Bart P. Vos

Cerebellar Golgi cells in the rat: receptive fields and timing of responses to facial stimulation

Golgi cells are the only elements within the cerebellar cortex that inhibit granule cells. Despite their unique position there is little information on how Golgi cells respond to afferent input. We studied responses of Golgi cells to mechanical stimulation of the face, in Crus I‐II of ketamine‐xylazine anaesthetized rats. In 41 rats, 87 putative Golgi cells were identified, based on spike characteristics and on location of electrolytic lesions in the granular layer. They displayed a slow firing rhythm at rest (8.4 spikes/s). Most Golgi cells (84%) showed excitatory responses to tactile input. Their receptive fields (RFs) included, in 78%, the entire ipsilateral infraorbital nerve territory, and extended, in 14%, to other trigeminal nerve branches and, in 48%, to the contralateral face. Excitatory responses consisted of multiple, precisely timed (± 1 ms) spikes. Most peristimulus time histograms (PSTHs) (69%) showed an early (5–10 ms) and a late (13–26 ms) excitatory component, with each component consisting of a single PSTH peak. In some PSTHs the early component was a double peak (< 4 ms interval). In others, only one, early or late, PSTH peak was observed. The excitatory components were followed by a silent period (28–69 ms latency), the duration of which (13–200 ms) varied with response amplitude. In single cells, response profiles changed with stimulus location. In simultaneously recorded cells, evoked profiles differed for identical stimuli. Differences in RF size between early ‘double’ and ‘single’ peaks suggested that they resulted from direct mossy fibre and parallel fibre input, respectively. Late PSTH peaks were assumed to reflect corticopontine activation.

Behavioral assessment of facial pain in rats : face grooming patterns after painful and non-painful sensory disturbances in the territory of the rat's infraorbital nerve

&NA; Noxious stimulation of the rats face evokes intense face grooming with face wash strokes almost exclusively directed to the stimulated area (e.g. Clavelou et al., Neurosci. Lett., 14 (1989) 3263–3270). Similar asymmetric face grooming behavior has been observed after transection (Berridge and Fentress, J. Neurosci., 6 (1986) 325–330) and chronic constriction of the infraorbital nerve (Vos et al., J. Neurosci., 14 (1994) 2708–2723). In the present study, the relation between unilateral facial pain and asymmetric face grooming was experimentally studied in normal, intact rats: face grooming patterns evoked by non‐painful sensory disturbances in the territory of the infraorbital nerve (i.c. unilateral vibrissae clipping, anesthetic infraorbital nerve blockade, application of mineral oil on vibrissae) were compared to those evoked by noxious facial stimulation (s.c. formalin injection in mystacial pad) and those observed in unstimulated control rats, using video‐analysis. Only formalin‐injected rats displayed significantly more face grooming activity directed to the affected infraorbital nerve territory than unstimulated control rats. Non‐painful sensory disturbances (especially mineral oil application) induced an initial bout of directed face grooming; this response was transient and short‐lasting. These observations suggest that directed face grooming can be used as a sign of unilateral facial pain in freely moving rodents; unilateral non‐painful facial sensory disturbances do not lead to intense and persistent directed face grooming.

Rat somatosensory cerebropontocerebellar pathways: spatial relationships of the somatotopic map of the primary somatosensory cortex are preserved in a three-dimensional clustered pontine map.

