the genesis and control of unconditioned and conditioned eyelid motor responses.

The eyelid motor system of the cat was used here for the study of the kinetic properties of reflex and conditioned lid movements, and of the role played by the cerebellum in the acquisition and/or performance of both types of motor responses. Spontaneous blinks, eyelid reflex responses, eye-guided lid movements and conditioned lid responses were recorded in alert cats in simultaneity with unitary and field electrical activity of cerebellar cortex and nuclear zones related to the eyelid motor system. Results indicate that nuclear unitary activity does not precede unconditioned or conditioned lid responses, but that cerebellar nuclei are directly involved in the performance of the late components of reflex lid movements and in the acquisition of conditioned lid responses.

Respiration-related neurons recorded in the deep cerebellar nuclei of the alert cat.

The activity of deep cerebellar nuclei neurons was recorded in the alert cat and related to the respiratory cycle. Respiration-related neurons (RRNs, n = 29), located in the rostral fastigial and interpositus nuclei, were classified as inspiratory (24%) or expiratory (76%). Nine RRNs were antidromically activated from the red nucleus, but none from the inferior olive. Half of the RRNs showed well defined proprioceptive inputs of a rather broad origin. Other RRNs (27%) showed a respiration-related pattern independent of respiratory movement performance. Repeated electrical stimulation of the inferior olive exerted a synchronizing effect on the firing rate of 24% of the RRNs. It is proposed that cerebellar nuclear RRNs are involved in locomotor re-adjustments of the respiratory musculature.

Response diversity of pontine and deep cerebellar nuclear neurons to air puff stimulation of the eye in the alert cat.

The discharge of antidromically identified brainstem and cerebellar nuclear neurons involved in the corneal reflex was recorded in the alert cat during corneal air puffs. Eye movements were measured with the search coil technique. Recorded sensory, motor, reticular formation and cerebellar nuclear neurons showed a wide diversity in latencies and patterns of response to air puff stimulation. This diversity suggests that each part of the circuit may contribute different properties to information processing for the corneal reflex, for sustained eyelid closure and, possibly, for the classical conditioning of the nictitating membrane response.

Kinematic analyses of classically-conditioned eyelid movements in the cat suggest a brain stem site for motor learning.

Upper eyelid movements were conditioned in the alert cat to the presentation of either tones or short, weak air puffs applied to the ipsi- or contralateral cornea followed by an unconditioned stimulus consisting of a long, strong air puff applied to the ipsilateral cornea. Eyelid movements were measured with the search-coil technique. Electromyographs of the orbicularis oculi muscle were also recorded. Quantitative analysis of the latencies and topographic profiles of eyelid conditioned responses suggests that the primary site for their initiation is the brain stem reflex circuit involved, depending on the sensory modality of and on the side where the conditioning stimulus was applied. However, the kinematic of the conditioned response indicates that other neural structures are involved in its acquisition and consolidation.

Scopolamine impairs information processing in the hippocampus and performance of a learned eyeblink response in alert cats.

The object of this work was to determine whether the changes in field activity and neuronal excitability recorded in the hippocampus during eyeblink classical conditioning are induced by the cholinergic input, and their relationships with the conditioned response performance. The pyramidal layer field activity, its response to fornix stimulation and eyelid responses were recorded during paired tone-air puff presentations, under scopolamine (25, 50 and 100 microg/kg) or saline administration, following well-established eyeblink conditioning in a trace paradigm in cats. Scopolamine impaired behavioral performance, and, in the hippocampus, disrupted conditioned stimulus-evoked field potential, high frequency shift in field activity, and paired presentation-induced hyperexcitability. These findings indicate that the cholinergic input participates in hippocampal information processing in a way that allows precise conditioned response performance and memory trace formation.

Harmaline induces different motor effects on facial vs. skeletal-motor systems in alert cats.

Harmaline's effects on reflex and classically conditioned eyelid responses and on tremor picked up by a coil attached to the back were measured in alert cats. Harmaline at a dose of 10 mg/kg produced skeletal muscle tremogenic effects that lasted 4h. Back movements presented a tremor-like displacement with a frequency peak at 10 Hz, but lid responses oscillated as in controls, at 20 Hz during both reflex and conditioned eyelid movements, with no increase in oscillation amplitude or frequency. The learning curves of harmaline-injected animals remained as in controls, but eyelid conditioned responses showed longer latencies, and smaller amplitude and peak velocity. Reflex and already-learned eyelid responses were not modified by harmaline. These results imply that neuronal control systems for skeletal-motor and facial responses are differentially affected by harmaline.

