Neal H. Barmack

Functions of Interneurons in Mouse Cerebellum

The output signal of Purkinje cells is conveyed by the modulated discharge of simple spikes (SSs) often ascribed to mossy fiber–granule cell–parallel fiber inputs to Purkinje cell dendrites. Although generally accepted, this view lacks experimental support. We can address this view by controlling afferent signals that reach the cerebellum over climbing and mossy fiber pathways. Vestibular primary afferents constitute the largest mossy fiber projection to the uvula-nodulus. The discharge of vestibular primary afferent mossy fibers increases during ipsilateral roll tilt. The discharge of SSs decreases during ipsilateral roll tilt. Climbing fiber discharge [complex spikes (CSs)] increases during ipsilateral roll tilt. These observations suggest that the modulation of SSs during vestibular stimulation cannot be attributed directly to vestibular mossy fiber afferents. Rather we suggest that interneurons driven by vestibular climbing fibers may determine SS modulation. We recorded from cerebellar interneurons (granule, unipolar brush, Golgi, stellate, basket, and Lugaro cells) and Purkinje cells in the uvula-nodulus of anesthetized mice during vestibular stimulation. We identified all neuronal types by juxtacellular labeling with neurobiotin. Granule, unipolar brush, stellate, and basket cells discharge in phase with ipsilateral roll tilt and in phase with CSs. Golgi cells discharge out of phase with ipsilateral roll tilt and out of phase with CSs. The phases of stellate and basket cell discharge suggests that their activity could account for the antiphasic behavior of CSs and SSs. Because Golgi cells discharge in phase with SSs, Golgi cell activity cannot account for SS modulation. The sagittal array of Golgi cell axon terminals suggests that they contribute to the organization of discrete parasagittal vestibular zones.

Activity of neurons in the beta nucleus of the inferior olive of the rabbit evoked by natural vestibular stimulation

The inferior olive (IO) appears to be organized functionally in discrete subnuclei that receive transmitter-specific inputs. In particular, the IO receives a GABAergic input that is most densely concentrated in the β -nucleus. In this experiment, we examined the functional specificity of neurons in the β -nucleus of the IO of rabbits by recording their activity during natural vestibular and optokinetic stimulation. Rabbits were anesthetized and positioned in a triaxial servo- controlled rate table with the head fixed at the center of rotation. Contour-rich visual stimuli were rear-projected onto a 70 deg tangent screen and moved at constant velocities. Recording sites in the β -nucleus were verified by subsequent histological analysis of marking microlesions. Neurons in the β -nucleus responded to roll vestibular stimulation about the longitudinal axis. These neurons were excited when the rabbit was rolled onto the side which was contralateral to the recording site, and inhibited when the rabbit was rolled ipsilaterally. Thirty-eight of the 75 β -nucleus neurons that were responsive to roll vestibular stimulation also responded to static tilt, indicating an otolithic as well as a vertical semicircular canal origin of the vestibular input. The modulated activity of none of the neurons could be attributed to stimulation of the horizontal semicircular canals. All the recorded neurons were found in a region of the β -nucleus that was retrogradely labeled following HRP injections into the cerebellar nodulus. Using a “null point” technique, we found that there was a differential projection of information from the anterior and posterior semicircular canals onto to the β -nucleus. Stimulation of the ipsilateral anterior-contralateral posterior semicircular canals modulates activity of the neurons in the caudal 500 μm of the β -nucleus. Stimulation of the ipsilateral posterior-contralateral anterior semicircular canals modulates activity of neurons located more rostrally. β -nucleus neurons and the olivocerebellar circuits in which they participate may constitute an important pathway for the control and adaptive modification of postural reflexes.

Zonal organization of olivocerebellar projections to the uvula in rabbits

Olivocerebellar projections to the uvula were studied by means of retrograde axonal transport of horseradish peroxidase (HRP) in pigmented rabbits. The distribution pattern of labeled cells in the inferior olive was compared among cases following large- and microinjections of HRP into the uvula. Findings indicate topographically organized projections to longitudinally oriented zones. There are at least 6 zones in the rabbits uvula. The caudal part of the nucleus beta projects contralaterally to a most medially located zone (caudal beta zone). The rostral part of the nucleus beta projects to a little more laterally located zone (rostral beta zone) at a distance of about 1 mm from the midline of the uvula. The caudolateral part of the MAO projects to a zone (caudolateral MAO zone) located laterally to the rostral beta zone. The dorsomedial cell column projects to a zone (dorsomedial cell column zone) located in the intermediate part of the uvula at about 2 mm from the midline. The rostrolateral part of the MAO projects to the most lateral zone (rostrolateral MAO zone) of the uvula. Finally, the ventral lamella of the PO projects to a zone (ventral lamella of PO zone) located between the rostrolateral MAO zone and the dorsomedial cell column zone.

THE HORIZONTAL AND VERTICAL CERVICO-OCULAR REFLEXES OF THE RABBIT

Horizontal and vertical cervico-ocular reflexes of the rabbit (HCOR, VCOR) were evoked by sinusoidal oscillation of the body about the vertical and longitudinal axes while the head was fixed. These reflexes were studied over a frequency range of 0.005-0.800 Hz and at stimulus amplitudes of +/- 10 degrees. When the body of the rabbit was rotated horizontally clockwise around the fixed head, clockwise conjugate eye movements were evoked. When the body was rotated about the longitudinal axis onto the right side, the right eye rotated down and the left eye rotated up. The mean gain of the HCOR (eye velocity/body velocity) rose from 0.21 and 0.005 Hz to 0.27 at 0.020 Hz and then declined to 0.06 at 0.3Hz. The gain of the VCOR was less than the gain of the HCOR by a factor of 2-3. The HCOR was measured separately and in combination with the horizontal vestibulo-ocular reflex (HVOR). These reflexes combine linearly. The relative movements of the first 3 cervical vertebrae during stimulation of the HCOR and VCOR were measured. For the HCOR, the largest angular displacement (74%) occurs between C1 and C2. For the VCOR, the largest relative angular displacement (45%) occurs between C2 and C3. Step horizontal clockwise rotation of the head and body (HVOR) evoked low velocity counterclockwise eye movements followed by fast clockwise (resetting) eye movements. Step horizontal clockwise rotation of the body about the fixed head (HCOR) evoked low velocity clockwise eye movements which were followed by fast clockwise eye movements. Step horizontal clockwise rotation of the head about the fixed body (HCOR + HVOR) evoked low velocity counterclockwise eye movements which were not interrupted by fast clockwise eye movements. These data provide further evidence for a linear combination of independent HCOR and HVOR signals.

Parasolitary nucleus: a source of GABAergic vestibular information to the inferior olive of rat and rabbit.

At least two subnuclei of the inferior olive, the β‐nucleus, and the dorsomedial cell column (dmcc), contain vestibularly responsive neurons that receive a dense descending projection that uses γ‐aminobutyric acid (GABA) as the transmitter. In contrast to the GABAergic innervation of other olivary subnuclei, the terminal boutons that terminate on neurons in the β‐nucleus and the dorsomedial cell column remain intact after cerebellectomy, ruling out both the cerebellum and the cerebellar nuclei as afferent sources. By using both immunohistochemical as well as orthograde and retrograde tracer methods, we have identified the source of the GABAergic pathway to the β‐nucleus and dmcc in both rat and rabbit. Under physiologic recording of single olivary neurons to guide electrode placement, we injected the bidirectional tracer, wheat germ agglutinin‐conjugated horseradish peroxidase (WGA‐HRP) into the β‐nucleus and dmcc of the inferior olive. These injections retrogradely labeled neurons in the parasolitary nucleus (Psol) near the vestibular complex. Psol neurons were identified as GABAergic with an antibody to glutamic acid decarboxylase (GAD). In the rat, Psol neurons are small (5–7 μm in diameter) and number approximately 1,800. In the rabbit, they are slightly larger (6–9 μm in diameter) and number approximately 2,200. WGA‐HRP injections in conjunction with GAD immunohistochemistry double labeled a high percentage of neurons in both the rat and rabbit Psol. Injection of the orthograde tracer Phaseolus vulgaris‐leucoagglutinin into the area of the Psol revealed a projection from this region to both the β‐nucleus and dmcc. Subtotal electrolytic lesions of this division of the Psol caused a substantial reduction in GAD‐positive synaptic terminals in both the ipsilateral β‐nucleus and dmcc. The location of these GABAergic neurons, bordering both the nucleus solitarius and caudal vestibular complex, emphasizes the importance of the Psol in the processing of both vestibular and autonomic information pertinent to postural control. J. Comp. Neurol. 392: 352–372, 1998.

The influence of bilateral labyrinthectomy on horizontal and vertical optokinetic reflexes in the rabbit.

Vestibular, visual and proprioceptive information converge onto the vestibular nuclei 11-13 and these nuclei participate in the control of reflexive eye movements evoked by each of these sensory inputs. Damage to the labyrinth or to the vestibular nerve not only impairs vestibulo-ocular reflexes, but also reduces the gain of optokinetic reflexes and the duration of optokinetic afternystagmus in cats 5, monkeys 6, humans a4 and rabbits 2,a. Destruction of the vestibular nuclei abolishes optokinetic nystagmus in guinea pigs 1. Previously, we have demonstrated that the horizontal vestibulo-ocular reflex has a lower gain (eye velocity/head velocity) than the vertical vestibulo-ocular reflex at low frequencies of sinusoidal stimulation, 0.005-0.050 Hz 3. This difference in low frequency gain can be ascribed to an otolithic contribution to the vertical vestibuloocular reflex. Conversely, the horizontal optokinetic reflex and the horizontal cervicoocular reflex have higher gains than the vertical optokinetic reflex and the vertical cervico-ocular reflex4, ~0. It appears that the higher gains of the horizontal optokinetic and cervico-ocular reflexes, both of which are maximal at lower stimulus frequencies, compensate in part for the absence of a low frequency otolithic contribution to the horizontal vestibulo-ocular reflex. Since the horizontal and vertical optokinetic reflexes have different gains, it seemed possible that the functional role of the vestibular nuclei in these orthogonal reflexes would also be different. If so, then these reflexes would be affected differentially by bilateral labyrinthectomies. Therefore, we have compared the open-loop gains of the monocularly evoked horizontal and vertical optokinetic reflexes before and after bilateral labyrinthectomies in rabbits. The horizontal and vertical optokinetic reflexes evoked by monocular open-loop stimulation were measured in 5 albino rabbits. During optokinetic reflex testing, the head of the rabbit was fixed with previously implanted bolts to a restraint rod at the

Influence of long-term optokinetic stimulation on eye movements of the rabbit

Two kinds of optokinetic afternystagmus (OKAN) have been studied in rabbits; positive and negative OKAN. Positive OKAN is the persistence of eye movements evoked by optokinetic stimulation following the termination of the stimulus, with the slow phase of the eye movements in the same direction as the inducing stimulus. Negative OKAN is evoked by long duration optokinetic stimulation, and has a slow phase of opposite direction to the inducing stimulus. The stimulus conditions which are optimal for inducing and maintaining negative OKAN were characterized. Rabbits were placed in an optokinetic drum for periods of 12-96 h (with appropriate intervening periods for food and water). Eye movements were recorded during and after the termination of optokinetic stimulation. The optimum optokinetic stimulus velocity for the induction of negative OKAN was 5 degrees/s. The minimum duration of stimulation for the induction of negative OKAN of maximum velocity was 48 h. Once induced, the slow phase of negative OKAN attained velocities of 50-100 degrees/s. Three conditions of restraint of the rabbits were studied after negative OKAN was induced during the intervening periods when eye movements were not being recorded. These conditions were: (1) unrestrained (full freedom of movement) without visual stimulation (in a dark enclosure); (2) restrained (horizontal head and body movement prevented) without visual stimulation; and (3) restrained with visual stimulation (in the stationary optokinetic drum). Conditions 1 and 2 caused negative OKAN to dissipate within 24 h. Condition 3 caused negative OKAN to be maintained for more than 70 h. The velocity imbalance of the horizontal vestibuloocular reflex (HVOR) was measured at different times following the induction of negative OKAN. It provided a more sensitive index of the central imbalance which caused negative OKAN, than did spontaneous nystagmus. One of the consequences of optokinetic stimulation measured over a 16 h period was a decrease in the gain of the optokinetic reflex. This reduction in gain could represent a central adaptation to maintained stimulation which in the absence of continued optokinetic stimulation is expressed as a nystagmus.

