Role of cortical feedback in the receptive field structure and nonlinear response properties of somatosensory thalamic neurons.

Previous studies have suggested that the descending pathway from the primary somatosensory (SI) cortex to the ventral posterior nucleus of the thalamus has only a mild facilitative influence over thalamic neurons. Given the large numbers of corticothalamic terminations within the rat somatosensory thalamus and their complex topography, we sought to examine the role of corticothalamic feedback in the genesis of spatiotemporal receptive fields and the integration of complex tactile stimuli in the thalamus. By combining focal cortical inactivation (produced by microinjection of the GABA(A) agonist muscimol), with chronic multielectrode recordings, we observed that feedback from the rat SI cortex has multiple influences on its primary thalamic relay, the ventral posterior medial (VPM) nucleus. Our data demonstrate that, when single-whisker stimuli were used, the elimination of cortical feedback caused significant changes in the spatiotemporal structure of the receptive fields of VPM neurons. Cortical feedback also accounted for the nonlinear summation of VPM neural responses to simultaneously stimulated whiskers, in effect "linearizing" the responses. These results argue that the integration and transmission of tactile information through VPM are strongly influenced by the state of SI cortex.

Behavioral properties of the trigeminal somatosensory system in rats performing whisker-dependent tactile discriminations.

To address several fundamental questions regarding how multiwhisker tactile stimuli are integrated and processed by the trigeminal somatosensory system, a novel behavioral task was developed that required rats to discriminate the width of either a wide or narrow aperture using only their large mystacial vibrissae. Rats quickly acquired this task and could accurately discriminate between apertures of very similar width. Accurate discriminations required a large number of intact facial whiskers. Systematic removal of individual whiskers caused a decrease in performance that was directly proportional to the number of whiskers removed, indicating that tactile information from multiple whiskers is integrated as rats gauge aperture width. In different groups of rats, different sets of whiskers were removed in patterns that preferentially left whisker rows or whisker arcs intact. These different whisker removals caused similar decreases in performance, indicating that individual whiskers within the vibrissal array are functionally equivalent during performance of this task. Lesions of the barrel cortex abolished the ability of rats to discriminate, demonstrating that this region is critically involved in this tactile behavior. Interestingly, sectioning the facial nerve, which abolished whisker movements, did not affect the ability to perform accurate discriminations, indicating that active whisker movements are not necessary for accurate performance of the task. Collectively, these results indicate that the trigeminal somatosensory system forms internal representations of external stimuli (in this case, aperture width) by integrating tactile input from many functionally equivalent facial whiskers and that the vibrissal array can function as a fine-grained distance detector without active whisker movements.

Bilateral integration of whisker information in the primary somatosensory cortex of rats.

The isomorphic representation of the contralateral whisker pad in the rodent cerebral cortex has served as a canonical example in primary somatosensory areas that the contralateral body surface is spatially represented as a topographic map. By characterizing responses evoked by multiwhisker stimuli, we provide direct evidence that the whisker region of the rat primary somatosensory cortex (SI) integrates information from both contralateral and ipsilateral whisker pads. The proportions of SI neurons responsive to ipsilateral whisker stimuli, as well as their response probabilities, increased with the number of ipsilateral whiskers stimulated. Under bilateral whisker stimulation, the responses of 95% of neurons recorded were affected by stimulation of ipsilateral whiskers. Contralateral tactile responses of SI neurons were profoundly influenced by preceding ipsilateral stimuli and vice versa. This effect depended on both the spatial location and the relative timing of bilateral whisker stimuli, leading to both spatial and temporal asymmetries of interaction. Bilateral whisker stimulation resulted in only modest changes in evoked response latency. Previous ipsilateral stimulation was also shown to affect tactile responses evoked by later ipsilateral stimuli. Inactivation of the opposite SI abolished ipsilaterally evoked responses as well as their influence on subsequently evoked contralateral responses in the intact SI. Based on these results, we conclude that the rat SI integrates information from both whisker pads and propose that such interactions may underlie the ability of rats to discriminate bilateral tactile stimuli.

