Alan Woodruff

SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators

Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a significant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a “scanless” microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the flexibility of laser microscopy, as it can substitute traditional simple fixed lenses with any calculated lens function.

Networks of parvalbumin-positive interneurons in the basolateral amygdala.

The amygdala is a temporal lobe structure that is required for processing emotional information. Polymodal sensory information enters the amygdala at the level of the basolateral amygdala (BLA) and undergoes local processing, after which the behavioral and autonomic responses that accompany emotions are initiated. Two main neuron types are present in the BLA, pyramidal-like principal neurons that use glutamate as their transmitter, and local circuit interneurons that use GABA as their transmitter. Although the properties of principal neurons are known in some detail, very little is known about the properties of BLA interneurons or the local circuits in which they are involved. Using mice in which EGFP (enhanced green fluorescent protein) is expressed under the control of the parvalbumin promoter, we characterized the properties of parvalbumin-positive interneurons in the BLA. By making recordings from interneuron–interneuron and interneuron–principal neuron pairs, we analyzed the intrinsic circuitry of the BLA. We show that parvalbumin-positive interneurons can be divided into four subtypes as defined by their firing properties. Interneurons are electrically coupled in subtype-specific networks and exhibit subtype-specific heterogeneities in their synaptic dynamics and patterns of connectivity. We propose that these properties allow networks of parvalbumin-expressing neurons to perform an array of information-processing tasks within the BLA.

Depolarizing effect of neocortical chandelier neurons

Chandelier (or axo-axonic) cells are one of the most distinctive types of GABAergic interneurons in the cortex. Although they have traditionally been considered inhibitory neurons, data from rat and human neocortical preparations suggest that chandelier cells have a depolarizing effect on pyramidal neurons at resting membrane potential, and could even activate synaptic chains of neurons. At the same time, recent results from rat hippocampal chandeliers indicate a predominantly inhibitory effect on their postsynaptic targets. To better understand the function of chandelier neurons, we generated Nkx2.1Cre MADM mice, a strain of genetically engineered animals that, by expressing GFP in a subset of neocortical interneurons, enable the identification and targeting of chandelier cells in living brain slices. Using these mice, we characterized the basic electrophysiological properties of a homogeneous population of chandelier neurons from upper layers of somatosensory cortical slices. These chandelier cells have characteristic axon cartridges and stereotypical electrophysiological features, distinguishable from basket cells. To investigate the effect of chandelier cells on target neurons, we performed paired recordings from chandeliers and postsynaptic pyramidal cells. In both perforated patch and cell-attached configurations, chandelier PSPs have in every case a reversal potential that is depolarized from rest. Our results support the idea that chandelier cells depolarize pyramidal neurons and could potentially have an excitatory effect on the network at rest.

State-Dependent Function of Neocortical Chandelier Cells

Chandelier (axoaxonic) cells (ChCs) are a distinct group of GABAergic interneurons that innervate the axon initial segments of pyramidal cells. However, their circuit role and the function of their clearly defined anatomical specificity remain unclear. Recent work has demonstrated that chandelier cells can produce depolarizing GABAergic PSPs, occasionally driving postsynaptic targets to spike. On the other hand, other work suggests that ChCs are hyperpolarizing and may have an inhibitory role. These disparate functional effects may reflect heterogeneity among ChCs. Here, using brain slices from transgenic mouse strains, we first demonstrate that, across different neocortical areas and genetic backgrounds, upper Layer 2/3 ChCs belong to a single electrophysiologically and morphologically defined population, extensively sampling Layer 1 inputs with asymmetric dendrites. Consistent with being a single cell type, we find electrical coupling between ChCs. We then investigate the effect of chandelier cell activation on pyramidal neuron spiking in several conditions, ranging from the resting membrane potential to stimuli designed to approximate in vivo membrane potential dynamics. We find that under quiescent conditions, chandelier cells are capable of both promoting and inhibiting spike generation, depending on the postsynaptic membrane potential. However, during in vivo-like membrane potential fluctuations, the dominant postsynaptic effect was a strong inhibition. Thus, neocortical chandelier cells, even from within a homogeneous population, appear to play a dual role in the circuit, helping to activate quiescent pyramidal neurons, while at the same time inhibiting active ones.

GABAergic Excitation in the Basolateral Amygdala

GABA-containing interneurons are a diverse population of cells whose primary mode of action in the mature nervous system is inhibition of postsynaptic target neurons. Using paired recordings from parvalbumin-positive interneurons in the basolateral amygdala, we show that, in a subpopulation of interneurons, single action potentials in one interneuron evoke in the postsynaptic interneuron a monosynaptic inhibitory synaptic current, followed by a disynaptic excitatory glutamatergic synaptic current. Interneuron-evoked glutamatergic events were blocked by antagonists of either AMPA/kainate or GABAA receptors, and could be seen concurrently in both presynaptic and postsynaptic interneurons. These results show that single action potentials in a GABAergic interneuron can drive glutamatergic principal neurons to threshold, resulting in both feedforward and feedback excitation. In interneuron pairs that both receive glutamatergic inputs after an interneuron spike, electrical coupling and bidirectional GABAergic connections occur with a higher probability relative to other interneuron pairs. We propose that this form of GABAergic excitation provides a means for the reliable and specific recruitment of homogeneous interneuron networks in the basal amygdala.

The Enigmatic Function of Chandelier Cells

Chandelier (or axo-axonic) cells are one of the most distinctive GABAergic interneurons in the brain. Their exquisite target specificity for the axon initial segment of pyramidal neurons, together with their GABAergic nature, long suggested the possibility that they provide the ultimate inhibitory control of pyramidal neuron output. Recent findings indicate that their function may be more complicated, and perhaps more interesting, than initially believed. Here we review these recent developments and their implications. We focus in particular on whether chandelier cells may provide a depolarizing, excitatory effect on pyramidal neuron output, in addition to a powerful inhibition.

Two-photon microscopy with diffractive optical elements and spatial light modulators.

Two-photon microscopy is often performed at slow frame rates due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE) generates a fixed number of beamlets that are scanned in parallel resulting in a corresponding increase in speed or in signal-to-noise ratio in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM) to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions including light path corrections as adaptive optics.

Of mice and men, and chandeliers.

How does the human neocortex reliably propagate information through neural circuits? One mechanism appears to involve relying on strong connections from pyramidal neurons to interneurons and a depolarizing action of cortical chandelier cells.

Spatial Light Modulator Microscopy

The use of spatial light modulators (SLMs) for two-photon laser microscopy is described. SLM phase modulation can be used to generate nearly any spatiotemporal pattern of light, enabling simultaneous illumination of any number of selected regions of interest. We take advantage of this flexibility to perform fast two-photon imaging or uncaging experiments on dendritic spines and neocortical neurons. By operating in the spatial Fourier plane, an SLM can effectively mimic any arbitrary optical transfer function and thus replace, in software, many of the functions provided by hardware in standard microscopes, such as focusing, magnification, and aberration correction.

Interneurons in the basolateral amygdala: A diversity of form and function

S01–01 LONG-TERM DEPRESSION, A FORM OF HIPPOCAMPAL SYNAPTIC PLASTICITY, IS RELATED TO STRESSINDUCED IMPAIRMENT OF MEMORY RETRIEVAL Wong, T.P., Howland, J.R., Phillips, A.G., Wang, Y.T. Department of Psychiatry, McGill University, Douglas Mental Health University Institute, Quebec, Canada Brain Research Centre, University of British Columbia, British Columbia, Canada Department of Psychology, University of Saskatchewan, Saskatchewan, Canada