Interactions of cervico-ocular and vestibulo-ocular fast-phase signals in the control of eye position in rabbits

1. Eye movements in unanaesthetized rabbits were studied during horizontal neck‐proprioceptive stimulation (movement of the body with respect to the fixed head), when this stimulation was given alone and when it was given simultaneously with vestibular stimulation (rotation of the head‐body). The effect of neck‐proprioceptive stimulation on modifying the anticompensatory fast‐phase eye movements (AFPs) evoked by vestibular stimulation was studied with a ‘conditioning‐test’ protocol; the ‘conditioning’ stimulus was a neck‐proprioceptive signal evoked by a step‐like change in body position with respect to the head and the ‘test’ stimulus was a vestibular signal evoked by a step rotation of the head‐body. 2. The influence of eye position and direction of slow eye movements on the occurrence of compensatory fast‐phase eye movements (CFPs) evoked by neck‐proprioceptive stimulation was also examined. 3. The anticompensatory fast phase (AFP) evoked by vestibular stimulation was attenuated by a preceding neck‐proprioceptive stimulus which when delivered alone evoked compensatory slow‐phase eye movements (CSP) in the same direction as the CSP evoked by vestibular stimulation. Conversely, the vestibularly evoked AFP was potentiated by a neck‐proprioceptive stimulus which evoked CSPs opposite to that of vestibularly evoked CSPs. 4. Eccentric initial eye positions increased the probability of occurrence of midline‐directed compensatory fast‐phase eye movements (CFPs) evoked by appropriate neck‐proprioceptive stimulation. 5. The gain of the horizontal cervico‐ocular reflex (GHCOR) was measured from the combined changes in eye position resulting from AFPs and CSPs. GHCOR was potentiated during simultaneous vestibular stimulation. This enhancement of GHCOR occurred at neck‐proprioceptive stimulus frequencies which, in the absence of conjoint vestibular stimulation, do not evoke CSPs.

Topsy Turvy: Functions of Climbing and Mossy Fibers in the Vestibulo-Cerebellum

The cerebellum’s role in sensory-motor control and adaptation is undisputed. However, a key hypothesis pertaining to the function of cerebellar circuitry lacks experimental support. It is universally assumed that the discharge of mossy fibers accounts for modulation of Purkinje cell “simple spikes” (SSs). This assumption acts as a prism through which all other functions of cerebellar circuitry are viewed. The vestibulo-cerebellum (nodulus and uvula) receives a large, unilateral, vestibular primary afferent mossy fiber projection. We can test its role in modulating Purkinje cell SSs by recording the modulated activity of both mossy fiber terminals and Purkinje cell SSs evoked by identical natural vestibular stimulation. Sinusoidal rotation about the longitudinal axis (roll) modulates the activity of vestibular primary afferent mossy and climbing fibers as well as Purkinje cell SSs and complex spikes (CSs). Remarkably, vestibular primary afferent mossy fibers discharge 180 degrees out of phase with SSs. This indicates that mossy fibers cannot account for SS modulation unless an inhibitory synapse is interposed between mossy fibers or vestibular climbing fibers and Purkinje cells. The authors review several experiments that address the relative contributions of mossy and climbing fiber afferents to the modulation of SSs. They conclude that climbing fibers, not mossy fibers, are primarily responsible for the modulation of SSs as well as CSs and they propose revised functions for these two afferent systems.

Activity-Dependent Expression of Acyl-Coenzyme A-Binding Protein in Retinal Muller Glial Cells Evoked by Optokinetic Stimulation

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior→anterior (preferred) direction with mRNAs from the retina stimulated in the anterior→posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the α1-subunit of the GABAA receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the α1-subunit of the GABAA receptor using an antibody to GABAAα1. The interaction was also confirmed by a “pull-down” assay in which histidine-tagged ACBP was used to pull down the GABAAα1. ACBP does not cross the blood–brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons. Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABAA receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.