Formation of new cortico-rubral synapses as a possible mechanism for classical conditioning mediated by the red nucleus in cat

1.) By extracellular and intracellular recordings of the red nucleus (RN) cell activity, we investigated enhancement of signaling effectiveness at the cortico-rubral synapses underlying the establishment of classical conditioning mediated by RN in the cat. The classical conditioning of forelimb flexion was produced by pairing the conditioned stimulus (CS) to the cerebral peduncle (CP) with the unconditioned stimulus (US) to the forelimb skin at an interval of 100 msec for about a week. 2.) The increased responsiveness of RN cells to the CS was correlated with acquisition of the conditioned forelimb flexion, i.e. RN cells responded to the CS with higher firing probability in the animals which received the paired conditioning than those in the animals which received the CS alone or pairing of the CS and the US at random intervals or those in the naive animals which did not receive any training. 3.) Monosynaptic excitation of RN cells in response to the single pulse to CP was most enhanced in the animals which received the paired conditioning. By contrast, response of RN cells, as well as the behavioral response, induced by stimulation of the cerebellar interpositus nucleus (IP) was not enhanced after the paired conditioning. The difference between the responses to the stimulation of CP and IP suggested that the primary site of neuronal change is the cortico-rubral synapses. 4.) In the animals that received the paired conditioning, the excitatory postsynaptic potentials (EPSPs) induced by stimulation of CP had fast-rising components superimposed on the normal slow-rising EPSPs. On the other hand, most of the CP-EPSPs recorded in the naive animals showed a slow time course. The slow time course of the CP-EPSPs has been attributed to the peripheral localization of the cortico-rubral synapses on the dendrites of RN cells. 5.) The electrotonic length of RN cells in the animals which received the paired conditioning was not shorter than that in the naive animals. Therefore, it was suggested that the appearance of the fast-rising component in the CP-EPSPs is cause by formation of the new cortico-rubral synapses on proximal portions of the soma-dendritic membrane of RN cells. 6.) Since it has been established that new synapses formed by collateral sprouting are retained for more than several months, the formation of new synaptic connections could underlie long-lasting behavioral modification.     

剌激中缝背核压抑大鼠小脑浦肯野细胞对苔状...

In anaesthetized and paralyzed rats, the effect of dorsal raphe (DR) conditioning stimulation on cerebellar Purkinje cell (PC) responses to mossy fiber and climbing fiber inputs were examined. The main results are as follows: (1) Stimulation of cerebral sensorimotor cortex elicits widespread activation of mossy and climbing fiber inputs to PCs in contralateral VI and VII lobules of the cerebellum and generates two kinds of evoked responses, i.e. the simple spike (SS) and the complex spike (CS) responses with respectively a latency 8-25 and 12-30 ms. (2) These PC responses could be markedly suppressed by stimulation of DR at intensities which by themselves were subthreshold for directly affecting PC's spontaneous SS and CS activities. (3) This DR-induced depressive effects on evoked PC's SS and CS excitations could be attenuated or blocked by systemic administration of 5-HT receptor blocker methysergide. These results demonstrate that serotonergic fiber input from DR can suppress the efficacy of mossy and climbing fiber synaptic action on PC, or decrease the responsiveness of PC itself to afferent synaptic action. The findings of this study also suggest that the raphe-cerebellar serotonergic fiber afferent system may be involved in some of the important neuronal processing in the cerebellum.     

Potentiation of mossy fiber EPSCs in the cerebellar nuclei by NMDA receptor activation followed by postinhibitory rebound current

Behavioral and computational studies predict that synaptic plasticity of excitatory mossy fiber inputs to cerebellar nuclear neurons is required for associative learning, but standard tetanization protocols fail to potentiate nuclear cell EPSCs in mouse cerebellar slices. Nuclear neurons fire action potentials spontaneously unless strongly inhibited by Purkinje neurons, raising the possibility that plasticity-triggering signals in these cells differ from those at classical Hebbian synapses. Based on predictions of neuronal activity during delay eyelid conditioning, we developed quasi-physiological induction protocols consisting of high-frequency mossy fiber stimulation and postsynaptic hyperpolarization. Robust, NMDA receptor-dependent potentiation of nuclear cell EPSCs occurred with protocols including a 150-250 ms hyperpolarization in which mossy fiber stimulation preceded a postinhibitory rebound depolarization. Mossy fiber stimulation potentiated EPSCs even when postsynaptic spiking was prevented by voltage-clamp, as long as rebound current was evoked. These data suggest that Purkinje cell inhibition guides the strengthening of excitatory synapses in the cerebellar nuclei.     

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.     

Specific Relationship between Excitatory Inputs and Climbing Fiber Receptive Fields in Deep Cerebellar Nuclear Neurons

Many mossy fiber pathways to the neurons of the deep cerebellar nucleus (DCN) originate from the spinal motor circuitry. For cutaneously activated spinal neurons, the receptive field is a tag indicating the specific motor function the spinal neuron has. Similarly, the climbing fiber receptive field of the DCN neuron reflects the specific motor output function of the DCN neuron. To explore the relationship between the motor information the DCN neuron receives and the output it issues, we made patch clamp recordings of DCN cell responses to tactile skin stimulation in the forelimb region of the anterior interposed nucleus in vivo. The excitatory responses were organized according to a general principle, in which the DCN cell responses became stronger the closer the skin site was located to its climbing fiber receptive field. The findings represent a novel functional principle of cerebellar connectivity, with crucial importance for our understanding of the function of the cerebellum in movement coordination.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal X-irradiation.

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilpateral forelimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 +/- 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Thermostimulation-Induced Modulation of Conditioned Reflex Movement-Related Reactions of Cortical Neurons of the Cat

In awake cats trained to perform a food-procuring conditioned operant reflex (placing movement), we studied impulse reactions of 86 neurons of the motor cortex (field 4) related to realization of the above movements. As conditioning stimuli (CS) initiating the reflex, we used either non-noxious electrocutaneous stimulation (ECS) of the contralateral forelimb or an acoustic stimulus (sound click). Impulsation of cortical neurons was recorded under conditions of (i) isolated presentation of the CS (control), (ii) presentation of the CS (either ECS or acoustic stimulus) combined with thermostimulation (heating with a miniature electric bulb) of the skin of the working forelimb, and (iii) the same, but with stimulation of the resting forelimb. When we recorded spike activity of neurons within the projection motor zone of the resting limb subjected to ESC, alternating thermostimulation of both forelimbs resulted in considerable intensification and an increase in the duration of neuronal responses, especially in cases where thermostimulation was applied to the working limb ipsilateral to the recording site (a two- to threefold increase). When spike reactions were recorded within the motor cortex of the working forelimb, thermostimulation resulted in a considerable increase in the intensity of these reactions and a decrease in their latency, but only when such stimulation was applied to the working forelimb. Thermostimulation of the resting (ipsilateral, subjected to ESC) limb evoked opposite effects (the intensity of neuronal reactions dropped). In both situations, placing movements remained within the control limits. When sound click was used as a distant CS, thermostimulation of the working limb enhanced neuronal responses, increased their duration by 50-100%, and also increased the time of forestalling of the movement initiation by spike neuronal reactions. Thermostimulation of the resting forelimb in this situation also suppressed neuronal reactions. We conclude that foreign stimulations directed toward modifications of the receptor model of the operant reflex experimental situation formed in the animal result in a decrease in the intensity of the spike responses of field-4 neurons and prolongation of the latencies of these responses, while stimulations promoting the inflow of afferent information to the cortical projection of the working limb evoke opposite effects, an increase in the intensity of neuronal spike responses and a decrease in their latencies.     

Neuron activities of the motor cortex during conditioned eye blink reflex in rabbits (author's transl)]

Effects of the tone (CS) on neurons of the motor cortex were investigated in naive, pseudoconditioned, and conditioned rabbits. Conditioning to eye blink reflex was made by a combination of CS and air puff (US). Effects of electrical stimulation of the subcortical structures were also observed on the cortical neurons associated with the conditioned reflex. The results were as follows. (1) Proportion of neurons which significantly increased the firing rate in response to the CS, type E, was higher in the conditioned group than in other two groups. On the other hand, no group difference was found in the proportion of neurons which significantly decreased the firing rate to the stimulus, type I. (2) Most of the type E neurons in the conditioned rabbits began to fire at latencies of about 50 to 100 msec after the CS, preceding about 200 msec to the appearance of the peripheral conditioned responses (EMG). (3) Most of the type E neurons in the conditioned animals were more easily affected by stimulation of the medial geniculate body and the brain stem reticular formation. Based on the results mentioned above, it is concluded that in the rabbits conditioned to the eye blink reflex, excitability of neurons in the motor cortex is enhanced by the tone (CS), and by electrical stimulation to the medial geniculate body and the brain stem reticular formation.     

Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex

We show that information flow through the adult cerebellar cortex, from the mossy fiber input to the Purkinje cell output, is controlled by furosemide-sensitive, diazepam- and neurosteroid-insensitive GABA(A) receptors on granule cells, which are activated both tonically and by GABA spillover from synaptic release sites. Tonic activation of these receptors contributes a 3-fold larger mean inhibitory conductance than GABA released synaptically by high-frequency stimulation. Tonic and spillover inhibition reduce the fraction of granule cells activated by mossy fiber input, generating an increase of coding sparseness, which is predicted to improve the information storage capacity of the cerebellum.     

Cerebellar Cortex Granular Layer Interneurons in the Macaque Monkey Are Functionally Driven by Mossy Fiber Pathways through Net Excitation or Inhibition

The granular layer is the input layer of the cerebellar cortex. It receives information through mossy fibers, which contact local granular layer interneurons (GLIs) and granular layer output neurons (granule cells). GLIs provide one of the first signal processing stages in the cerebellar cortex by exciting or inhibiting granule cells. Despite the importance of this early processing stage for later cerebellar computations, the responses of GLIs and the functional connections of mossy fibers with GLIs in awake animals are poorly understood. Here, we recorded GLIs and mossy fibers in the macaque ventral-paraflocculus (VPFL) during oculomotor tasks, providing the first full inventory of GLI responses in the VPFL of awake primates. We found that while mossy fiber responses are characterized by a linear monotonic relationship between firing rate and eye position, GLIs show complex response profiles characterized by “eye position fields” and single or double directional tunings. For the majority of GLIs, prominent features of their responses can be explained by assuming that a single GLI receives inputs from mossy fibers with similar or opposite directional preferences, and that these mossy fiber inputs influence GLI discharge through net excitatory or inhibitory pathways. Importantly, GLIs receiving mossy fiber inputs through these putative excitatory and inhibitory pathways show different firing properties, suggesting that they indeed correspond to two distinct classes of interneurons. We propose a new interpretation of the information flow through the cerebellar cortex granular layer, in which mossy fiber input patterns drive the responses of GLIs not only through excitatory but also through net inhibitory pathways, and that excited and inhibited GLIs can be identified based on their responses and their intrinsic properties.     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning

We recorded neurons from the hippocampus of freely behaving rats during an auditory fear conditioning task. Rats received either paired or unpaired presentations of an auditory conditioned stimulus (CS) and an electric shock unconditioned stimulus (US). Hippocampal neurons (place and theta cells) acquired responses to the auditory CS in the paired but not in the unpaired group. After CS-US pairing, rhythmic firing of theta cells became synchronized to the onset of the CS. Conditioned responses of place cells were gated by their location-specific firing, so that after CS-US pairing, place cells responded to the CS only when the rat was within the cell's place field. These findings may help to elucidate how the hippocampus contributes to context-specific memory formation during associative learning.     

Conditioned augmentation of natural killer cell activity. Independence from nociceptive effects and dependence on interferon-beta

Injection of mice with 20 micrograms polyinosinic: polycytidylic acid (Poly I:C) after exposure to camphor odor results in a conditioned augmentation of natural killer cell (NK) activity. In this study, we show that the conditioned response is not the result of nociceptive stimulation and that interferon-beta (IFN), but not IFN-alpha can replace Poly I:C as the unconditioned stimulus (US). Two conditioned stimuli (CS) were used with equivalent results. A combination CS consisting of a novel taste (saccharin) and a 125 mg/kg injection of LiCl that induces gastric upset was paired with a Poly I:C or IFN-beta (1 X 10(4) IU) injection. This resulted in an augmentation of NK activity when the conditioned animals were reexposed to the saccharin-LiCl CS. In addition, an identical conditioned response was elicited when a camphor odor CS was paired with either of these US. To test whether the conditioned response might be an artifact not detected by our controls, a mock conditioning experiment was performed, which assessed the differential effect of multiple exposures to the saccharin-LiCl CS without a CS/US pairing. The mock conditioned group was significantly suppressed relative to saline treated controls, whereas the mock nonconditioned group and the mock conditioned group that was not reexposed to the CS after conditioning did not show significant suppression. This indicates that the augmentation observed in the conditioned group after CS/US pairing was not the result of exposure to the CS itself. Small doses of Poly I:C (5 micrograms or 2.5 micrograms) given on days 3 and 5 (or on day 5 only) to boost NK activity had the effect of increasing the magnitude of the conditioned response measured on day 6. In addition, an identical conditioned response was observed when the interval between the CS/US pairing and the later CS exposures was changed, which places the test for the conditioned response either on day 6 (CS given on days 3 and 5) or day 10 (CS given on days 7 and 9). These results show that the observed conditioned enhancement of NK activity in conditioned animals is not caused by any nociceptive properties of the CS itself and is dependent on the IFN-beta produced after Poly I:C injection in the conditioned paradigm.     