In the primary somatosensory cortex (SI), the body surface is mapped in a relatively continuous fashion, with adjacent body regions represented in adjacent cortical domains. In contrast, somatosensory maps found in regions of the cerebellar hemispheres, which are influenced by the SI through a monosynaptic link in the pontine nuclei, are discontinuous (“fractured”) in organization. To elucidate this map transformation, the authors studied the organization of the first link in the SI‐cerebellar pathway, the SI‐pontine projection. After injecting anterograde axonal tracers into electrophysiologically defined parts of the SI, three‐dimensional reconstruction and computer‐graphic visualization techniques were used to analyze the spatial distribution of labeled fibers. Several target regions in the pontine nuclei were identified for each major body representation. The labeled axons formed sharply delineated clusters that were distributed in an inside‐out, shell‐like fashion. Upper lip and other perioral representations were located in a central core, whereas extremity and trunk representations were found more externally. The multiple clusters suggest that the pontine nuclei contain several representations of the SI map. Within each representation, the spatial relationships of the SI map are largely preserved. This corticopontine projection pattern is compatible with recently proposed principles for the establishment of subcortical topographic patterns during development. The largely preserved spatial relationships in the pontine somatotopic map also suggest that the transformation from an organized topography in SI to a fractured map in the cerebellum takes place primarily in the mossy fiber pontocerebellar projection. J. Comp. Neurol. 422:246–266, 2000.

Electrophysiological properties of ventromedial medulla neurons in response to noxious and non-noxious stimuli in the awake, freely moving rat: a single-unit study

The spontaneous and evoked activities of ventromedial medulla (VMM) neurons have been recorded in the chronic, awake, freely moving rat. The vast majority of neurons located at the level of the nucleus raphé magnus exhibited an irregular and variable (2-16 Hz) spontaneous activity and were activated by either cutaneous or auditory stimuli. Within this convergent neuronal class the neurons were activated by either cutaneous noxious and non-noxious inputs. The threshold for cutaneous activation was likely very low since a majority of units responded to air puffs, but the application of controlled brushing and pin-prick revealed that the VMM convergent neurons responded more for the noxious mechanical stimulation. Similar findings were found with pinch application. For both innocuous and noxious stimuli, the cutaneous receptive field was extremely extensive (almost all of the body); however, the application of the controlled brushing showed that for this innocuous stimulation, the most sensitive regions were the tail, back, snout and vibrissae and, to a lesser extent, the flank and paws. Preliminary experiments indicated that both the spontaneous and evoked activities of VMM convergent neurons were inhibited during stressful manipulations such as scruff lifting or defense reactions. These data contrast with other studies on VMM single unit recordings in anesthetized rats since the majority of these studies did not emphasize the VMM convergent group; in addition, with one exception, we did not find neurons exclusively driven by noxious inputs. Without excluding a role of the VMM convergent group in pain descending control systems, we proposed that this neuronal class is perhaps also involved in pain transmission or in general processess such as alertness and stress. Experiments are proposed in order to precisely determine the involvement of the VMM convergent neurons in alertness versus sensory discriminative aspects of nociception in the awake, freely moving rat.

The function of cerebellar Golgi cells revisited.

The inhibitory interneurons of the cerebellar cortex have received very little attention compared to the granule and Purkinje cells, and Golgi cells are no exception. Theoretical considerations of the function of Golgi cell functions have evolved little since from the late sixties and experimental studies were sparse until the last few years. Recent modeling and in vivo experimental studies by our group, combined with in vitro experimental studies by others, have provided new insights into the properties of these cells which necessitate a revisiting of their function. The connectivity of the Golgi cell The anatomical facts are rather simple. The numerically most important input to the cerebellum is the mossy fiber system (Murphy and Sabah, 1971; Brodal and Bjaalie, 1997). If we limit ourselves to this input, the anatomy of cerebellar cortex can be described as a two-layered network. The input layer, corresponding to the granular layer, processes the incoming mossy fiber signals and transmits them by the parallel fiber system to the output layer, consisting mainly of the Purkinje cells. In both layers activity is controlled by inhibitory neurons, the Golgi cells in the input layers, and the basket and stellate cells in the output layer. Mossy fibers activate both the excitatory granule cells and the inhibitory Golgi cells (Fig. 1A). The granule cell axon forms the parallel fibers, which not only transmit information to the output layer, but also provide additional excitatory input to Golgi cells. Each Golgi cell in turns inhibits the many granule cells present within the range of its axonal arbor (Eccles et al., 1966) with probably some overlap between adjacent Golgi cells. The combination of the parallel fiber excitation of Golgi cells with their inhibition of granule cells constitutes a feedback inhibition circuit (Fig. 1C). The direct excitation of Golgi cells by mossy fibers (Fig. 1B) provides a feed-forward connection. It should be noted, however, that the existence of mossy fiber contacts onto Golgi cells couldwere not be confirmed found in electron microscopal reconstructions of cerebellar glomeruli (Jakab and Hamori, 1988).

