Parallel Fiber Activity Research Articles

Mechanisms underlying input-specific expression of endocannabinoid-mediated synaptic plasticity in the dorsal cochlear nucleus

A hallmark of brain organization is the integration of primary and modulatory pathways by principal neurons. Primary sensory inputs are usually not plastic, while modulatory inputs converging to the same principal neuron can be plastic. However, the mechanisms determining this input-specific expression of synaptic plasticity remain unknown. We investigated this problem in the dorsal cochlear nucleus (DCN), where principal cells integrate primary auditory nerve input with plastic, parallel fiber input. Our previous DCN studies have shown that parallel fiber inputs exhibit short- and long-term plasticities mediated by endocannabinoid signaling. Here we show that auditory nerve inputs to principal cells do not show short- or long-term endocannabinoid-mediated synaptic plasticity. Electrophysiological and electron microscopy studies indicate that input specificity arises from selective expression of presynaptic cannabinoid (CB1) receptors in parallel fiber terminals, but not in auditory nerve terminals. However, pairing of parallel fiber activity with auditory nerve activity elicits plasticity in parallel fiber inputs, thus suggesting a role for synaptic plasticity in multisensory integration.

Purkinje Cell NMDA Receptors Assume a Key Role in Synaptic Gain Control in the Mature Cerebellum

A classic view in cerebellar physiology holds that Purkinje cells do not express functional NMDA receptors and that, therefore, postsynaptic NMDA receptors are not involved in the induction of long-term depression (LTD) at parallel fiber (PF) to Purkinje cell synapses. Recently, it has been demonstrated that functional NMDA receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in mice, reaching full expression levels at ∼2 months after birth. Here, we show that in the mature mouse cerebellum LTD (induced by paired PF and CF activation), but not long-term potentiation (LTP; PF stimulation alone) at PF to Purkinje cell synapses is blocked by bath application of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV). A blockade of LTD, but not LTP, was also observed when the noncompetitive NMDA channel blocker MK-801 was added to the patch-pipette saline, suggesting that postsynaptically expressed NMDA receptors are required for LTD induction. Using confocal calcium imaging, we show that CF-evoked calcium transients in dendritic spines are reduced in the presence of D-APV. This observation confirms that NMDA receptor signaling occurs at CF synapses and suggests that NMDA receptor-mediated calcium transients at the CF input site might contribute to LTD induction. Finally, we performed dendritic patch-clamp recordings from rat Purkinje cells. Dendritically recorded CF responses were reduced when D-APV was bath applied. Together, these data suggest that the late developmental expression of postsynaptic NMDA receptors at CF synapses onto Purkinje cells is associated with a switch toward an NMDA receptor-dependent LTD induction mechanism.

Molecular layer inhibitory interneurons provide feedforward and lateral inhibition in the dorsal cochlear nucleus.

In the outer layers of the dorsal cochlear nucleus, a cerebellum-like structure in the auditory brain stem, multimodal sensory inputs drive parallel fibers to excite both principal (fusiform) cells and inhibitory cartwheel cells. Cartwheel cells, in turn, inhibit fusiform cells and other cartwheel cells. At the microcircuit level, it is unknown how these circuit components interact to modulate the activity of fusiform cells and thereby shape the processing of auditory information. Using a variety of approaches in mouse brain stem slices, we investigated the synaptic connectivity and synaptic strength among parallel fibers, cartwheel cells, and fusiform cells. In paired recordings of spontaneous and evoked activity, we found little overlap in parallel fiber input to neighboring neurons, and activation of multiple parallel fibers was required to evoke or alter action potential firing in cartwheel and fusiform cells. Thus neighboring neurons likely respond best to distinct subsets of sensory inputs. In contrast, there was significant overlap in inhibitory input to neighboring neurons. In recordings from synaptically coupled pairs, cartwheel cells had a high probability of synapsing onto nearby fusiform cells or other nearby cartwheel cells. Moreover, single cartwheel cells strongly inhibited spontaneous firing in single fusiform cells. These synaptic relationships suggest that the set of parallel fibers activated by a particular sensory stimulus determines whether cartwheel cells provide feedforward or lateral inhibition to their postsynaptic targets.