Analysis of spike discharge of a neuron population in the cat motor cortex during a conditioned change of posture reflex

Spike responses of area 4 neurons in the projection area of the contralateral forelimb to acoustic stimulation (1 sec), which became the conditioned stimulus after training, and to dropping of the platform beneath the test limb, which served as reinforcing stimulus, were studied in trained and untrained cats. Responses only of those neurons which were activated during a passive movement caused by dropping of the platform were studied. In trained animals the number of these neurons which responded to the conditioned stimulus if a reflex occurred was 100%, and in the absence of conditioned-reflex movements to the conditioned stimulus it was 70%, much greater than the number of neurons responding to the same acoustic stimulus in untrained animals (45%). On peristimulus histograms of responses of the test neuron population in untrained and trained animals to acoustic stimulation (in the absence of movements) only the initial spike response with a latent period of under 50 msec and a duration of up to 100 msec could be clearly distinguished. In the presence of reflex movement multicomponent spike responses were observed: an initial spike response and early and late after-responses linked with performance of conditioned-reflex limb flexion. Early after-responses 100–200 msec in duration, appearing after a latent period of 100–150 msec, were linked to the time of application of the conditioned stimulus, whereas the appearance and duration of late after-responses were determined by the time of onset of conditioned-reflex movement. The magnitude of the neuronal response to reinforcement in trained animals does not depend on the appearance of the conditioned movement.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 93–102, January–February, 1985.     

Neural mechanisms of classical conditioning in mammals

Evidence supports the view that 'memory traces' are formed in the hippocampus and in the cerebellum in classical conditioning of discrete behavioural responses. In the hippocampus learning results in long-lasting increases in excitability of pyramidal neurons that resemble the phenomenon of long-term potentiation. Although it plays a role in certain aspects of conditioning, the hippocampus is not necessary for learning and memory of the basic conditioned responses. The cerebellum and its associated brain-stem circuitry, on the other hand, does appear to be essential (necessary and sufficient) for learning and memory of the conditioned response. Evidence to date supports the view that mossy fibre convey conditioned stimulus information and that climbing fibres conveys the critical 'reinforcement' information to the cerebellum and that 'memory traces' appear to be formed in cerebellar cortex and interpositus nucleus.     

Activity of neurons in the pedunculopontine nucleus during defensive instrumental conditioning

The activity of 109 neurons in the compact and diffuse parts of the pedunculopontine tegmental nucleus (PPTg) was recorded in freely moving rabbits during the acquisition and actualization of the defensive instrumental conditioned reflex. It was found that 47% of the recorded neurons responded to a conditioned stimulus (CS). This finding suggests the involvement of the PPTg in the instrumental conditioning. A significant prevalence of the excitatory conditioned responses to the CS suggests the predominantly activating influence of the PPTg on its projection structures during conditioning. The neuronal responses to the CS were classified into several basic patterns reflecting stimulus effects, the structure of the behavioral act, and the nature of the reinforcement. They indicated the involvement of the PPTg in attention, motor learning, and reinforcement. A significant decrease in the reactivity of the CS of the PPTg neurons as a result of learning specialization was shown. The revealed differences in the associative and reactive properties (with respect to CS) between the neurons of the compact and diffuse PPTg parts testify to the functional heterogeneity of this structure and suggest the leading role of the cholinergic compact part of the PPTg in the instrumental defensive conditioning. Thus, the obtained evidence suggests the involvement of the PPTg in the mechanisms of attention and acquisition of the active defensive motor conditioning.     

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

Extension of the CS past the US can facilitate conditioning of the rabbit's nictitating membrane response

Five experiments were conducted in which the onset of a tone conditioned stimulus (CS) preceded the unconditioned stimulus (US) by 500 ms. Across experiments, the offset of the CS was extended past the offset of the US by values ranging from 0 ms to 40000 ms. Extensions of the CS of 2000 ms or greater produced acquisition of a conditioned response (CR) that was as fast or faster than in the no-extension condition (0 ms). While extension of a forward tone CS after the US enhanced excitatory conditioning, insertion of another CS (light) in a purely backward relationship with the US passed only a retardation test, indicative of latent inhibition, and not a summation test needed for conditioned inhibition. The results add to the evidence that excitatory and inhibitory processes are both engaged following US offset. Alternative theories of CS processing are discussed.