Bidirectional modulation of deep cerebellar nuclear cells revealed by optogenetic manipulation of inhibitory inputs from Purkinje cells.

In the mammalian cerebellum, deep cerebellar nuclear (DCN) cells convey all information from cortical Purkinje cells (PCs) to premotor nuclei and other brain regions. However, how DCN cells integrate inhibitory input from PCs with excitatory inputs from other sources has been difficult to assess, in part due to the large spatial separation between cortical PCs and their target cells in the nuclei. To circumvent this problem we have used a Cre-mediated genetic approach to generate mice in which channelrhodopsin-2 (ChR2), fused with a fluorescent reporter, is selectively expressed by GABAergic neurons, including PCs. In recordings from brain slice preparations from this model, mammalian PCs can be robustly depolarized and discharged by brief photostimulation. In recordings of postsynaptic DCN cells, photostimulation of PC axons induces a strong inhibition that resembles these cells' responses to focal electrical stimulation, but without a requirement for the glutamate receptor blockers typically applied in such experiments. In this optogenetic model, laser pulses as brief as 1 ms can reliably induce an inhibition that shuts down the spontaneous spiking of a DCN cell for ∼50 ms. If bursts of such brief light pulses are delivered, a fixed pattern of bistable bursting emerges. If these pulses are delivered continuously to a spontaneously bistable cell, the immediate response to such photostimulation is inhibitory in the cell's depolarized state and excitatory when the membrane has repolarized; a less regular burst pattern then persists after stimulation has been terminated. These results indicate that the spiking activity of DCN cells can be bidirectionally modulated by the optically activated synaptic inhibition of cortical PCs.

Synaptic dynamics and long-term plasticity at synapses of Purkinje cells onto neighboring Purkinje cells of a mormyrid fish: a dual cell recording study.

The input synapses of cerebellar Purkinje cells (PCs) have been extensively studied and much has been learned about their dynamics, plasticity and functionality. In contrast there is limited information available about PC output synapses. This study uses dual cell recording methods to investigate synaptic dynamics and plasticity at individual PC synapses onto neighboring PCs in in vitro preparations of the mormyrid cerebellum. This synaptic connectivity may be strong or weak. For strong connections, inhibitory postsynaptic potentials (IPSPs) or currents (IPSCs) are synchronized with the action potentials of the presynaptic cell. For weak connections, however, the pre- and postsynaptic potentials are no longer synchronized, and presynaptic burst firing at intraburst rates of ∼50 Hz or higher is required to reliably induce the postsynaptic inhibition. A depression of this postsynaptic inhibition was observed for both types of connectivity following repeated presynaptic bursts, which was subsequently largely reversed following pairings of the presynaptic burst-induced IPSPs/IPSCs with evoked burst firing of the postsynaptic cell. Moreover, the original postsynaptic depression was found to be either augmented or reversed depending on the temporal order of each pair of additional pre- and postsynaptic cell activations, hence demonstrating a reversible and spike timing-dependent plasticity (STDP) at this synapse.

Functional circuitry of a unique cerebellar specialization: the valvula cerebelli of a mormyrid fish.

The valvula cerebelli of the mormyrid electric fish is a useful site for the study of cerebellar function. The valvula forms a part of the electrosensory-electromotor system of this fish, a system that offers many possibilities for the study of sensory-motor integration. The valvula also has a number of histological features not present in mammals which facilitate investigation of cerebellar circuitry and its plasticity. This initial study characterizes the basic physiology and pharmacology of cells in the valvula using an in vitro slice preparation. Intrinsic properties and synaptic responses of Purkinje cells and other cell types were examined. We found that Purkinje cells fire a small narrow Na(+) spike and a large broad Ca(2+) spike, generated in the axon initial segment and dendritic-soma region, respectively. Purkinje cells respond to parallel fiber inputs with graded excitatory postsynaptic potentials (EPSPs) and to climbing fiber inputs with all-or-none EPSPs. Efferent cells, Golgi cells, and deep stellate cells all fire a single type of large narrow spike and respond only to parallel fiber inputs. Both parallel fiber and climbing fiber responses in Purkinje cells appear to be entirely mediated by AMPA-type glutamate receptors, whereas parallel fiber responses in efferent cells and stellate cells include AMPA and NMDA components. In addition, a strong synaptic inhibition was uncovered in both Purkinje cells and efferent cells in response to the focal stimulation of parallel fibers. Dual cell recordings indicate that deep stellate cells contribute at least partially to this inhibition. We conclude that despite its unique histology, the local functional circuitry of the mormyrid valvula cerebelli is largely similar to that of the mammalian cerebellum. Thus, what is learned concerning the functioning of the mormyrid valvula cerebelli may be expected to be informative about cerebellar function in general.

Electrophysiological characteristics of cells in the anterior caudal lobe of the mormyrid cerebellum.

