Effect of cerebellar inactivation by lidocaine microdialysis on the vestibuloocular reflex in goldfish.

Vestibuloocular reflex performance and adaptation were examined during vestibulocerebellar inactivation by localized lidocaine microdialysis or injection in goldfish. In the light, eye velocity perfectly compensated for head velocity (Vis-VOR) during sinusoidal yaw rotation (1/8 Hz +/- 20 degrees). In the dark, the reflex (VOR) gain was slightly reduced (gain approximately 0.8-0.9). In neither Vis-VOR nor VOR, was gain altered after 1 h of lidocaine microdialysis in the vestibulocerebellum. Before adaptation of reflex gain, the initial suppression or augmentation of Vis-VOR reflex gain produced by in-phase or out-of-phase visual-vestibular stimulation was also unaffected by cerebellar inactivation. Subsequently, 3 h of adaptive reflex training in either the in-phase or out-of-phase paradigm (acquisition phase) respectively decreased (0.30 +/- 0.09) or increased (1.60 +/- 0.08) VOR gain during artificial cerebral spinal fluid (CSF) microdialysis. However, microdialysis of lidocaine completely blocked adaptive gain changes during a 3-4 h period of continuous application. This effect was reversible because VOR gain changes were produced 1 h after lidocaine was replaced with CSF as the dialysate. After adaptive training, bilateral CSF injections (0.25 microl/side) into the vestibulocerebellum did not alter the normal retention or decay of adapted gain changes during a 3 h period in the dark (retention phase). However, injection of lidocaine into the vestibulocerebellum completely blocked retention of the adapted VOR gain returning the gain to values recorded before adaptation. In contrast to either acute or chronic surgical removal, lidocaine inactivation of the cerebellum by microdialysis did not alter either Vis-VOR and VOR behavior or interactive Vis-VOR performance over a wide range of gain extending from 0.3 to 1.4. Thus short-term VOR motor learning is a dynamic process requiring either continuous operation of brain stem cerebellar loops or, alternatively, modifiable sites within or directly influenced by the cerebellum. Our data supports the latter hypothesis, because the direct brain stem VOR pathways appear to be unaltered after cerebellar inactivation, and, hence, independent of the VOR-adapted state.

Modulation of GAP-43 mRNA by GABA and glutamate in cultured cerebellar granule cells.

Expression of GAP-43 in the cerebellum and selected regions of the brain has been shown to be developmentally regulated. Localization of GAP-43 mRNA within granule cells of the immature and mature rat cerebellum has been demonstrated by in situ hybridization. Higher levels are detected in the neonate compared to the adult. To determine if the cerebellar neurotransmitters, GABA (gamma-amino-butyric acid) and glutamate are involved in the modulation of GAP-43 expression, cultured cerebellar granule cells were exposed to these transmitters. Cultures were treated with glutamate, GABA, or the agonists/antagonists to their receptors in serum-free media for 5-7 days. Analysis of the levels of GAP-43 mRNA by in situ hybridization indicated that a 7-day exposure to GABA (25 and 50 microM) significantly lowered levels of granule cell GAP-43 mRNA. Specific agonists to the GABAA (muscimol) and GABAB (baclofen) receptors produced a decrease similar to that observed for GABA. Results from these studies also indicated that exposure to non-NMDA (CNQX) and NMDA (CPP, MK-801) glutamate receptor antagonists, and a metabotropic receptor glutamate agonist (ACPD), decreased the level of GAP-43 mRNA. The involvement of GABA and glutamate in the modulation of GAP-43 expression was corroborated by Northern hybridization. These studies revealed that a 5-day exposure to GABA decreased the cellular content of GAP-43 mRNA by 21% whereas exposure to glutamate resulted in a 37% increase. Findings from the studies reported here, using an in vitro cerebellar granule cell model, suggest that levels of GAP-43 mRNA, in vivo, are modulated by input from both excitatory glutamatergic mossy fibers and inhibitory GABAergic Golgi interneurons. Thus, modulation of GAP-43 mRNA by these neurotransmitters may influence granule cell maturation during development in the neonate and neuroplasticity in the adult, possibly at the parallel fiber-Purkinje cell synapse.