A multi-channel whisker stimulator for producing spatiotemporally complex tactile stimuli.

A system is described that delivers complex, biologically realistic, tactile stimuli to the rat's facial whisker pad by independently stimulating up to 16 individual facial whiskers in a flexible yet highly controlled and repeatable manner. The system is technically simple and inexpensive to construct. The system consists of an array of 16 miniature-solenoid driven actuators that are attached to 16 individual facial whiskers via very small (130 microm dia.) Teflon-coated stainless steel wires. When individual solenoids are energized, the wire is rapidly retracted, resulting in a deflection of individual whiskers. The rise time of deflection is approx. 1 mm/ms. Repeatable stimulation of individual whiskers can be achieved without touching adjacent whiskers, thereby allowing a very high density of stimulators to be attached within the spatially restricted region of the facial whisker pad. Complex patterns of whisker stimulation (designed to mimic biologically realistic stimuli) are delivered to the whisker pad by activating individual solenoid actuators in precisely controlled temporal patterns. These stimulations can be combined with multi-electrode single-unit ensemble recordings at multiple sites within the rat trigeminal somatosensory system. Analysis of neuronal population responses to these complex stimuli is intended to examine how the trigeminal somatosensory system encodes and processes spatiotemporally complex stimuli.

Fluorescent detection of Zn(2+)-rich vesicles with Zinquin: mechanism of action in lipid environments.

High concentrations of free Zn2+ ions are found in certain glutamatergic synaptic vesicles in the mammalian brain. These terminals can be visualized histochemically with quinoline sulfonamide compounds that form fluorescent complexes with Zn2+. The present study was undertaken to examine the interaction of the water-soluble quinoline sulfonamide probe, Zinquin (2-methyl-8-(toluene-p-sulfonamido)-6-quinolyloxyacetic acid) with the complex heterogeneous cellular environment. Experiments on rat hippocampal and neocortical slices gave indications that Zinquin in its free acid form was able to diffuse across the plasma and synaptic vesicle membranes. Further experiments were undertaken on unilamellar liposomes to study the interaction of Zinquin and its metal complexes in membranes. These experiments confirmed that Zinquin is able to diffuse across lipid bilayers. Steady-state and time-resolved fluorimetric studies showed that Zinquin in aqueous solution mainly forms a 1:2 (metal:ligand) complex with small amounts of a 1:1 complex. Formation of the 1:1 complex was favored by the presence of lipid, suggesting that it partitions into membranes. Evidence is presented that Zinquin can act as a Zn(2+)-ionophore, exchanging Zn2+ for two protons. The presence of a pH gradient across vesicles traps the Zn(2+)-probe complex within the vesicles. Zinquin is useful as a qualitative probe for detecting the presence of vesicular Zn2+; however, its tendency to partition into membranes and to serve as an ionophore should be borne in mind.

Techniques for long-term multisite neuronal ensemble recordings in behaving animals.

Advances in our understanding of neural systems will go hand in hand with improvements in the experimental techniques used to study these systems. This article describes a series of methodological developments aimed at enhancing the power of the methods needed to record simultaneously from populations of neurons over broad regions of the brain in awake, behaving animals. First, our laboratory has made many advances in electrode design, including movable bundle and array electrodes and smaller electrode assemblies. Second, to perform longer and more complex multielectrode implantation surgeries in primates, we have modified our surgical procedures by employing comprehensive physiological monitoring akin to human neuroanesthesia. We have also developed surgical implantation techniques aimed at minimizing brain tissue damage and facilitating penetration of the cortical surface. Third, we have integrated new technologies into our neural ensemble, stimulus and behavioral recording experiments to provide more detailed measurements of experimental variables. Finally, new data analytical techniques are being used in the laboratory to analyze increasingly large quantities of data.

