Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo.

Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 µm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (80) populations of apical dendrites and follow these dendrites down to their somata of origin.

Toxicity assessment of intravitreal triamcinolone and bevacizumab in a retinal explant mouse model using two-photon microscopy.

PURPOSE: Intravitreal drug administration leads to high intraocular concentrations with potentially toxic effects on ocular tissues. This study was an assessment of the toxicity of triamcinolone and bevacizumab in living retinal explants using two-photon (2P) microscopy. METHODS: Wild-type mice received intravitreal injections of triamcinolone, bevacizumab, or vehicle. Ten and 45 days after injection, wholemounted retinal explants were incubated with the fluorescent dye sulforhodamine 101 (SR101) to analyze morphology and tissue damage with 2P microscopy ex vivo. Retinas that received the same treatment were stained for apoptosis (TUNEL) and glial activation (GFAP). An intravitreal injection of NMDA (N-methyl-d-aspartate) was used as a positive control to ensure the fidelity of detection of retinal damage with ex vivo 2P microscopy. RESULTS: Overall retinal morphology was undisturbed after all procedures and time points. NMDA injection resulted in a strong increase in the number of SR101-labeled cells and increased apoptosis and glial activation when compared with sham-injected eyes. This result was in contrast to exposure to bevacizumab, which caused no appreciable damage. After triamcinolone treatment, marked damage in the inner retina was observed. However, damaged cells were restricted to sharply demarcated areas, and only mild changes in TUNEL-positive cells and GFAP activation was observed when compared to sham-injected eyes. CONCLUSIONS: 2P microscopy in combination with SR101 staining allows fast morphologic assessment of living retinal explants and can be used to evaluate adverse effects on retinal viability of test substances. Bevacizumab treatment did not cause any detectable retinal damage, whereas triamcinolone was associated with substantial, although spatially restricted, damage.

Linking synaptic plasticity and spike output at excitatory and inhibitory synapses onto cerebellar Purkinje cells.

Understanding the relationship between synaptic plasticity and neuronal output is essential if we are to understand how plasticity is encoded in neural circuits. In the cerebellar cortex, motor learning is thought to be implemented by long-term depression (LTD) of excitatory parallel fiber (PF) to Purkinje cell synapses triggered by climbing fiber (CF) input. However, theories of motor learning generally neglect the contribution of plasticity of inhibitory inputs to Purkinje cells. Here we describe how CF-induced plasticity of both excitatory and inhibitory inputs is reflected in Purkinje cell spike output. We show that coactivation of the CF with PF input and interneuron input leads not only to LTD of PF synapses but also to comparable, "balanced" LTD of evoked inhibitory inputs. These two forms of plasticity have opposite effects on the spike output of Purkinje cells, with the number and timing of spikes sensitively reflecting the degree of plasticity. We used dynamic clamp to evaluate plasticity-induced changes in spike responses to sequences of excitation and feedforward inhibition of varied relative and absolute amplitude. Balanced LTD of both excitatory and inhibitory components decreased the net spike output of Purkinje cells only for inputs with small inhibitory components, whereas for inputs with a larger proportion of feedforward inhibition CF-triggered LTD resulted in an increase in the net spike output. Thus, the net effect of CF-triggered plasticity on Purkinje cell output depends on the balance of excitation and feedforward inhibition and can paradoxically increase cerebellar output, contrary to current theories of cerebellar motor learning.

Cerebellar LTD and pattern recognition by Purkinje cells.

Many 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.

Feed-forward inhibition shapes the spike output of cerebellar Purkinje cells.

Although the cerebellum is thought to play a key role in timing of movements on the time scale of milliseconds, it is unclear how such temporal fidelity is ensured at the cellular level. We have investigated the timing of feed-forward inhibition onto interneurons and Purkinje cells activated by parallel fibre stimulation in slices of cerebellar cortex from P18-25 rats. Feed-forward inhibition was activated within approximately 1 ms after the onset of excitation in both cell types. The rapid onset of feed-forward inhibition sharply curtailed EPSPs and increased the precision of the resulting action potentials. The time window for summation of EPSPs was reduced to 1-2 ms in the presence of feed-forward inhibition, which could inhibit the efficacy of asynchronous EPSPs for up to 30 ms. Our findings demonstrate how the inhibitory microcircuitry of the cerebellar cortex orchestrates synaptic integration and precise timing of spikes in Purkinje cells, enabling them to act as coincidence detectors of parallel fibre input.

Neuronal microcircuits: frequency-dependent flow of inhibition.

New work suggests that feedback inhibition of neurons in the hippocampus is mediated by two distinct microcircuits. Interneurons targeting a neuron's soma are triggered by onset of activity, while those targeting distal dendrites are recruited by sustained activity. These circuits may thus convey information about the timing and rate of activity, respectively.