The importance of N-methyl-d-aspartate (NMDA) receptors in subtraction of electrosensory reafference in the dorsal nucleus of skates

The dorsal nucleus of the little skate is a cerebellum-like sensory structure that adaptively filters out predictable electrosensory inputs. The filter's plasticity is mediated by anti-Hebbian associative depression at the synapses between parallel fibers and ascending efferent neurons (AENs). Changes in synaptic strength are indicated by the formation of a cancellation signal which is initiated by co-activation of parallel fibers and AENs, and can be reversed by parallel fiber activity in the absence of AEN activation. In other cerebellum-like sensory structures, the formation of the cancellation signal requires activation of postsynaptic NMDA receptors on the principal neurons. We demonstrate here by immunohistochemistry that the somas and the initial portion of both apical and basal dendrites of the AENs are labeled with antibodies raised against the NR1 subunit of NMDA receptors from a South American electric fish. In in vivo physiological experiments, we show that the formation of the cancellation signal induced by coupling an electrosensory stimulus to ventilatory movements or direct parallel fiber stimulation is blocked when either of the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid (APV) or MK801 is injected into the molecular layer above the recorded AEN. Blocking NMDA receptors prevented formation of a cancellation signal in 79% (15/19; APV) and 60% (3/5; MK801) of the AENs. This blockage was reversible in 40% (6/15) of the AENs after APV removal. Thus, in the dorsal nucleus, the activity-dependent, long-lasting but reversible change in synaptic strength of the parallel fiber-AEN synapses appears to be an NMDA receptor-dependent process.

Comparative evolutionary computational analysis of cerebellar purkinje cell structure and function

Most computational efforts to understand the functional significance of neurons and networks are based on data obtained in a single species. In this report, we describe the use of comparative computational analysis to test a specific hypothesis arising from our computational studies of the mammalian cerebellum. Specifically, our previous experimental and model-based investigations have suggested that the inhibitory basket connections to the Purkinje cell (PC) serve functionally to limit the influence of parallel fiber activity on somatic output [1]. Because basket connections are not found in non-mammalian vertebrates, we hypothesized that either the electrical properties and/or some aspect of PC relationships to the parallel fibers would differ between mammals and non-mammals.

Noradrenergic Control of Associative Synaptic Plasticity by Selective Modulation of Instructive Signals

Synapses throughout the brain are modified through associative mechanisms in which one input provides an instructive signal for changes in the strength of a second coactivated input. In cerebellar Purkinje cells, climbing fiber synapses provide an instructive signal for plasticity at parallel fiber synapses. Here, we show that noradrenaline activates alpha2-adrenergic receptors to control short-term and long-term associative plasticity of parallel fiber synapses. This regulation of plasticity does not reflect a conventional direct modulation of the postsynaptic Purkinje cell or presynaptic parallel fibers. Instead, noradrenaline reduces associative plasticity by selectively decreasing the probability of release at the climbing fiber synapse, which in turn decreases climbing fiber-evoked dendritic calcium signals. These findings raise the possibility that targeted presynaptic modulation of instructive synapses could provide a general mechanism for dynamic context-dependent modulation of associative plasticity.

Genetic analyses of mechanisms for memory loss in Caenorhabditis elegans

A hallmark of brain organization is the integration of primary and modulatory pathways by principal neurons. Primary sensory inputs are usually not plastic, while modulatory inputs converging to the same principal neuron can be plastic. However, the mechanisms determining this input-specific expression of synaptic plasticity remain unknown. We investigated this problem in the dorsal cochlear nucleus (DCN), where principal cells integrate primary auditory nerve input with plastic, parallel fiber input. Our previous DCN studies have shown that parallel fiber inputs exhibit short- and long-term plasticities mediated by endocannabinoid signaling. Here we show that auditory nerve inputs to principal cells do not show short- or long-term endocannabinoid-mediated synaptic plasticity. Electrophysiological and electron microscopy studies indicate that input specificity arises from selective expression of presynaptic cannabinoid (CB1) receptors in parallel fiber terminals, but not in auditory nerve terminals. However, pairing of parallel fiber activity with auditory nerve activity elicits plasticity in parallel fiber inputs, thus suggesting a role for synaptic plasticity in multisensory integration.