Acute isolation of neurons from the mature mammalian central nervous system.

The acute dissociation procedure provides a simple means of isolating neurons from the mature mammalian central nervous system. The method was primarily devised to isolate neurons for patch-clamp electrophysiology. It may also prove useful for single-cell PCR, immunocytochemistry, sorting of fluorescently labeled cells, or long-term tissue culture of mature neurons. Dissociation is brought about by a combination of proteolysis and an ionic environment that encourages breakdown of the tissue. The method allows the isolation of neurons free of glial ensheathments in as little as 45 min after the sacrifice of the animal. Neurons so isolated lose fine dendritic branches, although the structure proximal to the cell body is often maintained, allowing identification of the morphological type of the neuron. The preparation has the following advantages: (1) the neurons are fully differentiated; (2) the cells are electronically compact, which improves the fidelity of the voltage clamp; (3) the cells are removed from the influence of surrounding cells; and (4) neurons can be isolated from small, circumscribed loci within the adult central nervous system.

Immediate thalamic sensory plasticity depends on corticothalamic feedback.

Multiple neuron ensemble recordings were obtained simultaneously from both the primary somatosensory (SI) cortex and the ventroposterior medial thalamus (VPM) before and during the combined administration of reversible inactivation of the SI cortex and a reversible subcutaneous block of peripheral trigeminal nerve fibers. This procedure was performed to quantify the contribution of descending corticofugal projections on (i) the normal organization of thalamic somatosensory receptive fields and (ii) the thalamic somatosensory plastic reorganization that immediately follows a peripheral deafferentation. Reversible inactivation of SI cortex resulted in immediate changes in receptive field properties throughout the VPM. Cortical inactivation also significantly reduced but did not completely eliminate the occurrence of VPM receptive field reorganization resulting from the reversible peripheral deafferentation. This result suggests that the thalamic plasticity that is seen immediately after a peripheral deafferentation is dependent upon both descending corticofugal projections and ascending trigeminothalamic projections.

Potential circuit mechanisms underlying concurrent thalamic and cortical plasticity.

During the last two decades, plastic reorganization of both sensory and motor representations in the adult central nervous system has been demonstrated following a large variety of manipulations, ranging from partial lesions of the sensory receptor surface to modifications in sensory experience (see /14/ for review). Yet, little is known about the neural circuit mechanisms underlying such reorganization process. Despite the difficulty in addressing this issue, recent studies have provided some insights into this fundamental question. Altogether, these studies suggest that the process of plastic reorganization is a system-wide phenomenon, involving both cortical and subcortical representations. Contrary to classical beliefs, recent work also suggests that the final outcome of the reorganization process is not necessarily beneficial, since it can lead to abnormal perceptual experiences /31/, such as the phantom limb sensation, and even pain /31,32/. In this review, we focus on recent insights into the possible circuit mechanisms underlying sensory plasticity and discuss the potential implications of these findings. We then present physiological evidence supporting the view that the process of plasticity observed at the cortical level may reflect simultaneous changes in many subcortical structures.

The nature of reinforcement in cerebellar learning.

In a now classic study, W. J. Brogden and W. H. Gantt (1942, American Journal of Physiology, 119, 277-278) demonstrated that movements (limbs, head, eyelid) elicited by direct electrical stimulation of certain regions of the cerebellum (particularly the ansiform lobe) could be trained to respond to neutral auditory or visual conditioned stimuli with appropriate pairing. In recent work we have replicated these results in detail and presented considerable evidence that the reinforcing or "teaching" pathway so activated for the learning of discrete movements is the inferior olive-climbing fiber projection system to the cerebellum. Very strong evidence now indicates that the memory traces for this "skilled response" learning are formed and stored in the cerebellum. The climbing fiber system and inhibitory pathway from the interpositus nucleus to the inferior olive appear to form a neural instantiation of the Resorla-Wagner formulation of classical conditioning and accounts for the "cognitive" phenomenon of blocking. It is concluded that reinforcement in this form of learning is not due simply to contiguity/contingency or to unconditioned stimulus aversiveness, per se, but rather to activation of a particular brain circuit, here the climbing fiber system, a circumstance that may apply to other forms of learning, with other reinforcement circuits, as well.