We have examined the basic electrophysiology and pharmacology of cells in the anterior caudal lobe (CLa) of the mormyrid cerebellum. Intracellular recordings were performed in an in vitro slice preparation using the whole-cell patch recording method. The responses of cells to parallel fiber (PF) and climbing fiber (CF) stimulation and to somatic current injection were recorded, and then characterized by bath application of receptor and ion channel blockers. Using biocytin or neurobiotin, these cells were also morphologically identified after recording to ensure their classification. Efferent cells and two subtypes of Purkinje cells were identified on the basis of their physiology and morphology. While the majority of Purkinje cells fire a single type of spike that is mediated by Na(+), some fire a large broad spike mediated by Ca(2+) and a narrow spike mediated by Na(+) at resting potential levels. By patching one recording electrode to the soma and another to one of the proximal dendrites of the same cell simultaneously, it was found that the Na(+) spike has an axonal origin and the Ca(2+) spike is generated in the soma-dendritic region of Purkinje cells. Efferent cells fire a single type of Na(+) spike only. Despite variations in their physiology and morphology, all cell types responded to PF stimulation with graded excitatory postsynaptic potentials (EPSPs) mediated by AMPA receptors. However, none of the efferent cells and only some of the Purkinje cells responded to CF activation with a large, AMPA receptor-mediated all-or-none EPSPs. We conclude that the functional circuitry of the CLa resembles that of other regions of the mormyrid cerebellum and is largely similar to that of the mammalian cerebellum.

Membrane current-based mechanisms for excitability transitions in neurons of the rat mesencephalic trigeminal nuclei.

Since Hodgkin's first description of three classes of excitability in crustacean nerve axons (1948), theoretical studies have used mathematical models to demonstrate that small changes in the parameters describing ionic currents could result in transitions between classes of membrane excitability. However, these transitions have rarely been investigated experimentally. Here, we show that states of excitability in rat mesencephalic V (Mes V) neurons can be classified into three groups, with manipulations of the 4-aminopyridine sensitive K(+) current (I(4-AP)) or persistent Na(+) current (I(NaP)) leading to the corresponding transitions. However, alterations in the hyperpolarization-activated cation current (I(h)), tetraethylammonium (TEA)-sensitive K(+) current, or Cd(2+)-sensitive Ca(2+) current were ineffective in causing these transitions. These results provide experimental evidence for the excitability transitions predicted by Hodgkin and characterize their ionic mechanisms in Mes V neurons.

Reversible associative depression and nonassociative potentiation at a parallel fiber synapse.

The electrosensory lobe (ELL) of mormyrid electric fish is one of several cerebellum-like sensory structures in fish that remove predictable features of the sensory inflow. This adaptive process obeys anti-Hebbian rules and appears to be mediated by associative depression at the synapses between parallel fibers and Purkinje-like cells of ELL. We show here that there is also a nonassociative potentiation at this synapse that depends only on the repeated occurrence of the EPSP. The depression can be reversed by the potentiation and vice versa. Finally, we show that the associative depression requires NMDA receptor activation, changes in postsynaptic calcium, and the occurrence of a postsynaptic dendritic spike within a few milliseconds following EPSP onset.

Rapid activation of GABAergic interneurons and possible calcium independent GABA release in the mormyrid electrosensory lobe.

The primary afferent fibers from the electroreceptors of mormyrid electric fish terminate centrally in the granular layer of the electrosensory lobe (ELL). This study examines the excitatory and inhibitory processes that take place in this layer using an in vitro slice preparation and field potentials evoked by stimulation of primary afferent fibers in the deep fiber layer of ELL. The postsynaptic response to stimulation of the afferent fibers was still present after blocking chemical transmission in three different ways: by adding glutamate receptor antagonists to the medium, by substituting a nominally calcium-free medium for normal medium, and by blocking calcium channels with cadmium. Blockade of chemical transmission was demonstrated by disappearance of control responses to parallel fiber stimulation. The continued presence of a postsynaptic response in the absence of chemical excitation is consistent with previous anatomic and physiological evidence for electrical synapses between afferent fibers and granular cells in ELL. Granular cell activation by primary afferent fibers was followed by a powerful, short-latency inhibition mediated by GABA and GABA(A) receptors, as indicated by a large increase in the postsynaptic response to afferent fiber stimulation following application of the GABA(A) receptor antagonist, bicuculline. Bicuculline caused a marked increase of the postsynaptic response even after chemical synaptic excitation had been blocked by glutamate receptor antagonists, by a calcium-free medium, or by cadmium. Thus activation of the inhibitory interneurons responsible for GABA release did not require chemical excitation. Nonchemical excitation of the inhibitory interneurons could be mediated either by electrical synapses between afferent fibers and inhibitory interneurons, or by nonsynaptic activation of the large GABAergic terminals that are known to be present on granular cells. The marked increase of the postsynaptic response caused by bicuculline in a calcium-free medium or in the presence of cadmium suggests that the release of GABA by inhibitory terminals was not entirely dependent on calcium influx. This effect of bicuculline on the postsynaptic response in a calcium-free medium or in the presence of cadmium was markedly reduced by prior addition of the GABA transporter antagonist, nipecotic acid. Thus calcium-independent release of GABA may occur in ELL and may be partly dependent on reversal of a GABA transporter. Rapid and powerful inhibition at the first stage in the processing of electrosensory information could serve to enhance the small differences in latency among afferent fibers that appear to encode small differences in stimulus intensity.