A Positive Feedback Signal Transduction Loop Determines Timing of Cerebellar Long-Term Depression

Synaptic activity produces short-lived second messengers that ultimately yield a long-term depression (LTD) of cerebellar Purkinje cells. Here, we test the hypothesis that these brief second messenger signals are translated into long-lasting biochemical signals by a positive feedback loop that includes protein kinase C (PKC) and mitogen-activated protein kinase. Histochemical "epistasis" experiments demonstrate the reciprocal activation of these kinases, and physiological experiments--including the use of a light-activated protein kinase--demonstrate that such reciprocal activation is required for LTD. Timed application of enzyme inhibitors reveals that this positive feedback loop causes PKC to be active for more than 20 min, allowing sufficient time for LTD expression. Such regenerative mechanisms may sustain other long-lasting forms of synaptic plasticity and could be a general mechanism for prolonging signal transduction networks.

Morphological analysis of the mormyrid cerebellum using immunohistochemistry, with emphasis on the unusual neuronal organization of the valvula

This study used immunohistochemistry, Golgi impregnation, and electron microscopy to examine the circuitry of the cerebellum of mormyrid fish. We used antibodies against the following antigens: the neurotransmitters glutamate and gamma-aminobutyric acid (GABA); the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N-methyl-D-aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1alpha and mGluR2/3; the intracellular signaling molecules calcineurin and calcium calmodulin kinase IIalpha (CAMKIIalpha); and the receptor for inositol triphosphate (IP3RIalpha). Purkinje cells are immunoreactive to anti-IP3R1alpha, anticalcineurin, and anti-mGluR1alpha. Cerebellar efferent cells (eurydendroid cells) are anticalretinin and anti-NR1 positive in the valvula but not in the corpus and caudal lobe. In contrast, climbing fibers are anticalretinin and anti-NR1 immunopositive in the corpus and caudal lobe but not in the valvula. Purkinje cells, Golgi cells, and stellate cells are GABA positive, whereas efferent cells are glutamate positive. Unipolar brush cells are immunoreactive to anti-mGluR2/3, anticalretinin, and anticalbindin. We describe a "new" cell type in the mormyrid valvula, the deep stellate cell. These cells are GABA, calretinin, and calbindin positive. They are different from superficial stellate cells in having myelinated axons that terminate massively with GAD- and gephyrin-positive terminals on the cell bodies and proximal dendrites of efferent cells. We discuss how the valvula specializations described here may act in concert with the palisade pattern of Purkinje cell dendrites for analyzing spatiotemporal patterns of parallel fiber activity.

Determinants of pattern recognition by cerebellar Purkinje cells

Many theories of cerebellar function assume that longterm depression (LTD) of parallel fiber (PF) synapses enables Purkinje cells (PCs) to learn to recognize PF activity patterns. According to the classic view, a PC can store and learn to distinguish PF activity patterns that have been presented repeatedly together with climbing fibre (CF) input to the cell. The resulting LTD of the PF synapses is often assumed to lead to a decreased rate of PC simple spike firing, a reduction in the inhibition of their target neurons in the deep cerebellar nuclei and thus an increased output from the cerebellum. We have recently shown by combining computer simulations with electrophysiological recordings in slices and in awake behaving mice that the readout of learned patterns in PCs may operate in a fundamentally different way. Our simulations and experiments predict that the best criterion to distinguish between learned and novel patterns is the duration of a pause in firing that occurs after presentation of a pattern, with shorter pauses in response to learned patterns [1].

