Tahl Holtzman

Different responses of rat cerebellar Purkinje cells and Golgi cells evoked by widespread convergent sensory inputs

While the synaptic properties of Golgi cell‐mediated inhibition of granule cells are well studied, less is known of the afferent inputs to Golgi cells so their role in information processing remains unclear. We investigated the responses of cerebellar cortical Golgi cells and Purkinje cells in Crus I and II of the posterior lobe cerebellar hemisphere to activation of peripheral afferents in vivo, using anaesthetized rats. Recordings were made from 70 Golgi cells and 76 Purkinje cells. Purkinje cells were identified by the presence of climbing fibre responses. Golgi cells were identified by both spontaneous firing pattern and response properties, and identification was confirmed using juxtacellular labelling of single neurones (n= 16). Purkinje cells in Crus II showed continuous firing at relatively high rates (25–60 Hz) and stimulation of peripheral afferents rarely evoked substantial responses. The most common response was a modest, long‐latency, long‐lasting increase in simple spike output. By comparison, the most common response evoked in Golgi cells by the same stimuli was a long‐latency, long‐lasting depression of firing, found in ∼70% of the Golgi cells tested. The onsets of Golgi cell depressions had shorter latencies than the Purkinje cell excitations. Brief, short‐latency excitations and reductions in firing were also evoked in some Golgi cells, and rarely in Purkinje cells, but in most cases long‐lasting depressions were the only significant change in spike firing. Golgi cell responses could be evoked using air puff or tactile stimuli and under four different anaesthetic regimens. Long‐lasting responses in both neurone types could be evoked from wide receptive fields, in many cases including distal afferents from all four limbs, as well as from trigeminal afferents. These Golgi cell responses are not consistent with the conventional feedback inhibition or ‘gain control’ models of Golgi cell function. They suggest instead that cerebellar cortical activity can be powerfully modulated by the general level of peripheral afferent activation from much of the body. On this basis, Golgi cells may act as a context‐specific gate on transmission through the mossy fibre–granule cell pathway.

A wireless multi-channel recording system for freely behaving mice and rats.

To understand the neural basis of behavior, it is necessary to record brain activity in freely moving animals. Advances in implantable multi-electrode array technology have enabled researchers to record the activity of neuronal ensembles from multiple brain regions. The full potential of this approach is currently limited by reliance on cable tethers, with bundles of wires connecting the implanted electrodes to the data acquisition system while impeding the natural behavior of the animal. To overcome these limitations, here we introduce a multi-channel wireless headstage system designed for small animals such as rats and mice. A variety of single unit and local field potential signals were recorded from the dorsal striatum and substantia nigra in mice and the ventral striatum and prefrontal cortex simultaneously in rats. This wireless system could be interfaced with commercially available data acquisition systems, and the signals obtained were comparable in quality to those acquired using cable tethers. On account of its small size, light weight, and rechargeable battery, this wireless headstage system is suitable for studying the neural basis of natural behavior, eliminating the need for wires, commutators, and other limitations associated with traditional tethered recording systems.

Electrophysiological Localization of Eyeblink-Related Microzones in Rabbit Cerebellar Cortex

The classically conditioned eyeblink response in the rabbit is one of the best-characterized behavioral models of associative learning. It is cerebellum dependent, with many studies indicating that the hemispheral part of Larsells cerebellar cortical lobule VI (HVI) is critical for the acquisition and performance of learned responses. However, there remain uncertainties about the distribution of the critical regions within and around HVI. In this learning, the unconditional stimulus is thought to be carried by periocular-activated climbing fibers. Here, we have used a microelectrode array to perform systematic, high-resolution, electrophysiological mapping of lobule HVI and surrounding folia in rabbits, to identify regions with periocular-evoked climbing fiber activity. Climbing fiber local field potentials and single-unit action potentials were recorded, and electrode locations were reconstructed from histological examination of brain sections. Much of the sampled cerebellar cortex, including large parts of lobule HVI, was unresponsive to periocular input. However, short-latency ipsilateral periocular-evoked climbing fiber responses were reliably found within a region in the ventral part of the medial wall of lobule HVI, extending to the base of the primary fissure. Small infusions of the AMPA/kainate receptor antagonist CNQX into this electrophysiologically defined region in awake rabbits diminished or abolished conditioned responses. The known parasagittal zonation of the cerebellum, supported by zebrin immunohistochemistry, indicates that these areas have connections consistent with an essential role in eyeblink conditioning. These small eyeblink-related areas provide cerebellar cortical targets for analysis of eyeblink conditioning at a neuronal level but need to be localized with electrophysiological identification in individual animals.

