Current and Calcium Responses to Local Activation of Axonal NMDA Receptors in Developing Cerebellar Molecular Layer Interneurons

In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca2+ channels (VDCCs). Using Ca2+ imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg2+ or by the addition of APV. Similar paradigms yielded restricted Ca2+ transients in interneurons loaded with a Ca2+ indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca2+ elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca2+-induced Ca2+ release process mediated by presynaptic Ca2+ stores. Such a mechanism is likely to exert a crucial role in various forms of Ca2+-mediated synaptic plasticity

Rebound Discharge in Deep Cerebellar Nuclear Neurons In Vitro

Neurons of the deep cerebellar nuclei (DCN) play a critical role in defining the output of cerebellum in the course of encoding Purkinje cell inhibitory inputs. The earliest work performed with in vitro preparations established that DCN cells have the capacity to translate membrane hyperpolarizations into a rebound increase in firing frequency. The primary means of distinguishing between DCN neurons has been according to cell size and transmitter phenotype, but in some cases, differences in the firing properties of DCN cells maintained in vitro have been reported. In particular, it was shown that large diameter cells in the rat DCN exhibit two phenotypes of rebound discharge in vitro that may eventually help define their functional roles in cerebellar output. A transient burst and weak burst phenotype can be distinguished based on the frequency and pattern of rebound discharge immediately following a hyperpolarizing stimulus. Work to date indicates that the difference in excitability arises from at least the degree of activation of T-type Ca2+ current during the immediate phase of rebound firing and Ca2+-dependent K+ channels that underlie afterhyperpolarizations. Both phenotypes can be detected following stimulation of Purkinje cell inhibitory inputs under conditions that preserve resting membrane potential and natural ionic gradients. In this paper, we review the evidence supporting the existence of different rebound phenotypes in DCN cells and the ion channel expression patterns that underlie their generation

Spike-Timing Precision and Neuronal Synchrony Are Enhanced by an Interaction between Synaptic Inhibition and Membrane Oscillations in the Amygdala

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2–6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine

Determinants of synaptic integration and heterogeneity in rebound firing explored with data-driven models of deep cerebellar nucleus cells

Significant inroads have been made to understand cerebellar cortical processing but neural coding at the output stage of the cerebellum in the deep cerebellar nuclei (DCN) remains poorly understood. The DCN are unlikely to just present a relay nucleus because Purkinje cell inhibition has to be turned into an excitatory output signal, and DCN neurons exhibit complex intrinsic properties. In particular, DCN neurons exhibit a range of rebound spiking properties following hyperpolarizing current injection, raising the question how this could contribute to signal processing in behaving animals. Computer modeling presents an ideal tool to investigate how intrinsic voltage-gated conductances in DCN neurons could generate the heterogeneous firing behavior observed, and what input conditions could result in rebound responses. To enable such an investigation we built a compartmental DCN neuron model with a full dendritic morphology and appropriate active conductances. We generated a good match of our simulations with DCN current clamp data we recorded in acute slices, including the heterogeneity in the rebound responses. We then examined how inhibitory and excitatory synaptic input interacted with these intrinsic conductances to control DCN firing. We found that the output spiking of the model reflected the ongoing balance of excitatory and inhibitory input rates and that changing the level of inhibition performed an additive operation. Rebound firing following strong Purkinje cell input bursts was also possible, but only if the chloride reversal potential was more negative than −70 mV to allow de-inactivation of rebound currents. Fast rebound bursts due to T-type calcium current and slow rebounds due to persistent sodium current could be differentially regulated by synaptic input, and the pattern of these rebounds was further influenced by HCN current. Our findings suggest that active properties of DCN neurons could play a crucial role for signal processing in the cerebellum

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b+Gr-1+ myeloid cells without compromising CD8+ T cell immune responses

BACKGROUND: Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. METHODS: We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. RESULTS: We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b(+)Gr-1(+) myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8(+) T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. CONCLUSIONS: Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF