James G. McElligott

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.

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.

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.

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.

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

SummaryThe 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.

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.

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.

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.

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.

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.