Enzyme-based choline and l-glutamate biosensor electrodes on silicon microprobe arrays

Brain-implantable microprobe arrays, 6.5 mm shaft-length, incorporating several recessed Pt microelectrodes (50 μm×150 μm) and an integrated Ag/AgCl reference electrode fabricated by silicon micromachining dry etching techniques (DRIE) are described. The microelectrodes are coated by an enzyme membrane and a semi-permeable m-phenylenediamine layer for the selective detection of the neurotransmitters choline and L-glutamate at physiologically relevant concentrations. The functionalisation is based on electrochemically aided adsorption (EAA) combined with chemical co-cross-linking using glutaraldehyde and electrochemical polymerisation, respectively. These deposition methods are fully compatible with the fabricated microprobe arrays for the simultaneous detection of several analytes in different brain target areas. They are spatially controlled and allow fabricating biosensors on several microelectrodes in parallel or providing a cross-talk-free coating of closely spaced microelectrodes with different enzyme membranes. A sensitivity of 132±20 μA mM(-1) cm(-2) for choline and 95±20 μA mM(-1) cm(-2) for L-glutamate with limits of detections below 0.5 μM was obtained. The results of in vitro and in vivo experiments confirm the functional viability of the choline and l-glutamate biosensors.

Oscillatory Activity in the Medial Prefrontal Cortex and Nucleus Accumbens Correlates with Impulsivity and Reward Outcome

Actions expressed prematurely without regard for their consequences are considered impulsive. Such behaviour is governed by a network of brain regions including the prefrontal cortex (PFC) and nucleus accumbens (NAcb) and is prevalent in disorders including attention deficit hyperactivity disorder (ADHD) and drug addiction. However, little is known of the relationship between neural activity in these regions and specific forms of impulsive behaviour. In the present study we investigated local field potential (LFP) oscillations in distinct sub-regions of the PFC and NAcb on a 5-choice serial reaction time task (5-CSRTT), which measures sustained, spatially-divided visual attention and action restraint. The main findings show that power in gamma frequency (50–60 Hz) LFP oscillations transiently increases in the PFC and NAcb during both the anticipation of a cue signalling the spatial location of a nose-poke response and again following correct responses. Gamma oscillations were coupled to low-frequency delta oscillations in both regions; this coupling strengthened specifically when an error response was made. Theta (7–9 Hz) LFP power in the PFC and NAcb increased during the waiting period and was also related to response outcome. Additionally, both gamma and theta power were significantly affected by upcoming premature responses as rats waited for the visual cue to respond. In a subgroup of rats showing persistently high levels of impulsivity we found that impulsivity was associated with increased error signals following a nose-poke response, as well as reduced signals of previous trial outcome during the waiting period. Collectively, these in-vivo neurophysiological findings further implicate the PFC and NAcb in anticipatory impulsive responses and provide evidence that abnormalities in the encoding of rewarding outcomes may underlie trait-like impulsive behaviour.

Cerebellar Golgi cells in the rat receive multimodal convergent peripheral inputs via the lateral funiculus of the spinal cord

We recently showed that the activity of cerebellar Golgi cells can be powerfully modulated by stimulation of peripheral afferents, in a pattern different to local Purkinje cells. Here we have examined the pathways underlying these responses. Graded electrical stimulation of muscle and cutaneous nerves revealed that long‐lasting depressions and short‐lasting excitations of Golgi cells were evoked by stimulation of cutaneous nerves at stimulus intensities that activated large mechanoreceptive afferents, and grew as additional afferents were recruited. In contrast, none of the neurones responded to stimulation of muscle nerves at intensities that activated group I afferents, although about half responded with long‐lasting depressions, but not excitations, to stimuli that recruited group II and III afferents. Selective lesions of the spinal dorsal columns did not affect either of these types of response. After lesions of one lateral funiculus in the lumbar cord the responses evoked by stimulation of the hindlimb contralateral to the lesion were reduced or abolished, leaving responses evoked by ipsilateral hindlimb afferents unaltered. Since both ipsi‐ and contralateral afferents generate responses in Golgi cells, the convergence from the two sides must occur supraspinally. It is difficult to reconcile these properties with any of the direct spinocerebellar pathways or spinoreticulocerebellar pathways that have been described. Instead, it is likely that the responses are evoked via the multimodal ‘wide dynamic range’ neurones of the anterolateral system. Golgi cell activity may thus be powerfully enhanced or depressed during arousal via the anterolateral system.

Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell–granule cell loops

Non‐technical summary The cerebellar cortex contains complex neural circuits related to information processing for the learning and control of movements. We show that the interaction between the main input neurones, the granule cells and their inhibitory counterparts, the Golgi cells, is far more complex than previously thought. Traditionally, granule cells are considered to excite Golgi cells, thereby forming a negative feedback loop. In contrast, our study reveals that granule cell input to Golgi cells is predominantly inhibitory, through the action of specialised glutamate receptors expressed in Golgi cells. These results force a re‐evaluation of our current best theories of how the cerebellar circuitry processes information.

Probabilistic identification of cerebellar cortical neurones across species.

Despite our fine-grain anatomical knowledge of the cerebellar cortex, electrophysiological studies of circuit information processing over the last fifty years have been hampered by the difficulty of reliably assigning signals to identified cell types. We approached this problem by assessing the spontaneous activity signatures of identified cerebellar cortical neurones. A range of statistics describing firing frequency and irregularity were then used, individually and in combination, to build Gaussian Process Classifiers (GPC) leading to a probabilistic classification of each neurone type and the computation of equi-probable decision boundaries between cell classes. Firing frequency statistics were useful for separating Purkinje cells from granular layer units, whilst firing irregularity measures proved most useful for distinguishing cells within granular layer cell classes. Considered as single statistics, we achieved classification accuracies of 72.5% and 92.7% for granular layer and molecular layer units respectively. Combining statistics to form twin-variate GPC models substantially improved classification accuracies with the combination of mean spike frequency and log-interval entropy offering classification accuracies of 92.7% and 99.2% for our molecular and granular layer models, respectively. A cross-species comparison was performed, using data drawn from anaesthetised mice and decerebrate cats, where our models offered 80% and 100% classification accuracy. We then used our models to assess non-identified data from awake monkeys and rabbits in order to highlight subsets of neurones with the greatest degree of similarity to identified cell classes. In this way, our GPC-based approach for tentatively identifying neurones from their spontaneous activity signatures, in the absence of an established ground-truth, nonetheless affords the experimenter a statistically robust means of grouping cells with properties matching known cell classes. Our approach therefore may have broad application to a variety of future cerebellar cortical investigations, particularly in awake animals where opportunities for definitive cell identification are limited.

Characterization in vivo of bilaterally branching pontocerebellar mossy fibre to Golgi cell inputs in the rat cerebellum

Golgi cells regulate the flow of information from mossy fibres to the cerebellar cortex, through a mix of feedback and feedforward inhibitory actions on granule cells. The aim of the current study was to examine mossy fibre input to Golgi cells, in order to assess their impact on switching Golgi cells into feedforward behaviour. In urethane‐anaesthetized rats, extracellular recordings were made from Golgi cells in Crus II (n = 18). Spikes were evoked in all Golgi cells by microstimulation within the contralateral hemispheral cortex, via branches of mossy fibres that terminate in both cerebellar hemispheres. The latencies of these responses were very short, consistent with a monosynaptic mossy fibre contact [average onset latency 2.3 ± 0.1 ms (SEM)]. The same stimuli had no measurable effect on spike responses of nearby Purkinje cells (n = 12). Systematic mapping in the contralateral cerebellar hemisphere (Crus Ib, IIa, IIb and the paramedian lobule) usually revealed one low‐intensity stimulus ‘hotspot’ (12–35 μA) from which short‐latency spikes could be evoked in an individual Golgi cell. Microinjections of red and green retrograde tracers (latex beads, ∼50–150 nL injection volume) made at the recording site and the stimulation hotspot resulted in double‐labelled neurons within the pontine nuclei. Overall, this suggests that subsets of pontine neurons supply mossy fibres that branch to both hemispheres, some of which directly target Golgi cells. Such an arrangement may provide a common feedforward inhibitory link to temporally couple activity on both sides of the cerebellum during behaviour.

Compact wireless neural recording system for small animals using silicon-based probe arrays

This paper reports on a compact, small-scale neural recording system combining state-of-art silicon-based probe arrays with a light-weight 32-channel wireless head stage. The system is equipped with two- and four-shaft, comb-shaped probe arrays connected to highly flexible ribbon cables enabling a reliable and controlled insertion of probe arrays through the intact dura mater into the medial prefrontal cortex and nucleus accumbens of rats. The in vivo experiments applied the 5-choice serial reaction time task (5-CSRTT) using freely behaving rats in order to understand the neural basis of sustained visual attention and impulsivity. The long-term stability of the system allowed local field potential (LFP) activity to be recorded without a significant decrement in signal quality for up to 28 weeks, and similarly, we were able to follow single unit activity for up to 4 weeks.