Evidence of plasticity in the pontocerebellar conditioned stimulus pathway during classical conditioning of the eyeblink response in the rabbit.

Electrical stimulation thresholds required to elicit eyeblinks with either pontine or cerebellar interpositus stimulation were measured before and after classical eyeblink conditioning with paired pontine stimulation (conditioned stimulus, CS) and corneal airpuff (unconditioned stimulus, US). Pontine stimulation thresholds dropped dramatically after training and returned to baseline levels following extinction, whereas interpositus thresholds and input-output functions remained stable across training sessions. Learning rate, magnitude of threshold change, and electrode placements were correlated. Pontine projection patterns to the cerebellum were confirmed with retrograde labeling techniques. These results add to the body of literature suggesting that the pons relays CS information to the cerebellum and provide further evidence of synaptic plasticity in the cerebellar network.

Inhibitory cerebello-olivary projections and blocking effect in classical conditioning.

The behavioral phenomenon of blocking indicates that the informational relationship between the conditioned stimulus and the unconditioned stimulus is essential in classical conditioning. The eyeblink conditioning paradigm is used to describe a neural mechanism that mediates blocking. Disrupting inhibition of the inferior olive, a structure that conveys unconditioned stimulus information (airpuff) to the cerebellum prevented blocking in rabbits. Recordings of cerebellar neuronal activity show that the inferior olive input to the cerebellum becomes suppressed as learning occurs. These results suggest that the inferior olive becomes functionally inhibited by the cerebellum during conditioning, and that this negative feedback process might be the neural mechanism mediating blocking.

Reversible inactivation of the cerebellar interpositus nucleus completely prevents acquisition of the classically conditioned eye-blink response.

Numerous studies from several laboratories report that temporary inactivation of the cerebellar interpositus nucleus and regions of overlying cortex during eye-blink conditioning completely prevents acquisition of the conditioned eye-blink response (CR) without affecting the ability to learn the CR in subsequent training without inactivation. Recently, these results have been challenged by the suggestion that learning was not completely blocked in these studies. Instead, it has been suggested that low levels of responses on test sessions might represent a retarded form of learning caused by drug effects on cerebellar cortex. The present study was designed to address this issue directly. Very low doses of muscimol were used to selectively inactivate the interpositus nucleus of rabbits during five conditioning sessions. Animals performed no significant levels of CRs during those sessions. Training was continued four more sessions without any inactivations to test whether any learning had occurred during the previous five sessions. Detailed analysis of responses during session six revealed that learning was completely blocked by the low doses of muscimol infused into the interpositus during the first five sessions. Animals subsequently acquired the CR normally. These results confirm and extend the original findings that appropriate lesions (either temporary or permanent) of the interpositus nucleus completely prevent acquisition of the conditioned eye-blink response. Other issues regarding reversible inactivation studies are also discussed.

Associative learning.