Distribution of GAP-43 mRNA in the immature and adult cerebellum: a role for GAP-43 in cerebellar development and neuroplasticity.

Expression of GAP-43 mRNA in the rat cerebellum and inferior olivary nucleus was examined at birth, during postnatal development and in the adult by both Northern and in situ hybridization. Northern blot analysis revealed that cerebellar GAP-43 mRNA expression increases from birth to postnatal day (PD) 7 and then declines to a lower level in the adult. At birth, in situ hybridization experiments showed intense labeling of GAP-43 mRNA in the premigratory, but not the germinal, zone of the cerebellar external granule cell layer. Localization of GAP-43 within the premigratory zone, a layer containing post-mitotic granule cells, indicates that granule cells begin expressing GAP-43 mRNA after final mitosis and during axonal outgrowth of the parallel fibers. The deep cerebellar nuclei and the inferior olive were also intensely labeled at birth. GAP-43 mRNA was localized in granule cells during their migration through the molecular layer of the developing cerebellum and after their arrival in the internal granule cell layer. By PD 21, the pattern of GAP-43 expression was similar to that observed in the adult; GAP-43 mRNA was localized to the internal granule layer and the inferior olive with minimal to no hybridization in the deep cerebellar nuclei and none in the molecular layer. Purkinje cells were devoid of GAP-43 mRNA throughout the postnatal and adult periods. In light of our observations, we propose that GAP-43 is a critical factor in granule cell differentiation/migration, as well as in the parallel and climbing fiber axonal outgrowth and synaptogenesis during development. Localization of GAP-43 mRNA within granule and inferior olivary cells of adult animals indicates that GAP-43 protein observed in the molecular layer is transported from these cells to their terminals in the molecular layer suggesting that GAP-43 is also an intrinsic presynaptic determinant in cerebellar neuroplasticity.

Effect of temperature on the normal and adapted vestibulo-ocular reflex in the goldfish.

1. The vestibulo-ocular reflex, a sensorimotor process, operates in a similar manner for homeothermic (mammals) and poikilothermic (fish) animals. However, individual physiological, biochemical, and/or pharmacological thermolabile processes that underlie the operation of this reflex could alter the operation of this reflex in a poikilotherm. The object of this study was to determine what aspects of the vestibulo-ocular reflex are affected by temperature changes naturally experienced by a poikilothermic animal, the goldfish. 2. Experiments were conducted on the visuovestibulo-(Vis-VOR) and vestibulo-ocular reflex (VOR) during normal operation as well as during the acquisition (learning) and retention (memory) phases of adaptive gain change. These studies were carried out at temperatures to which goldfish had been acclimated over several weeks and after rapid (< 5 min) shifts from this acclimation temperature. 3. Normal sinusoidal Vis-VOR and VOR gains before adaptation were found to be independent of the acclimation temperature over a wide range. Acute temperature changes of up to 10 degrees C either above or below a 20 degrees C acclimation temperature (Ac degree C = 20 degrees C) did not significantly modify normal visual and/or vestibular oculomotor reflex gains. 4. Surprisingly, slight reductions in temperature, as small as 2.5 degrees C, noticeably reduced Vis-VOR and VOR gain adaptations. Both short (3 h) and intermediate (up to 48 h) term reflex modifications were affected. Loss of adaptation was observed 10 degrees C below the acclimation temperature (Ac - 10 degrees C); however, return to the original temperature immediately restored most (60-100%) of the previously acquired Vis-VOR and VOR gain changes. In contrast, elevation of temperature up to 10 degrees C above the acclimation temperature (Ac + 10 degrees C) did not alter either increases or decreases in the adapted Vis-VOR or VOR gain. 5. A decrease in temperature reduced the magnitude of an adapted VOR gain increase and elevated the magnitude of an adapted gain decrease, thus returning the VOR gain back toward its normal control gain before adaptation. Because both increases and decreases in VOR gain were affected by the same temperature reduction, the cold effect was not a generalized reflex suppression, but inactivation of a process responsible for maintaining VOR adaptation. 6. During the acquisition phase, the time course and magnitude of adaptive VOR gain increases at temperatures acutely set 8-10 degrees C below the acclimation temperature were similar to those obtained at the acclimation temperature. Because the same temperature decrease inactivated retention of adapted VOR gain changes, the neuronal processes underlying the acquisition and the retention phases of Vis-VOR or VOR adaptation are suggested to differ qualitatively. 7. With the use of velocity step stimuli, both the adapted dynamic (< 100 ms) and sustained (> 100 ms) components of VOR adaptation were reduced by cooling. This effect on the dynamic component demonstrates an alteration in the shortest latency pathway through the vestibular nucleus and indicates that one thermosensitive site resides in the brain stem. 8. These results also show that, over a wide range of temperatures (20 +/- 10 degrees C), the neuronal processing that is responsible for the normal operation of the visuovestibulo- and/or vestibulo-ocular reflex and for the retention of reflex adaptation functions by separate physiological processes within the same brain stem and cerebellar circuitry. 9. We conclude that temperature exhibits a unique, and unexpected, state-dependent effect on sensorimotor regulation and adaptation for periods up to 48 h. Temperature does not alter normal VOR or the acquisition phase of an adapted gain change. (ABSTRACT TRUNCATED)

Cerebellar nitric oxide is necessary for vestibulo-ocular reflex adaptation, a sensorimotor model of learning.

1. Nitric oxide (NO) production in the nervous system has been implicated in cellular mechanisms of learning and memory. Our study investigates an in vivo sensorimotor model of learning. It demonstrates that a localized vestibulocerebellar injection of the NO synthase inhibitor, L-NG-monomethyl-arginine (L-NMMA), which specifically blocks NO production, inhibited the acquisition of adaptive vestibulo-ocular reflex (VOR) gain increases but not gain decreases in the goldfish. 2. Restoration of NO production by concomitant administration of L-arginine (the substrate for NO synthase) and L-NMMA suppressed the inhibitory effect of L-NMMA on adaptive gain increases. 3. This effect of L-NMMA was stereospecific because injection of D-NMMA did not suppress adaptive VOR gain increases. 4. Injection of L-NMMA after VOR adaptation had no effect on retention, failing to alter the postadaptive recovery after a VOR gain increase. 5. In conclusion, acquisition of adaptive VOR gain increases are affected by cerebellar NO inhibition. However, because gain decreases are not, they may involve either non-NO cerebellar or extracerebellar mechanisms. In addition, different processes for acquisition and retention of gain increases may be operating, because inhibition of cerebellar NO affects the acquisition but not the retention phase.

Kainic acid administration in the fastigial nucleus produces differential cardiovascular effects in awake and anesthetized rats.

Kainic acid was microinjected or microdialyzed into the rostral medial aspect of the fastigial nucleus to determine its effect on mean arterial pressure and heart rate. This was carried out in both the awake and the anesthetized (alpha-chloralose) rat. In awake animals, kainic acid elicited an initial phasic pressor response which was followed by a long-term elevation of mean arterial pressure that lasted for the duration of the experiment (2 h). Rats anesthetized with alpha-chloralose exhibited only a tonic depressor response. This converted to a pressor response as the rats began to emerge from anesthesia after 2 h. Both the awake and the anesthetized rats exhibited regular phasic changes in mean arterial pressure that was superimposed on the longer term changes in the mean arterial pressure. Similar results were obtained in both the microinjected and the microdialyzed animals. Thus, stimulation of the intrinsic fastigial neurons by kainic acid evokes an elevation of the mean arterial pressure in the awake rat. This is manifested as a decrease in pressure in the anesthetized animal. Thus, stimulation of the cardiovascular region of the fastigial nucleus can increase or decrease mean arterial pressure. It is possible that the direction of the change in mean arterial pressure is dependent on the level of afferent or intrinsic fastigial neural activity.

A method for restraining awake rats using head immobilization.

A method for restraining awake rats using head implant immobilization is described. In order to reduce stress to the individual animal, rats were restrained side by side in pairs during the adaptation and experimental periods. This technique was used in studies of central regulation of cardiovascular function. In particular, microdialysis probes were placed stereotaxically in deep nuclei of the cerebellum while mean arterial pressure and heart rate were monitored in the awake rat. After initial habituation, normal levels of heart rate and blood pressure obtained during restraint indicated that this is essentially a nonstressful procedure. This technique could also be used for a variety of other experimental conditions where head movement is not desirable, such as during oculomotor or vestibular experiments.

Physical and chemical considerations in the in vitro calibration of microdialysis probes for biogenic amine neurotransmitters and metabolites.

The object of the present study was to examine the effects of temperature, oxidation, and pH on in vitro relative recovery of catecholamine and indoleamine neurotransmitters and their metabolites using microdialysis probes. Relative recovery of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), homovanillic acid (HVA), and 5-hydroxytryptamine (5HT) increased with temperature from 0 to 46 degrees C. For each compound, the increase in the amount recovered with increasing temperature was different. The stability of norepinephrine and dopamine was not affected at any temperature using deoxygenated calibration standard solutions containing ascorbic acid but was greatly reduced when exposed to ambient air without antioxidant treatment; catecholamine metabolites and the indole compounds were less affected. No change for in vitro relative recovery was observed by varying the pH of the perfusing solution from 6 to 8. Thus, temperature control in probe calibration as well as analyte stability using antioxidant treatment are important in reducing the error when estimating extracellular concentrations of neurotransmitter and metabolites.

Comparison of vestibulo-ocular reflex (VOR) modification methods in cats.

The vestibulo-ocular reflex (VOR) has been measured and optically modified in several animal species. The VOR gain can be increased optokinetically by rotating the animal's visual surround opposite to the animal's direction of rotation or a VOR increase can result from the use of magnifying lenses. We describe here a comparison of three methods for producing VOR increases in cats: (i) optokinetic drum; (ii) a pair of 2.2 x telescopic lenses; (iii) Fresnel lens goggles. The animals were put through several preliminary calibrations followed by a sequence of VOR modification periods alternating with 10 testing periods. The results of the comparison in 4 cats show that the Fresnel lens system produces a greater and more stable VOR gain increase than the other two methods.

Vestibulo-ocular reflex adaptation in cats before and after depletion of norepinephrine.

The vestibulo-ocular reflex (VOR) operates to stabilize the eyes in space during movements of the head. The system has been described as having a gain of approximately -1 since stimulation of the semi-circular canals brought about by head movements will have the effect of causing the eyes to rotate an equal amount in the opposite direction. Change in the gain of the VOR has been put forth as a model to study plasticity in the central nervous system. Since numerous studies have implicated norepinephrine (NE) in neuroplasticity and modifiability of neural circuits, we attempted to determine the effect of NE depletion (via 6-hydroxydopamine (6-OHDA) intra-cisternal injection) on the modifiability of the VOR. We have found that cats increase the gain of their VOR over a four hour period when rotated in the horizontal plane in a manner equal but opposite to the rotation of a surrounding opto-kinetic drum. The entire group of animals manifests a statistically significant decrement in their ability to increase VOR gain when central stores of norepinephrine are depleted via intra-cisternal injection of 6-OHDA. Individual animals manifest a wide variety of gain changes (0.98 to 1.62). We have found that there were two groups of cats--high and low gain modifiers. The greatest reduction in VOR gain increase after NE depletion was observed in the high gain modifiers. No difference was observed in the low gain modifiers. These same animals tested for VOR modification after amphetamine injection, produced similar results. Alertness during the VOR modification task, as estimated by saccadic eye movement counts, was unchanged after NE depletion NE levels, measured by HPLC-EC, after depletion were reduced to the greatest extent in the cerebellum. There was also a substantial reduction of NE in the visual cortex with less of a reduction in the brain stem.

Saccades to visual targets are uncompensated after cerebellar stimulation.

Electrical stimulation of the posterior vermis produces a saccadic perturbation of the eyes. If this stimulation occurs after presentation of a visual target but before a trained saccade is initiated, the ensuing movement misses the target by an amount approximating the perturbation (uncompensated). These observations differ from those obtained after stimulation of the superior colliculus or frontal eye fields which result in compensated saccades that land on target. It is suggested that vermal stimulation may be acting outside the pontine burst feedback system.

Reduction of cerebellar norepinephrine alters climbing fiber enhancement of mossy fiber input to the Purkinje cell.

Extra-cellular simple and complex spike activity from 58 Purkinje cells were recorded in cats that previously received an intracisternal injection of 6-OHDA which depletes brain catecholamines. The severest catecholamine depletion was noted for cerebellar norepinephrine (21.1% of controls). Less depletion occurred in the brainstem and the visual cortex. Past studies have shown that in normal non-depleted cats, somatosensory stimuli (forepaw tap) evoke both complex and simple spike responses. On those trials where complex spike or climbing fiber responses are evoked, there is an enhancement or increase in responsiveness in the majority of excitatory and inhibitory simple spike responses. In the norepinephrine depleted animal, there is a significant decrease in this climbing fiber enhancement only for the excitatory response components. Furthermore, on those trials where no complex spikes are evoked, there is a significant decrease in the excitatory but not in the inhibitory response amplitude. A slight but non-significant increase in Purkinje cell background firing rate is also observed in the depleted animals. Thus, depletion of norepinephrine is associated with a reduction of both response amplitude and climbing fiber induced enhancement of excitatory simple spike responses. The inhibitory responses in these same cells are unchanged when compared to those recorded in the normal non-depleted animals.

Cerebellar vermis involvement in monkey saccadic eye movements: microstimulation.

The cerebellar vermis (lobules V to VII) was focally stimulated (maximum current = 300 microA) through microelectrodes in alert monkeys trained to fixate on visual targets located at different positions in the eye movement field. Microstimulation of this area evoked saccades whose direction and amplitude were dependent on the spatial locus of the vermal point stimulated and on the position of the eye at the time of stimulation. Stimulation evoked saccades on most (70%) of the electrode penetrations. By alternatively stimulating and recording through the electrode as it advanced through the depth of the vermis, it was possible to map the threshold for evoking saccades along a penetration as well as to ascertain the type of tissue (white matter or Purkinje cell layer) situated at the stimulation points. On penetrations where saccades were evoked, there was generally one (90%) and sometimes two (10%) low-threshold region(s). These low-threshold regions were located in fiber tracks and not in the Purkinje or other cellular layers of the cerebellar cortex. The direction and size of the evoked saccades were dependent on position of the eye in the orbit. At a few sites, even the presence or the absence of an evoked saccade depended on the initial eye position. Postsaccadic drifts after termination of evoked saccades were also a common feature (50% of the tracks) associated with vermal microstimulation. The presence or absence as well as the direction of the postsaccadic drift were also dependent on initial eye position. These observations suggest that the vermal stimulations evoked saccades by the antidromic activation of mossy fiber afferent inputs that emanate from the brain stem saccadic burst generator. Furthermore, stimulation in this manner would actually bypass the cerebellar circuitry and produce saccades without the usual modifying influence of the cerebellum.

Cerebellar norepinephrine depletion and impaired acquisition of specific locomotor tasks in rats.