[Neuronal regeneration and functional recovery following peripheral nervous system lesions]. / Regeneracion neuronal y recuperacion funcional tras la lesion del sistema nervioso periferico.

INTRODUCTION: The complete traumatic sectioning of peripheral nerves start subcellular and molecular processes in the involved sensory and motor neurons that ends, in many cases, with a complete reinnervation of the sensory or muscular target. Nevertheless, the process is frequently disturbed, from a functional point of view, by the improper reinnervation of targets different from the original ones, a fact implying a partial or total lost of the involved sensory or motor function. METHOD AND AIMS: Results obtained with several types of axotomy and of experimental anastomosis carried out with the different brainstem motor nerves are shown. The aim was to analyze the capabilities of the different brainstem centers to adapt their physiology to the functional characteristics of a new motor target, with respect to their affinity with the motor tasks carried out by the new target. CONCLUSIONS: It is concluded that there is a gradient of functional adaptability in motoneurons to the role of new motor targets depending on their affinity in embryologic origins and functional properties. It is remarked the importance that, for a proper recovery of the lost function, have the compensatory processes started by synergistic motor systems not affected directly by the lesion.

Firing activities of identified posterior interpositus nucleus neurons during associative learning in behaving cats.

On the basis of stimulation and permanent or transient lesions of putatively involved structures, and using transgenic mice with defective functional circuits, it has been proposed that cerebellar cortex and/or nuclei could be the sites where classically conditioned nictitating membrane/eyelid responses are acquired and stored. Here, we review recent information regarding the electrical activities of deep cerebellar nuclei neurons recorded during the performance of reflex and acquired eyeblinks. In particular, the rostral pole of the dorsolateral region of the posterior interpositus nucleus contains neurons significantly related to reflexively evoked and classically conditioned eyelid responses. Thus, type A interpositus neurons increase their discharge rate during eyelid movements, modulating it depending upon eyelid motorics. In contrast, type B neurons decrease their firing, even to a stop, during the same eyelid responses. However, as these changes in firing start after the onset of eyelid conditioned responses (CRs), and because they do not seem to encode eyelid position and velocity during the CR, the interpositus nucleus cannot be conclusively considered the site where eyelid learned responses are generated and stored. Additional microstimulation and pharmacological blockage of the recorded sites support the suggestion that posterior interpositus neurons contribute to the enhancement of CRs. Moreover, interpositus neurons probably contribute to the proper damping of newly acquired eyelid responses. The contributing role of other neuronal centers and circuits related to the eyelid motor system are also discussed.

Cerebellar cortex and eyeblink conditioning: bilateral regulation of conditioned responses.

We examined the role of the cerebellum in classical conditioning of the nictitating membrane response (NMR) of rabbits by comparing the effects of unilateral and bilateral cerebellar cortical lesions. Using extended preoperative conditioning to ensure high levels of learning, we confirmed that unilateral lesions of lobules HVI and ansiform lobe impaired conditioned responses (CRs) previously established to an auditory conditioned stimulus, but did not prevent some relearning with post-operative retraining. Bilateral lesions of HVI and ansiform lobe produced similar impairments of CRs, but also prevented subsequent relearning. Unilateral cortical lesions produced significant enhancement of unconditioned response (UR) amplitudes to periorbital electrical stimulation. Bilateral cortical lesions enhanced UR amplitudes to a lesser extent. Because there was no correlation between the degree of CR impairment and UR enhancement across the unilateral and bilateral lesion groups, the suggestion that the lesions impaired CRs due to general effects upon performance, rather than due to losses of learning, is not supported. Both sides of the cerebellar cortex contribute towards learning a unilaterally trained CR. This finding is important for the re-interpretation of unilateral, reversible inactivation studies that have found no involvement of the cerebellar deep nuclei in the acquisition of NMR conditioning. In addition, we found conditioning-dependent modifications of unconditioned responses that were particularly apparent at low intensities of periorbital electrical stimulation. This finding is important for the re-interpretation of studies that have found apparent changes in the UR of conditioned subjects after cerebellar lesions.