This chapter reviews evidence demonstrating the essential role of the cerebellum and its associated circuitry in the learning and memory of classical conditioning of discrete behavioral responses (e.g., eyeblink, limb flexion, head turn). It now seems conclusive that the memory traces for this basic category of associative learning are formed and stored in the cerebellum. Lesion, neuronal recording, electrical microstimulation, and anatomical procedures have been used to identify the essential conditioned stimulus (CS) circuit, including the pontine mossy fiber projections to the cerebellum; the essential unconditioned stimulus (US) reinforcing or teaching circuit, including neurons in the inferior olive (dorsal accessory olive) projecting to the cerebellum as climbing fibers; and the essential conditioned response (CR) circuit, including the interpositus nucleus, its projection via the superior cerebellar peduncle to the magnocellular red nucleus, and rubral projections to premotor and motor nuclei. Each major component of the eyeblink CR circuit was reversibly inactivated both in trained animals and over the course of training. In all cases in trained animals, inactivation abolished the CR (and the UR as well when motor nuclei were inactivated). When animals were trained during inactivation (and not exhibiting CRs) and then tested without inactivation, animals with inactivation of the motor nuclei, red nucleus, and superior peduncle had fully learned, whereas animals with inactivation of a very localized region of the cerebellum (anterior interpositus and overlying cortex) had not learned at all. Consequently, the memory traces are formed and stored in the cerebellum. Several alternative possibilities are considered and ruled out. Both the cerebellar cortex and the interpositus nucleus are involved in the memory storage process, suggesting that a phenomenon-like long-term depression (LTD) is involved in the cerebellar cortex and long-term potentiation (LTP) is involved in the interpositus. The experimental findings reviewed in this chapter provide perhaps the first conclusive evidence for the localization of a basic form of memory storage to a particular brain region, namely the cerebellum, and indicate that the cerebellum is indeed a cognitive machine.

Inactivation of brainstem motor nuclei blocks expression but not acquisition of the rabbit's classically conditioned eyeblink response.

Rabbits were eyeblink conditioned while their accessory abducens nucleus (ACC), facial nucleus (FN), and surrounding reticular formation (RF) were temporarily inactivated with microinjections of muscimol to determine whether these structures are critically involved in acquisition of the conditioned eyeblink response (CR). Rabbits performed no CRs or unconditioned responses (URs) during inactivation training. Training was continued without inactivation and rabbits performed the CR at asymptotic levels from the start of training without inactivation. They had fully learned the CR while their ACC, FN, and RF were inactivated, despite performing no CRs or URs at all during inactivation. These results rule out any critical role for neurons within the ACC, FN, or surrounding RF in acquisition of the classically conditioned eyeblink response.

Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit's classically conditioned eye-blink response.

The localization of sites of memory formation within the mammalian brain has proven to be a formidable task even for simple forms of learning and memory. Recent studies have demonstrated that reversibly inactivating a localized region of cerebellum, including the dorsal anterior interpositus nucleus, completely prevents acquisition of the conditioned eye-blink response with no effect upon subsequent learning without inactivation. This result indicates that the memory trace for this type of learning is located either (i) within this inactivated region of cerebellum or (ii) within some structure(s) efferent from the cerebellum to which output from the interpositus nucleus ultimately projects. To distinguish between these possibilities, two groups of rabbits were conditioned (by using two conditioning stimuli) while the output fibers of the interpositus (the superior cerebellar peduncle) were reversibly blocked with microinjections of the sodium channel blocker tetrodotoxin. Rabbits performed no conditioned responses during this inactivation training. However, training after inactivation revealed that the rabbits (trained with either conditioned stimulus) had fully learned the response during the previous inactivation training. Cerebellar output, therefore, does not appear to be essential for acquisition of the learned response. This result, coupled with the fact that inactivation of the appropriate region of cerebellum completely prevents learning, provides compelling evidence supporting the hypothesis that the essential memory trace for the classically conditioned eye-blink response is localized within the cerebellum.

Localization of a memory trace in the mammalian brain.

The localization of sites of memory formation within the brain has proven to be a formidable task even for simple forms of learning and memory. In order to localize a particular site of memory formation within the brain, the rabbit eyeblink response was classically conditioned while regions of the cerebellum or red nucleus were temporarily inactivated by microinfusions of the gamma-aminobutyric acid agonist muscimol. Cerebellar inactivation completely blocked learning but had no effect on subsequent learning after inactivation, whereas red nucleus inactivation did not prevent learning but did block the expression of conditioned responses. The site of memory formation for this learned response thus appears to be localized within the cerebellum.