A patchy horizontal organization of the somatosensory activation of the rat cerebellum demonstrated by functional MRI.

Blood oxygenation level dependent contrast (BOLD) functional MRI (fMRI) responses, in a 7‐T magnet, were observed in the cerebellum of alpha‐chloralose anaesthetized rats in response to innocuous electrical stimulation of a forepaw or hindpaw. The responses were imaged in both coronal and sagittal slices which allowed for a clear delineation and localization of the observed activations. We demonstrate the validity of our fMRI protocol by imaging the responses in somatosensory cortex to the same stimuli and by showing reproducibility of the cerebellar responses. Widespread bilateral activations were found with mainly a patchy and mediolateral band organization, more pronounced ipsilaterally. Possible parasagittal bands were observed only in contralateral lobule VI. There was no overlap between the cerebellar activations caused by forepaw and hindpaw stimuli. The overall horizontal organization of these responses was quite remarkable. For both stimulation paradigms most of the activation patches were positioned in either a rostral or caudal broad plane running anteroposteriorly through both anterior and posterior cerebellum. The rostral planes were completely separated, with the forepaw activation closer to the surface, while the caudal plane was common to both stimulation protocols. We relate our findings to the known projection patterns of spinocerebellar and cuneocerebellar mossy fibres, and to human fMRI studies.

Weak common parallel fibre synapses explain the loose synchrony observed between rat cerebellar Golgi cells

In anaesthetized rats, pairs of cerebellar Golgi cells fired synchronously at rest, provided they were aligned along the parallel fibre axis. The observed synchrony was much less precise, however, than that which would be expected to result from common, monosynaptic parallel fibre excitation. To explain this discrepancy, the precision and frequency of spike synchronization (i.e. the width and area of the central peak on the spike train cross‐correlogram) were computed in a generic model for varying input, synaptic and neuronal parameters. Correlation peaks between model neurons became broader, and peak area smaller, when the number of afferents increased and each single synapse decreased proportionally in strength. Peak width was inversely proportional to firing rate, but independent of the percentage of shared afferents. Peak area, in contrast, scaled with the percentage of shared afferents but was almost firing rate independent. Broad correlation peaks between pairs of model neurons resulted from the loose spike timing between single model neurons and their afferents. This loose timing reflected a need for long‐term synaptic integration to fire the neurons. Model neurons could accomplish this through firing rate adaptation mediated by a Ca2+‐activated K+ channel. We conclude that loose synchrony may be entirely explained by shared input from monosynaptic, non‐synchronized afferents. The inverse relationship between peak width and firing rate allowed us to distinguish common parallel fibre input from firing rate covariance as a primary cause of loose synchrony between cerebellar Golgi cells in anaesthetized rats.