The role of interpositus nucleus in eyelid conditioned responses.

One of the most widely used experimental models for the study of learning processes in mammals has been the classical conditioning of nictitating membrane/eyelid responses, using both trace and delay paradigms. Mainly on the basis of permanent or transitory lesions of putatively-involved structures, and using other stimulation and recording techniques, it has been proposed that cerebellar cortex and/or nuclei could be the place/s where this elemental form of associative learning is acquired and stored. We have used here an output-to-input approach to review recent evidence regarding the involvement of the cerebellar interpositus nucleus in the acquisition of these conditioned responses (CRs). Eyelid CRs appear to be different in profile, duration, and peak velocity from reflexively-evoked blinks. In addition, CRs are generated in a quantum manner across conditioning sessions, suggesting a gradual neural process for their proper acquisition. Accessory abducens and orbicularis oculi motoneurons have different membrane properties and contribute differently to the generation of CRs, with significant species differences. In particular, facial motoneurons seem to encode eyelid velocity during reflexively-evoked blinks and eyelid position during CRs, two facts suggestive of a differential somatic versus dendritic arrival of specific motor commands for each type of movement. Identified interpositus neurons recorded in alert cats during classical conditioning of eyelid responses show firing properties suggestive of an enhancing role for CR performance. However, as their firing started after CR onset, and because they do not seem to encode eyelid position during the CR, the interpositus nucleus cannot be conclusively considered as the place where this acquired motor response is generated. More information is needed regarding neural signal transformations taking place in each involved neural center, and it its proposed that more attention should be paid to functional states (as opposed to neural sites) able to generate motor learning in mammals. The contribution of feedforward mechanisms normally involved in the processing activities of related centers and circuits, and the possible functional interactions within neural systems subserving the associative strength between the conditioned and unconditioned stimuli, are also considered.

Signalling properties of identified deep cerebellar nuclear neurons related to eye and head movements in the alert cat.

1. The spike activity of deep cerebellar nuclear neurons was recorded in the alert cat during spontaneous and during vestibularly and visually induced eye movements. 2. Neurons were classified according to their location in the nuclei, their antidromic activation from projection sites, their sensitivity to eye position and velocity during spontaneous eye movements, and their responses to vestibular and optokinetic stimuli. 3. Type I EPV (eye position and velocity) neurons were located mainly in the posterior part of the fastigial nucleus and activated antidromically almost exclusively from the medial longitudinal fasciculus close to the oculomotor complex. These neurons, reported here for the first time, increased their firing rate during saccades and eye fixations towards the contralateral hemifield. Their position sensitivity to eye fixations in the horizontal plane was 5.3 +/- 2.6 spikes s-1 deg-1 (mean +/- S.D.). Eye velocity sensitivity during horizontal saccades was 0.71 +/- 0.52 spikes s-1 deg-1 s-1. Variability of their firing rate during a given eye fixation was higher than that shown by abducens motoneurons. 4. Type I EPV neurons increased their firing rate during ipsilateral head rotations at 0.5 Hz with a mean phase lead over eye position of 95.3 +/- 9.5 deg. They were also activated by contralateral optokinetic stimulation at 30 deg s-1. Their sensitivity to eye position and velocity in the horizontal plane during vestibular and optokinetic stimuli yielded values similar to those obtained for spontaneous eye movements. 5. Type II neurons were located in both fastigial and dentate nuclei and were activated antidromically from the restiform body, the medial longitudinal fasciculus close to the oculomotor complex, the red nucleus and the pontine nuclei. Type II neurons were not related to spontaneous eye movements. These neurons increased their firing rate in response to contralateral head rotation and during ipsilateral optokinetic stimulation, and decreased it with the oppositely directed movements. 6. Saccade-related neurons were located mostly in the fastigial and dentate nuclei. Fastigial neurons were activated antidromically from the medial longitudinal fasciculus, while dentate neurons were activated from the red nucleus. These neurons fired a burst of spikes whose duration was significantly related to saccade duration. Dentate neurons responded during the omni-directional saccades, while some fastigial neurons fired more actively during contralateral saccades. 7. These three types of neuron represent the output channel for oculomotor signals of the posterior vermis and paravermis. It is proposed that type I EPV neurons correspond to a group of premotor neurons directly involved in oculomotor control.(ABSTRACT TRUNCATED AT 400 WORDS)