Kenneth C. Reinert

Cerebellar Cortical Molecular Layer Inhibition Is Organized in Parasagittal Zones

Molecular layer inhibitory interneurons generate on-beam and off-beam inhibition in the cerebellar cortex that is hypothesized to control the timing and/or spatial patterning of Purkinje cell discharge. On- and off-beam inhibition has been assumed to be spatially uniform and continuous within a folium. Using flavoprotein autofluorescence optical imaging in the mouse cerebellar cortex in vivo, this study demonstrates that the inhibition evoked by parallel fiber and peripheral stimulation results in parasagittal bands of decreases in fluorescence that correspond to zebrin II-positive bands. The parasagittal bands of decreased fluorescence are abolished by GABAA antagonists and reflect the activity of molecular layer interneurons on their targets. The same banding pattern was observed using Ca2+ imaging. The bands produce spatially specific decreases in the responses to peripheral input. Therefore, molecular layer inhibition is compartmentalized into zebrin II parasagittal domains that differentially modulate the spatial pattern of cerebellar cortical activity.

Flavoprotein autofluorescence imaging in the cerebellar cortex in vivo

Autofluorescence optical imaging is rapidly becoming a widely used tool for mapping activity in the central nervous system function in vivo and investigating the coupling among neurons, glia, and metabolism. This paper provides a brief review of autofluorescence and of our recent work using flavoprotein imaging in the cerebellar cortex. Stimulation of the parallel fibers evokes an intrinsic fluorescence signal that is tightly coupled to neuronal activation and primarily generated postsynaptically. The signal originates from mitochondrial flavoproteins. The signal is biphasic, with the initial increase in fluorescence (light phase) resulting from the oxidation of flavoproteins and the subsequent decrease (dark phase) from the reduction of flavoproteins. The light phase is primarily neuronal, and the dark phase is primarily glial. Exploiting the spatial properties of molecular layer inhibition in the cerebellar cortex, we show that flavoprotein autofluorescence can monitor both excitatory and inhibitory activity in the cerebellar cortex. Furthermore, flavoprotein autofluorescence has revealed that molecular layer inhibition is organized into parasagittal domains that differentially modulate the spatial pattern of cerebellar cortical activity. The reduction in flavoprotein autofluorescence occurring in the inhibitory bands most likely reflects a decrease in intracellular Ca2+ in the neurons inhibited by the molecular layer interneurons. Therefore, flavoprotein autofluorescence imaging is providing new insights into cerebellar cortical function and neurometabolic coupling.

Cellular and Metabolic Origins of Flavoprotein Autofluorescence in the Cerebellar Cortex in vivo

Flavoprotein autofluorescence imaging, an intrinsic mitochondrial signal, has proven useful for monitoring neuronal activity. In the cerebellar cortex, parallel fiber stimulation evokes a beam-like response consisting of an initial, short-duration increase in fluorescence (on-beam light phase) followed by a longer duration decrease (on-beam dark phase). Also evoked are parasagittal bands of decreased fluorescence due to molecular layer inhibition. Previous work suggests that the on-beam light phase is due to oxidative metabolism in neurons. The present study further investigated the metabolic and cellular origins of the flavoprotein signal in vivo, testing the hypotheses that the dark phase is mediated by glia activation and the inhibitory bands reflect decreased flavoprotein oxidation and increased glycolysis in neurons. Blocking postsynaptic ionotropic and metabotropic glutamate receptors abolished the on-beam light phase and the parasagittal bands without altering the on-beam dark phase. Adding glutamate transporter blockers reduced the dark phase. Replacing glucose with lactate (or pyruvate) or adding lactate to the bathing media abolished the on-beam dark phase and reduced the inhibitory bands without affecting the light phase. Blocking monocarboxylate transporters eliminated the on-beam dark phase and increased the light phase. These results confirm that the on-beam light phase is due primarily to increased oxidative metabolism in neurons. They also show that the on-beam dark phase involves activation of glycolysis in glia resulting in the generation of lactate that is transferred to neurons. Oxidative savings in neurons contributes to the decrease in fluorescence characterizing the inhibitory bands. These findings provide strong in vivo support for the astrocyte–neuron lactate shuttle hypothesis.