Local circuitry in the anterior caudal lobe of the mormyrid cerebellum: A study of intracellular recording and labeling

The caudal lobe of the mormyrid cerebellum includes the anterior portion, which is associated with the lateral line and eighth nerve senses, and the posterior portion, which is associated with the electrosense. This study examines the physiology and morphology of cells in the anterior portion in slice preparations. Two subtypes of Purkinje cells, efferent cells and stellate cells, are described. Multipolar Purkinje cells are located in the central region of the lobe, with large, multipolar, spiny dendrites and locally ending axons. Small Purkinje cells are located along its anterior border with the eminentia granularis anterior (EGa), with spiny dendrites in the molecular region. Axons of some small Purkinje cells end locally, whereas axons of other such cells are cut at the surface of the slices, suggesting that they project outside the lobe. Efferent cells are also distributed along the border with EGa. These cells have thin, smooth dendrites in the molecular region, and their axons are cut at the sliced surface. Stellate cells have thin, smooth dendrites and locally terminating axons. Physiologically, all types of cells respond to parallel fiber activation, but only multipolar Purkinje cells showed characteristic all-or-none climbing fiber responses. Although the majority of Purkinje cells fire a single type of spikes at resting level, a subset of small Purkinje cells fire small, narrow and large, broad spikes. Thus, the anterior caudal lobe of the mormyrid cerebellum is different from the mammalian cerebellum in having different subtypes of Purkinje cells and local termination of many Purkinje cell axons.

Control of neuronal firing by dynamic parallel fiber feedback:implications for electrosensory reafference suppression

The cancellation of self-generated components of sensory inputs is a key function of sensory feedback pathways. In many systems, cerebellar parallel fiber feedback mediates this cancellation through anti-Hebbian plasticity, resulting in the generation of a negative image of the reafferent inputs. Parallel fiber feedback involves direct excitation and disynaptic inhibition as well as synaptic plasticity on multiple time scales. How the dynamics of these processes interact with anti-Hebbian plasticity to shape synaptic inputs and provide a cancellation mechanism remains unclear. In the present study, we investigated the influence of parallel fiber feedback onto pyramidal neurons of the electrosensory lateral line lobe (ELL) in weakly electric fish under open loop conditions. We mimicked naturalistic parallel fiber inputs in an ELL brain slice by implementing an experimentally based model of this synaptic pathway using dynamic clamp. We showed that as parallel fiber activity increases, the effective input to ELL pyramidal neurons changes from net excitation to net inhibition, resulting in a non-monotonic firing response. Using a model neuron, we found that this robust non-monotonic response is due to a shift from balanced excitation and inhibition at low parallel fiber input rates, to dominant inhibition at high input rates. We then showed that this non-monotonic response provides a simple basis for negative image generation. Through changes in the mean activation rate of parallel fibers, the feedback can switch roles between enhancement and suppression of sensory inputs in a manner that is directly determined by the slope of the non-monotonic response curve.

Synaptic Plasticity and Calcium Signaling in Purkinje Cells of the Central Cerebellar Lobes of Mormyrid Fish

Climbing fiber (CF)-evoked calcium transients play a key role in plasticity at parallel fiber (PF) to Purkinje cell synapses in the mammalian cerebellum. Whereas PF activation alone causes long-term potentiation (LTP), coactivation of the heterosynaptic CF input, which evokes large dendritic calcium transients, induces long-term depression (LTD). This unique type of heterosynaptic interaction is a hallmark feature of synaptic plasticity in mammalian Purkinje cells. Purkinje cells in the cerebellum of mormyrid electric fish are characterized by a different architecture of their dendritic trees and by a more pronounced separation of CF and PF synaptic contact sites. We therefore examined the conditions for bidirectional plasticity at PF synapses onto Purkinje cells in the mormyrid cerebellum in vitro. PF stimulation at elevated frequencies induces LTP, whereas LTD results from PF stimulation at enhanced intensities and depends on dendritic calcium influx and metabotropic glutamate receptor type 1 activation. LTD can also be observed after pairing of low intensity PF stimulation with CF stimulation. Using a combination of whole-cell patch-clamp recordings and fluorometric calcium imaging, we characterized calcium transients in Purkinje cell dendrites. CF activation elicits calcium transients not only within the CF input territory (smooth proximal dendrites) but also within the PF input territory (spiny palisade dendrites). Paired PF and CF activation elicits larger calcium transients than stimulation of either input alone. A major source for dendritic calcium signaling is provided by P/Q-type calcium channels. Our data show that despite the spatial separation between the two inputs CF activity facilitates LTD induction at PF synapses.