Previous work in our laboratory has shown that norepinephrine (NE)-depleted rats manifested impaired acquisition of a locomotor task as measured in a new rod runway paradigm. This paradigm involved the initial training of water-deprived rats on an equally spaced regular rod arrangement (REG), and subsequent testing, after intracisternal 6-hydroxydopamine (6-OHDA; 3 X 25 micrograms/microliter free base) infusion, on a more difficult irregular rod arrangement (IRR). These NE-depleted animals manifested impaired acquisition of the task as measured by running times (RT, 25 trials/day) over a 4 day post-infusion test period (IRR). In this present study, this same REG/IRR paradigm was employed in combination with a localized 6-OHDA lesion of the coeruleo-cerebellar pathway. A bilateral infusion of 6-OHDA (8 micrograms/2 microliters) induced cerebellar noradrenergic deafferentation (26% of controls) and produced a significant impairment of 4 day post-infusion RT. Thus, the coeruleo-cerebellar-lesioned rats demonstrated acquisitional impairment when tested on the new locomotor task (IRR). Moreover, the degree of impaired acquisitional, but not initial post-infusion motor performance, was found to correlate directly with the degree of cerebellar noradrenergic deafferentation. Furthermore, these rats showed no arousal, motivational or general cognitive learning deficits since no significant differences were observed in runway intertrial interval times, open field behavior, or in reversal of a T-maze position habit. Thus, cerebellar NE appears to be strongly associated with the adaptive ability to coordinate and choreograph the movements necessary to perform in this locomotor task.

6-OHDA induced effects upon the acquisition and performance of specific locomotor tasks in rats.

The effect of norepinephrine (NE) depletion on acquisition and performance of locomotor tasks requiring precise paw placement was tested. Running times (RT, 25 trials/day, 4 consecutive days) of water-deprived rats trained to transverse horizontal rods in an equally spaced regular rod arrangement (REG) were obtained before and after (REG/REG) intracisternal 6-hydroxydopamine (6-OHDA, 3 X 25 micrograms free base) infusion. No significant differences from ascorbate (0.1%) vehicle controls were seen. Additional rats were tested using the same protocol except a more difficult, irregularly spaced rod arrangement (IRR) was used. These IRR/IRR rats also revealed no significant differences. However, testing on the REG task before, and the new IRR task after infusion produced impaired performance on days 3 and 4 when 6-OHDA and vehicle treated rats were compared. These REG/IRR rats also showed a significant difference in the slope of the line reflecting the decrease in RT over the 4 day post-infusion period. Since no differences in intertrial intervals or extinction behavior were seen, the effect was not attributed to differences in arousal or motivational state. This effect could not be attributed to a simple reduction in non-specific activity, since significant differences in spontaneous locomotor activity or open field behavior were not seen. Assays verified the severe reduction of cerebellar NE to 14.5% of vehicle controls, and the smaller reduction in limbic forebrain NE and dopamine (53.8% and 75.2% of controls respectively). These findings suggest that NE deafferentation of the cerebellum causes impaired acquisition of locomotor behavior rather than an impairment of post-acquisitional performance.

Simple spike activity of Purkinje cells in the posterior vermis of awake cats during spontaneous saccadic eye movements.

Extracellular recordings were made from 151 cerebellar cortical cells in the posterior vermis of 12 awake cats. Thirty-two percent (n = 48) of these cells modulated their activity with respect to the onset of spontaneous saccadic eye movements. Thirty-five cells in this group were positively identified as Purkinje cells and manifested changes in simple spike activity that were related to saccade onset. These included short excitatory, inhibitory, or biphasic changes that were superimposed on background tonic firing rates (avg. = 54 spikes/sec). Such changes were recorded before as well as after the onset of a saccade. Sixty-five percent (n = 22) of these cells were related to horizontal and vertical saccades in more than one direction of motion. These cells were randomly distributed throughout the posterior vermis and manifested no anatomical topographic organization with respect to the direction of saccadic eye movement. The results of this study suggest that lobules VI and VII of the cerebellar vermis participate in both the initiation and execution of spontaneous saccades in preferred directions.