Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean±SD: 0.84±0.17 vs 0.64±0.12 µm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82±0.34 µm, n=52) were slightly larger than on parallel fibers (1.43±0.24 µm, n=430). In addition, per unit length (100 µm) there were more swellings on ascending axons (11.2±4.2) than on parallel fibers (9.7±4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15–20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 µm²) to posteriorly (3090 µm²) and 104% from medially (1690 µm²) to laterally (3450 µm²). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.     

Fluctuation of extracellular potassium and calcium in the cerebellar cortex related to climbing fiber activity

It is known that repetitive stimulation of spino- and cortico-olivary pathways can lead to waxing and waning of the evoked climbing fiber responses of cerebellar Purkinje cells. Extracellular recordings performed with ion-selective microelectrodes showed that the amplitude fluctuations of climbing fiber-related field potentials were accompanied by corresponding changes in ion-activity levels. When the amplitude of the field potentials increased, potassium activity rose by up to 0.7 mmol.l^?1 and calcium activity fell by up to 0.3 mmol.l^?1. Ion activities returned to baseline values when climbing fiber responses failed.The rhythmic fluctuations in ion activities have cycle lengths of 10–20 s, or multiples of it. Presumably they are secondary consequences of slow variations of synaptic transmission in the inferior olive. The generator for the slow fluctuations of olivary synaptic transmission and the consequent variations in ion activities within the cerebellum is probably located within the inferior olive. The ionic modulations could have functional implications for cerebellar information processing. Possible mechanisms of the rhythmogenesis are discussed.     

Topographical investigations on the climbing fiber inputs from forelimb and hindlimb afferents to the cerebellar anterior lobe

Summary Volleys in group I and II fibers of muscle nerves and group II fibers of cutaneous, joint and fascial nerves have evoked CF responses in the anterior lobe of the cerebellum. In the pars intermedia there is a fairly sharp somatotopic localization of the forelimb CF responses to the Vth lobule (Larsell) and the hindlimb to the IVth and IIIrd lobules. In the vermis there is much more admixture, with the hindlimb-evoked responses tending to dominate in the lateral vermis of the Vth lobule, and the forelimb more medially. In the IVth and IIIrd lobules forelimb responses were rare and were never large. In the medial vermis up to 1–1.7 mm from the midline there were no CF-evoked responses from the limb nerves. These distributions of CF-evoked responses are remarkably different from those reported by Oscarsson, and consideration is given to the factors responsible for this discrepancy.A more detailed examination was made of the CF-evoked responses from a large variety of hindlimb and forelimb nerves. Observations were made along many tracks usually arranged in a transverse plane, and it was found that between different recording sites along the same track or along adjacent tracks, there was a great deal of variation in the relative magnitudes of the CF-evoked responses from the different nerves. These distributions have an ill-defined patchy character so that at any focus there is opportunity for the most diverse kinds of piecemeal integration.These findings on the CF-input are considered in relationship to the mossy fiber input. It is pointed out that the pathways conveying CF-input to the cerebellum have a level of discriminative input adequate for the operation of fine control.     

Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons.

1. Calcium transients related to climbing fiber (CF) and parallel fiber (PF) synaptic potentials were recorded from Purkinje cells in guinea pig cerebellar slices. Transients were measured using either absorbance changes of arsenazo III or fluorescence changes of fura-2, which were injected into individual cells in the slice. 2. All-or-none somatically recorded CF potentials elicited by white matter stimulation had all-or-none Ca transients. These signals began with a delay of > or = 2 ms from the start of the electrically recorded synaptic potential. The recovery time of CF-induced arsenazo III absorbance transients was < 50 ms in the fine dendrites in conditions that minimized the effects of dye buffering. 3. Ca2+ entry through voltage-gated Ca channels opened by Ca action potentials was the dominant source of the rise in [Ca2+]i after CF activation. There was no significant change in [Ca2+]i corresponding to the plateau potential that followed the large CF response. 4. The appearance and amplitude of distal CF-evoked Ca signals was more variable than proximal signals, suggesting that CF potentials do not reliably spread to the fine distal dendrites. The distal transient could be enhanced by intrasomatic depolarizing pulses, suggesting that it was a property of the postsynaptic membrane and not the presynaptic side of the CF synapse that was responsible for this variability. 5. Parallel fiber responses were evoked by electrical stimulation near the pial surface. Graded synaptic potentials and related Ca transients were reversibly blocked by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Small synaptic potentials induced small, localized Ca transients. With increasing stimulus intensity, the PF electrical response developed a regenerative component. Larger dendritic Ca transients were detected corresponding to this component. Ca transients evoked by the regenerative responses had the same rapid rise times and fall times as those related to somatically stimulated Ca action potentials, suggesting that they also were due to Ca2+ entry through voltage-sensitive channels. 6. During trains of PF responses, we observed an increase in the spatial extent of related Ca transients. This effect could be modulated by changes in the resting potential, suggesting that the same intrinsic mechanism was affecting the spread of both CF and PF signals.     

Sensory separation in climbing and mossy fiber inputs to cat vestibulocerebellum

Summary Electrical or flash stimulation of the visual pathway evoked in the vestibulocerebellum of barbiturate anesthetized cats, field and unitary potentials characteristic of climbing fiber (CF) activation of Purkinje cells. The latency of the CF field potentials was 11–15 msec in the flocculus and 14–19 msec in the nodulus/ventral uvula. Mossy fiber (MF) related field and unitary responses were not observed following visual stimulation. Conversely, electrical stimulation of the VIIIth nerve evoked in the vestibulocerebellum MF-related field and unitary potentials, exclusively. Despite this dichotomy, the field potentials evoked by visual and vestibular stimulation frequently overlapped within the cerebellar cortex. This overlap was shown at the level of individual Purkinje cells by means of extra- and intracellular recordings which demonstrated vestibulo-visual convergence. These observations indicate that a given sensory modality may reach specific cerebellar areas utilizing only one of the two cerebellar afferent systems. It is concluded that the MF and CF afferent systems, when considered as sensory inputs, can operate as independent information channels.     

Release of cerebellar inhibition by climbing fiber deafferentation

Summary Cerebellar units were recorded extracellularly in rats before and after an intraveinous injection of 3-acetylpyridine destroying selectively the IO. All the Purkinje cells show a loss of the complex discharge between 2 h 15 min and 2 h 45 min after treatment. This time, called the critical period corresponds to the degeneration of the neurons of the inferior olive as revealed by the decrease of their metabolic activity. The simple spikes of the Purkinje cells increase their discharge frequency soon after the climbing fibers cease firing. On the contrary the firing frequency of the inhibitory interneurons does not show significant changes after degeneration of the inferior olive. The efferent cerebellar neurons, including cells of the cerebellar and vestibular nuclei receiving the axon terminals of the Purkinje cells, decrease their discharge rate up to thirty times during and after the critical period. It is demonstrated that this effect is due to the increased inhibitory activity of the Purkinje cells deafferented from the climbing fibers, whereas the deafferentation of the efferent cerebellar neurones from the collaterals of the olivary cells has little impact.     

Imaging parallel fiber and climbing fiber responses and their short-term interactions in the mouse cerebellar cortex in vivo

A major question in the study of cerebellar cortical function is how parallel fiber and climbing fiber inputs interact to shape information processing. Emphasis has been placed on the long-term effects due to conjunctive stimulation of climbing fibers and parallel fibers. Much less emphasis has been placed on short-term interactions and their spatial nature. To address this question the responses to parallel fiber and climbing fiber inputs and their short-term interaction were characterized using optical imaging with Neutral Red in the anesthetized mouse in vivo. Electrical stimulation of the cerebellar surface evoked an increase in fluorescence consisting of a transverse optical beam. The linear relationship between the optical responses and stimulus parameters, high spatial resolution and close coupling to the electrophysiological recordings show the utility of this imaging methodology. The majority of the optical response was due to activation of postsynaptic alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) and metabotropic glutamate receptors with a minor contribution from the presynaptic parallel fibers. Stimulation of the inferior olive evoked parasagittal bands that were abolished by blocking AMPA glutamate receptors. Conjunctive stimulation of the cerebellar surface and inferior olive resulted in inhibition of the climbing fiber evoked optical responses. This lateral inhibition of the parasagittal bands extended out from both sides of an activated parallel fiber beam and was mediated by GABA(A) but not GABA(B) receptors. One hypothesized role for lateral inhibition of this type is to spatially focus the interactions between parallel fiber and climbing fiber input on Purkinje cells. In summary optical imaging with Neutral Red permitted visualization of cerebellar cortical responses to parallel fiber and climbing fiber activation. The GABA(A) dependent lateral inhibition of the climbing fiber evoked parasagittal bands by parallel fiber stimulation shows that cerebellar interneurons play a short-term role in shaping the responses of Purkinje cells to climbing fiber input.     

The distribution to the cerebellar anterior lobe of the climbing and mossy fiber inputs from the plantar and palmar cutaneous afferents

Summary Systematic examination has been made of the potentials evoked in the ipsilateral anterior lobe by single Group II volleys in different branches of cutaneous nerves to the fore-paw and hind-paw of the cat. Field potentials evoked by the mossy and climbing fiber inputs have been recorded along microelectrode tracks arranged so that there has been a comprehensive study through the whole branching foliated structure. In a previous investigation it was shown that large cutaneous nerves of the forelimb and hindlimb have wide fields of action for both the mossy fiber and climbing fiber inputs. In this present investigation it was found that small cutaneous nerves have more localized distributions within these wide fields. This discriminative distribution is exhibited for Group II volleys in the subdivisions of the nerves providing innervation to the palmar and plantar foot pads. It thus appears from this somatotopic investigation that there are pathways to the cerebellum sufficiently specific to give information about the part of the foot that is being stimulated in natural movements.     

Activation of mossy and climbing fiber pathways to the cerebellar cortex by stimulation of the fornix in the rat

Summary The fornix of the rat was electrically stimulated with bipolar concentric electrodes to determine the properties of single unit responses in Purkinje cells of the cerebellar cortex. Both climbing (CF) and mossy fiber (MF) pathways were activated by fornix stimulation. MF responses were indicated by single or double spike responses appearing at latencies of 5–10 ms. The MF spike responses, as quantified by histogram analysis, were further identified by appearance of graded responses with increasing stimulus strength and by following at frequencies up to and greater than 20/s. CF responses were identified by characteristic complex all-or-none burst responses with latencies usually between 10 and 20 ms and with following frequencies at no faster than 10/s. Experiments which involved movement of the stimulating electrode and production of lesions around it established that the activated fiber system was within the dorsal fornix and not in adjacent areas. The results indicate that hippocampal and other limbic areas can influence the cerebellar cortex by direct mossy and climbing fiber pathways, as has been demonstrated for other afferents. It is further suggested that motor patterns linked to hippocampal activity may be regulated by this system.This work was supported by N.S.F. Grant No. 77-01174 awarded to Dr. Donald J. Woodward, and an award from the Biological Humanics Foundation     

Mossy and climbing fiber collateral inputs in monkey cerebellar paraflocculus lobulus petrosus and hemispheric lobule VII and their relevance to oculomotor functions

Recent lesion studies on monkeys suggest that the cerebellar lobulus petrosus of the paraflocculus (LP) and crura I and II of hemispheric lobule VII (H-7) are involved in smooth pursuit eye movement control. To reveal the relationship between the LP and H-7, we studied mossy and climbing fiber collateral inputs to these areas in four cynamolgus monkeys. After unilateral injections of retrograde tracers into the LP, labeled mossy fibers were seen ipsilaterally in the crura I and II of H-7. A very small number of labeled mossy fiber collaterals were also seen in the dorsal paraflocculus (DP). Labeled climbing fibers were seen exclusively in the ipsilateral crus I. No labeled mossy/climbing fibers were seen in the flocculus, ventral paraflocculus and other cortical areas. Combined injections of fast blue in the LP and cholera toxin subunit B in the posterior crus I and crus II of H-7 resulted in a small number of the double-labeled pontine and principal olivary neurons. Combined injections in the LP and DP induced only a few double-labeled neurons in the pontine nuclei, and no double-labeled neurons in the olivary nuclei. These results suggest that the LP and crura I and II of H-7 may share some of their mossy and climbing fiber inputs and mediate similar functional roles involving smooth pursuit eye movement control.     

Maturation of evoked climbing fiber input to rat cerebellar Purkinje cells (I.)

Summary An analysis of evoked responses of Purkinje cells in developing rat cerebellum to climbing fiber input was conducted to determine which identifying properties of this afferent system are established early in development and which specific features mature with age. Rat pups at various ages were anesthetized with 0.5% halothane and unit recordings made with glass micropipettes. By the third postnatal day, electrical stimulation of the sensorimotor cortex and limbs at low stimulation rates (<1/sec) could elicit distinct burst responses appearing at long latencies (180 msec), indicating that pathways of both ascending and descending climbing fiber systems are intact early in cerebellar cortical development. A distinctive feature maturing over the first 1–1.5 weeks was the characteristic of the all-or-none burst response since before about day 11 the interspike interval, amount of inactivation, and number of spikes in evoked burst responses all varied from stimulation to stimulation. Mean latencies decreased from 180 msec at day 3 to 50 msec by day 10, but did not achieve the adult value of 20 msec until the fourth week. Typically, climbing fiber responses could only follow at stimulation rates of less than 0.2/sec at day 3, but by day 12 could follow up to 10/sec, which is the same as in the adult. The data indicate that the climbing fiber system establishes connectivity from diverse sources and exhibits identifying characteristics similar to the adult early in cerebellar development. Most aspects of the maturation of transmission can be explained if there were a decrease in the time scale of function of the synapses involved, mainly those in the inferior olive.Supported by N.I.H. Grant 5-R01-6M00133 and N.S.F. Grant GB 43301     

Cutaneous mechanoreceptors influencing impulse discharges in cerebellar cortex. III. In Purkyně cells by climbing fiber input

Summary An account is given of single Purkyn cell responses that are evoked by cutaneous mechanoreceptors acting via climbing fibers (CF). There was no complication by mossy fiber input, which was selectively depressed by light pentothal anesthesia.Brief mechanical pulses (taps) to the footpads and air jets on hairy skin were very effective in evoking CF responses, the usual range of latencies being 25–35 msec for the hindfoot and 19–30 msec for the forefoot. There was often a considerable difference in the effectiveness of the different pads of the same foot. For any particular Purkyn cell there was a close correspondence between the receptive fields for footpads and hairy skin.The threshold was often below 0.02 mm taps for the most sensitive pads, and was even lower for more prolonged mechanical displacements. Mechanical pulses of 80 to several hundred msec in duration were effective in evoking CF responses of Purkyn cells at off as well as at on. This finding and the very low threshold strongly suggest Pacinian corpuscles as the principal receptors concerned in activating the CF system.Repetitive taps to footpads were effective at slow frequencies, 5 or 10 Hz, but there was a response only to the first tap with a frequency of 65 Hz.     

Excitatory inputs to cerebellar dentate nucleus neurons from the cerebral cortex in the cat

Summary 1. In anesthetized cats, we investigated excitatory and inhibitory inputs from the cerebral cortex to dentate nucleus neurons (DNNs) and determined the pathways responsible for mediating these inputs to DNNs. 2. Intracellular recordings were made from 201 DNNs whose locations were histologically determined. These neurons were identified as efferent DNNs by their antidromic responses to stimulation of the contralateral red nucleus (RN). Stimulation of the contralateral pericruciate cortex produced excitatory postsynaptic potentials (EPSPs) followed by long-lasting inhibitory postsynaptic potentials (IPSPs) in DNNs. The most effective stimulating sites for inducing these responses were observed in the medial portion (area 6) and its adjacent middle portion (area 4) of the precruciate gyrus. Convergence of cerebral inputs from area 4 and area 6 to single DNNs was rare. 3. To determine the precerebellar nuclei responsible for mediation of the cerebral inputs to the dentate nucleus (DN), we examined the effects of stimulation of the pontine nucleus (PN), the nucleus reticularis tegmenti pontis (NRTP) and the inferior olive (IO). Systematic mapping was made in the NRTP and the PN to find effective low-threshold stimulating sites for evoking monosynaptic EPSPs in DNNs. Stimulation of either the PN or the NRTP produced monosynaptic EPSPs and polysynaptic IPSPs in DNNs. Using a conditioning-testing paradigm (a conditioning stimulus to the cerebral peduncle (CP) and a test stimulus to the PN or the NRTP) and intracellular recordings from DNNs, we tested cerebral effects on neurons in the PN and the NRTP making a monosynaptic connection with DNNs. Conditioning stimulation of the CP facilitated PN- and NRTP-induced monosynaptic EPSPs in DNNs. This spatial facilitation indicated that the excitatory inputs from the cerebral cortex to DNNs are at least partly relayed via the PN and the NRTP. 4. Stimulation of the contralateral IO produced monosynaptic EPSPs and polysynaptic IPSPs in DNNs. These monosynaptic EPSPs were facilitated by conditioning stimulation of the CP, strongly suggesting that the IO is partly responsible for mediating excitatory inputs from the cerebral cortex to the DN. A comparison was made between the latencies of IO-evoked IPSPs in DNNs and the latencies of IO-evoked complex spikes in Purkinje cells. Such a comparison indicated that the shortest-latency IPSPs evoked from the IO were not mediated via the Purkinje cells and suggested the pathway mediated by inhibitory interneurons in the DN. 5. The functional significance of the excitatory inputs from the PN and the NRTP to the DN is discussed in relation to the motor control mechanisms of the cerebellum.