Timing dependence of the induction of cerebellar LTD

Long-term depression (LTD) of the granule cell to Purkinje cell synapse is thought to contribute to motor learning. According to the Marr/Albus/Ito model, sensory inputs drive granule cells to fire, thereby exciting Purkinje cells and influencing motor output. Inappropriate motor output causes neurons in the inferior olive to fire and activate Purkinje cells via the powerful climbing fiber (CF) synapse. CF activity is an error signal and the association of CF and granule cell parallel fiber (PF) activity results in LTD at coactivated PF synapses. Here we examine the timing dependence of LTD by using an induction protocol consisting of a single CF activation paired with a PF burst, with the relative timing of CF and PF activation systematically varied. LTD was most prominent when PF activation occurred before CF activation. A plot of LTD magnitude as a function of PF and CF timing was well approximated by a fit in which LTD peaked for PF activity ∼80ms before CF activation and the half width was ∼300ms. This indicates that the timing dependence of LTD is well suited to allow a CF to depress preceding PF inputs that generated inappropriate motor outputs. We also find that LTD induction and endocannabinoid release have a similar dependence on PF and CF timing. This suggests that the properties of endocannabinoid release may underlie the timing dependence of some forms of motor learning.

Cerebellar LTD and Pattern Recognition by Purkinje Cells

SummaryMany theories of cerebellar function assume that long-term depression (LTD) of parallel fiber (PF) synapses enables Purkinje cells to learn to recognize PF activity patterns. We have studied the LTD-based recognition of PF patterns in a biophysically realistic Purkinje-cell model. With simple-spike firing as observed in vivo, the presentation of a pattern resulted in a burst of spikes followed by a pause. Surprisingly, the best criterion to distinguish learned patterns was the duration of this pause. Moreover, our simulations predicted that learned patterns elicited shorter pauses, thus increasing Purkinje-cell output. We tested this prediction in Purkinje-cell recordings both in vitro and in vivo. In vitro, we found a shortening of pauses when decreasing the number of active PFs or after inducing LTD. In vivo, we observed longer pauses in LTD-deficient mice. Our results suggest a novel form of neural coding in the cerebellar cortex.

Short- and long-term depression of rat cerebellar parallel fibre synaptic transmission mediated by synaptic crosstalk.

Cerebellar granule cell to Purkinje cell synapses have been reported to show plasticity when stimulating the parallel fibres, but not when granule cell axons are stimulated in the granular layer. The latter absence of plasticity has been attributed either to the synapses made by ascending granule cell axons lacking some feature needed to evoke plasticity, such as metabotropic glutamate receptors, or to spillover of glutamate between adjacent active synapses being essential for plasticity to occur and having a greater effect for parallel fibre stimulation than for granular layer stimulation. Here we show that both long-term depression (LTD) and endocannabinoid plasticity can depend on interaction between adjacent synapses. These results focus attention on the need to characterize the spatial pattern of parallel fibre activity evoked by physiological stimuli, in order to assess the conditions under which synaptic plasticity will occur in vivo.