Synaptic plasticity in the mormyrid electrosensory lobe.

The mormyrid electrosensory lateral line lobe (ELL) is one of several different sensory structures in fish that behave as adaptive sensory processors. These structures generate negative images of predictable features in the sensory inflow which are added to the actual inflow to minimize the effects of predictable sensory features. The negative images are generated through a process of association between centrally originating predictive signals and sensory inputs from the periphery. In vitro studies in the mormyrid ELL show that pairing of parallel fiber input with Na+ spikes in postsynaptic cells results in synaptic depression at the parallel fiber synapses. The synaptic plasticity observed at the cellular level and the associative process of generating negative images of predicted sensory input at the systems level share a number of properties. Both are rapidly established, anti-Hebbian, reversible, input-specific and tightly restricted in time. These common properties argue strongly that associative depression at the parallel fiber synapse contributes to the adaptive generation of negative images in the mormyrid ELL.

Mormyrid electrosensory lobe in vitro: morphology of cells and circuits.

The electrosensory lobe (ELL) of mormyrid electric fish is a cerebellum-like brainstem structure that receives the primary afferent fibers from electroreceptors in the skin. The ELL and similar sensory structures in other fish receive extensive input from other central sources in addition to the peripheral input. The responses to some of these central inputs are adaptive and serve to minimize the effects of predictable sensory inputs. Understanding the interaction between peripheral and central inputs to the mormyrid ELL requires knowledge of its functional circuitry, and this paper examines this circuitry in the in vitro slice preparation and describes the axonal and dendritic morphology of major ELL cell types based on intracellular labeling with biocytin. The cells described include medium ganglion cells, large ganglion cells, large fusiform cells, thick-smooth dendrite cells, small fusiform cells, granule cells, and primary afferent fibers. The medium ganglion cells are Purkinje-like interneurons that terminate on the two types of efferent cells, i.e., large ganglion and large fusiform cells, as well as on each other. These medium ganglion cells fall into two morphologically distinct types based on the distributions of basal dendrites and axons. These distributions suggest hypotheses about the basic circuit of the ELL that have important functional consequences, such as enhancement of contrast between "on" elements that are excited by increased afferent activity and "off" elements that are inhibited.

The mormyrid electrosensory lobe in vitro: physiology and pharmacology of cells and circuits.

This paper is concerned with the electrosensory lobe (ELL) of mormyrid electric fish as examined in in vitro slices. Intracellular recordings from morphologically identified cells and field potential recordings were used to characterize the physiology and pharmacology of ELL cells. Most intracellular recordings were from the Purkinje-like interneurons that are known as medium ganglion cells and from the two types of efferent neurons, large ganglion and large fusiform cells. Stimulation of primary afferent fibers elicits both excitatory and inhibitory effects in these cells, with the excitatory effects being mediated by both the AMPA and NMDA types of glutamate receptors and the inhibitory effects being mediated by both GABAA and glycine receptors. Parallel-fiber stimulation evokes an EPSP-IPSP sequence, with the EPSPs being mediated by both AMPA and NMDA receptors and the IPSPs being mediated by GABAA receptors only. The parallel fiber-evoked EPSPs and IPSPs show marked paired-pulse facilitation. A large and unusually broad spike is recorded inside medium ganglion cells, and field potential responses suggest that this spike is propagated into the apical dendrites. The results provide essential information for understanding how peripheral and central inputs are integrated in ELL.

Synaptic plasticity in a cerebellum-like structure depends on temporal order.

Cerebellum-like structures in fish appear to act as adaptive sensory processors, in which learned predictions about sensory input are generated and subtracted from actual sensory input, allowing unpredicted inputs to stand out. Pairing sensory input with centrally originating predictive signals, such as corollary discharge signals linked to motor commands, results in neural responses to the predictive signals alone that are 'negative images' of the previously paired sensory responses. Adding these 'negative images' to actual sensory inputs minimizes the neural response to predictable sensory features. At the cellular level, sensory input is relayed to the basal region of Purkinje-like cells, whereas predictive signals are relayed by parallel fibres to the apical dendrites of the same cells. The generation of negative images could be explained by plasticity at parallel fibre synapses. We show here that such plasticity exists in the electrosensory lobe of mormyrid electric fish and that it has the necessary properties for such a model: it is reversible, anti-hebbian (excitatory postsynaptic potentials (EPSPs) are depressed after pairing with a postsynaptic spike) and tightly dependent on the sequence of pre- and postsynaptic events, with depression occurring only if the postsynaptic spike follows EPSP onset within 60 ms.