IMAGING PARALLEL FIBER AND CLIMBING FIBER RESPONSES AND THEIR SHORT-TERM INTERACTIONS IN THE MOUSE CEREBELLAR CORTEX IN VIVO

A major question in the study of cerebellar cortical function is how parallel fiber and climbing fiber inputs interact to shape information processing. Emphasis has been placed on the long-term effects due to conjunctive stimulation of climbing fibers and parallel fibers. Much less emphasis has been placed on short-term interactions and their spatial nature. To address this question the responses to parallel fiber and climbing fiber inputs and their short-term interaction were characterized using optical imaging with Neutral Red in the anesthetized mouse in vivo. Electrical stimulation of the cerebellar surface evoked an increase in fluorescence consisting of a transverse optical beam. The linear relationship between the optical responses and stimulus parameters, high spatial resolution and close coupling to the electrophysiological recordings show the utility of this imaging methodology. The majority of the optical response was due to activation of postsynaptic alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) and metabotropic glutamate receptors with a minor contribution from the presynaptic parallel fibers. Stimulation of the inferior olive evoked parasagittal bands that were abolished by blocking AMPA glutamate receptors. Conjunctive stimulation of the cerebellar surface and inferior olive resulted in inhibition of the climbing fiber evoked optical responses. This lateral inhibition of the parasagittal bands extended out from both sides of an activated parallel fiber beam and was mediated by GABA(A) but not GABA(B) receptors. One hypothesized role for lateral inhibition of this type is to spatially focus the interactions between parallel fiber and climbing fiber input on Purkinje cells. In summary optical imaging with Neutral Red permitted visualization of cerebellar cortical responses to parallel fiber and climbing fiber activation. The GABA(A) dependent lateral inhibition of the climbing fiber evoked parasagittal bands by parallel fiber stimulation shows that cerebellar interneurons play a short-term role in shaping the responses of Purkinje cells to climbing fiber input.

Optical imaging of cerebellar functional architectures: parallel fiber beams, parasagittal bands and spreading acidification.

Publisher Summary An intriguing feature of the cerebellar cortex is its highly ordered circuitry and architectures, and understanding the function of this stereotypic circuitry and neuronal architectures is needed to unravel the nature of the neural computations performed by the cerebellar cortex. This chapter outlines the efforts put to develop and use optical imaging methodologies in the cerebellar cortex in vivo and emphasizes the significance of optical imaging in studying the interactions among the parallel fiber and climbing fiber systems, monitoring synaptic plasticity at the circuitry level, and observing novel phenomena such as spreading acidification and depression. Neutral red imaging has been used to map cerebellar cortical neuronal activity in vivo and has several useful properties for monitoring spatial patterns of neuronal activation in the cerebellar cortex. Thus, neutral red imaging provides a dramatic visualization that the cerebellar cortex is activated in parasagittal zones. Autofluorescence of flavoproteins and nicotinamide adenine dinucleotide has been used as an indirect measure of neuronal activity in isolated cell cultures and brain slice, but only to a limited extent in vivo . Spreading acidification and depression (SAD) appears to be a unique type of propagating activity, and multiple factors are likely to contribute to the spread of the acidification and depression such as hyperexcitability of the cerebellar circuitry. Conjunctive stimulation of climbing fiber and parallel fiber inputs results in long term depression at the parallel fiber–Purkinje cell synapse. Spreading acidification and depression in the cerebellar cortex is a newly described propagating activity in the central nervous system; however, much is still to be learned about SAD, including the mechanism of spread and the nature of the regenerative process.

Autofluorescence imaging in the mouse cerebellar cortex in vivo

Activity dependent intrinsic autofluorescence was studied in mouse cerebellar cortex in vivo. The properties and flavoprotein origin of this autofluorescence signal were systematically evaluated, including the unique capability of differentiating between neuronal excitation and inhibition.