Peripheral stimuli excite coronal beams of Golgi cells in rat cerebellar cortex

Cerebellar granule cells constitute the largest neurone population of the brain. Their axons run as parallel fibres along the coronal axis, and the one-dimensional spread of excitation that is expected to result from this arrangement is a key assumption of theories of cerebellar function. In many studies using various techniques, however, it was not possible to evoke such a beam-like propagation of excitation with natural stimuli. We recorded, in Crus I and II of anaesthetised rats, pairs of Golgi cells aligned along the parallel fibre axis and synchronising spontaneously. Each pair was subjected to two stimulation protocols: punctate and semi-continuous. Local punctate facial stimulation evoked distinct fast and late responses of variable strength and latency (fast: 4.0-10.2 ms; late: 13.6-22.7 ms). Semi-continuous stimulation with a brush increased the firing rate, and modified the precision and phase of synchronisation. Differences between a pair in response strength and phase to brush stimulation correlated strongly with the difference in latency to punctate stimulation. These observations were reproduced in a model of the granular layer. The stimulus activated a central patch of mossy fibres, and Golgi cells received short- and long-range excitation from mossy and parallel fibres, respectively. The strength and latency of the punctate response of a model Golgi cell were found to vary with its position, reflecting a systematic change in the contribution of mossy and parallel fibres to its excitation with distance from the activated patch. During brush stimulation, model Golgi cells inside the patch fired more precisely synchronised, whereas the other Golgi cells responded with a lag proportional to their distance from the patch, thereby reproducing the experimentally observed changes in synchronisation. Taken together with the previously reported large receptive fields of Golgi cells and with their spontaneous synchronisation, the variable, position-dependent latency of evoked Golgi cell responses indicates a beam-like spread of excitation along the parallel fibres in rat cerebellar cortex.

Miniature carrier with six independently moveable electrodes for recording of multiple single-units in the cerebellar cortex of awake rats

Ensemble recording in cerebellar cortex of awake rats presents unique methodological challenges not encountered when recording from the cerebral cortex or from deep brain structures with more homogeneous cell populations. Compared to the cerebral cortex, removal of dura over the cerebellum evokes pronounced swelling, and insertion of multiple closely spaced electrodes in the cerebellar cortex causes considerable dimpling (Welsh JP, Schwartz C. Multielectrode recording from the cerebellum. In: Nicolelis MAL, editor. Methods for Neural Ensemble Recordings, CRC Methods in Neuroscience Series. Boca Raton, FL: CRC Press LLC, 1999, pp. 79-100). Also, a repetitious and well-defined neural circuit characterizes the cerebellar cortex across its entire surface. With conventional multi-electrode methods, such as chronically implanted bundles or arrays of microwires, the risk of disrupting the cerebellar cytoarchitecture is high. In most conventional multi-electrode systems, electrodes have rather low impedance and cannot be moved independently after implantation. These limitations make proper unit isolation, necessary to identify each of the recorded cerebellar units, very difficult. We designed a lightweight (14 g), miniature (base plate: 19 x 23 mm; total height: 16 mm) multi-electrode system to allow for the chronic implantation of six independently moveable sharp electrodes with high impedance, in the cerebellar cortex. The six electrodes are arranged in a 2 x 3 matrix (inter-electrode distance: 0.6 mm). At any time after the implantation the vertical position of each individual electrode can be adjusted by screwing spring-loaded electrode heads up or down. The system preserves the integrity of the cerebellar cytoarchitecture, and enables easy isolation and identification of individual cerebellar units in awake, freely moving rats.

A single-unit recording system, contact thermal probe and electromechanical stimulator for studying cellular mechanisms related to nociception at brain stem level of awake, freely moving rats.

The purpose of this paper is to describe a simple, light-weight (3 g) device bearing a fine platinum-irridium Teflon-coated wire (50 microns) used to record single-unit activity extracellularly at brain stem level in the totally conscious freely moving rat. The up and down movements of the electrode through a guide cannula are insured by a small nut and a spring; the distance between the electrode and the end of the guide cannula is measured with a nut index. The system is directly connected to an amplifier (no FET or preamplifier) and allows for long term recordings necessary for a complete neuronal characterization and pharmacological experiments. The device is easy to make, entirely recoverable, and can be implanted from an animal to another. Further improvements are possible such as tungsten microelectrodes and telemetric or microinjection systems. In order to study some neuronal brain stem mechanisms involved in nociception, we have also designed a contact thermal probe and an electromechanical stimulator. The thermode is stuck to the shaved skin on the back of the rat, allowing heat pulses up to 51 degrees C to be applied. The mechanical stimulator is used manually and delivers reproducible innocuous stimuli to the skin. The fact that both types of stimulations are driven electrically enables the elaboration of cumulated peristimulus histograms which will reflect the neuronal activities in response to the application of noxious and non noxious stimuli.