Nitric Oxide Regulates Input Specificity of Long-Term Depression and Context Dependence of Cerebellar Learning

Recent studies have shown that multiple internal models are acquired in the cerebellum and that these can be switched under a given context of behavior. It has been proposed that long-term depression (LTD) of parallel fiber (PF)–Purkinje cell (PC) synapses forms the cellular basis of cerebellar learning, and that the presynaptically synthesized messenger nitric oxide (NO) is a crucial “gatekeeper” for LTD. Because NO diffuses freely to neighboring synapses, this volume learning is not input-specific and brings into question the biological significance of LTD as the basic mechanism for efficient supervised learning. To better characterize the role of NO in cerebellar learning, we simulated the sequence of electrophysiological and biochemical events in PF–PC LTD by combining established simulation models of the electrophysiology, calcium dynamics, and signaling pathways of the PC. The results demonstrate that the local NO concentration is critical for induction of LTD and for its input specificity. Pre- and postsynaptic coincident firing is not sufficient for a PF–PC synapse to undergo LTD, and LTD is induced only when a sufficient amount of NO is provided by activation of the surrounding PFs. On the other hand, above-adequate levels of activity in nearby PFs cause accumulation of NO, which also allows LTD in neighboring synapses that were not directly stimulated, ruining input specificity. These findings lead us to propose the hypothesis that NO represents the relevance of a given context and enables context-dependent selection of internal models to be updated. We also predict sparse PF activity in vivo because, otherwise, input specificity would be lost.

In vivo calcium imaging from genetically specified target cells in mouse cerebellum

Genetically encoded fluorescent calcium indicator proteins provide the potential to monitor activity from genetically specified target cells without a need for single cell resolution. Here we report the use of transgenic mice expressing the fluorescent calcium indicator protein GCaMP2 in cerebellar granule cells to image parallel fiber activity transcranially in vivo. We demonstrated reliable measurements of calcium transients from beams of parallel fibers in response to electrical stimulation in the molecular layer through the intact skull. These parallel fiber calcium transients differed from intrinsic postsynaptic autofluorescence signals in their faster kinetics and resistance to blockers of synaptic transmission. Finally, we used 2P laser-scanning microscopy to demonstrate reliable measurements of calcium transients from beams of parallel fibers at high spatial resolution in living mice. We expect that genetically targeted fluorescent calcium indicator proteins along with optical imaging techniques will be instrumental for the construction of macroscopic and microscopic maps of the function of specific brain circuits.

Postsynaptic inositol 1,4,5-trisphosphate signaling maintains presynaptic function of parallel fiber–Purkinje cell synapses via BDNF

The maintenance of synaptic functions is essential for neuronal information processing, but cellular mechanisms that maintain synapses in the adult brain are not well understood. Here, we report an activity-dependent maintenance mechanism of parallel fiber (PF)-Purkinje cell (PC) synapses in the cerebellum. When postsynaptic metabotropic glutamate receptor (mGluR) or inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically inhibited in vivo, PF-PC synaptic strength decreased because of a decreased transmitter release probability. The same effects were observed when PF activity was inhibited in vivo by the suppression of NMDA receptor-mediated inputs to granule cells. PF-PC synaptic strength similarly decreased after the in vivo application of an antibody against brain-derived neurotrophic factor (BDNF). Furthermore, the weakening of synaptic connection caused by the blockade of mGluR-IP(3) signaling was reversed by the in vivo application of BDNF. These results indicate that a signaling cascade comprising PF activity, postsynaptic mGluR-IP(3) signaling and subsequent BDNF signaling maintains presynaptic functions in the mature cerebellum.

Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells

Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long-term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors (mGluR1) and CB1 receptors. This depression is induced by postsynaptic increases in Ca(2+) concentration ([Ca(2+)]) and is limited to activated synapses. To understand how synapse-specific plasticity is induced by diffusible second messengers in aspiny dendrites, we examined diffusion of Ca(2+) and small molecules within stellate cell dendrites. Activation of a single parallel fiber opened CP-AMPARs, generating long-lived Ca(2+) transients that were confined to submicron dendritic stretches. The diffusion of Ca(2+) was severely retarded due to interactions with parvalbumin and a general restriction of small molecule mobility. Thus stellate cell dendrites spatially restrict signaling cascades that lead from CP-AMPAR activation to endocannabinoid production and trigger the selective regulation of active synapses.