Dopamine Increases the Intrinsic Excitability of Parvalbumin-Expressing Fast-Spiking Cells in the Piriform Cortex.

The piriform cortex (PCx) is essential for the adaptive processing of olfactory information. Neuromodulatory systems, including those utilizing serotonin, acetylcholine, noradrenaline, and dopamine, innervate and regulate neuronal activity in the PCx. Previous research has demonstrated the importance of acetylcholine, noradrenaline and serotonin in odor learning and memory. In contrast, the role of dopamine in the PCx remains under-explored. Here we examined how dopamine modulates the intrinsic electrical properties of identified classes of neurons in the PCx. We found that dopamine had no consistent effect on the intrinsic electrical properties of two types of glutamatergic neurons (semilunar and superficial pyramidal cells) or three types of GABAergic interneurons (horizontal, neurogliaform and somatastatin-expressing regular-spiking cells). However, dopamine had a striking effect on the intrinsic excitability of the parvalbumin-expressing fast-spiking (FS) class of GABAergic interneuron. Dopamine depolarized the resting potential, increased the input resistance and increased the firing frequency of FS cells. Co-application of dopamine with the D1-class dopamine receptor antagonist SCH 23390 blocked the effects of dopamine modulation on FS cells. Conversely, co-application of dopamine with the D2-class antagonist RS-(±)-sulpiride had no effect on dopamine modulation of these cells. Our results indicate that dopamine binds to D1-class dopamine receptors to increase the intrinsic excitability of FS cells. These findings suggest that dopamine has a highly targeted effect in the PCx and reveal how dopamine may modulate the balance between excitation and inhibition, with consequences for odor processing. In addition, our findings provide clues for understanding why neurodegenerative disorders that modify the dopamine system, such as Parkinson's disease, have a deleterious effect on the sense of smell, and may suggest novel diagnostics for the early detection of such disorders.

Fast and slow feedforward inhibitory circuits for cortical odor processing.

Feedforward inhibitory circuits are key contributors to the complex interplay between excitation and inhibition in the brain. Little is known about the function of feedforward inhibition in the primary olfactory (piriform) cortex. Using in vivo two-photon-targeted patch clamping and calcium imaging in mice, we find that odors evoke strong excitation in two classes of interneurons - neurogliaform (NG) cells and horizontal (HZ) cells - that provide feedforward inhibition in layer 1 of the piriform cortex. NG cells fire much earlier than HZ cells following odor onset, a difference that can be attributed to the faster odor-driven excitatory synaptic drive that NG cells receive from the olfactory bulb. As a result, NG cells strongly but transiently inhibit odor-evoked excitation in layer 2 principal cells, whereas HZ cells provide more diffuse and prolonged feedforward inhibition. Our findings reveal unexpected complexity in the operation of inhibition in the piriform cortex.

Autaptic Cultures: Methods and Applications.

Neurons typically form daisy chains of synaptic connections with other neurons, but they can also form synapses with themselves. Although such self-synapses, or autapses, are comparatively rare in vivo, they are surprisingly common in dissociated neuronal cultures. At first glance, autapses in culture seem like a mere curiosity. However, by providing a simple model system in which a single recording electrode gives simultaneous access to the pre- and postsynaptic compartments, autaptic cultures have proven to be invaluable in facilitating important and elegant experiments in the area of synaptic neuroscience. Here, I provide detailed protocols for preparing and recording from autaptic cultures (also called micro-island or microdot cultures). Variations on the basic procedure are presented, as well as practical tips for optimizing the outcomes. I also illustrate the utility of autaptic cultures by reviewing the types of experiments that have used them over the past three decades. These examples serve to highlight the power and elegance of this simple model system, and will hopefully inspire new experiments for the interrogation of synaptic function.

Sodium and potassium conductances in principal neurons of the mouse piriform cortex: a quantitative description.

KEY POINTS: The primary olfactory (or piriform) cortex is a promising model system for understanding how the cerebral cortex processes sensory information, although an investigation of the piriform cortex is hindered by a lack of detailed information about the intrinsic electrical properties of its component neurons. In the present study, we quantify the properties of voltage-dependent sodium currents and voltage- and calcium-dependent potassium currents in two important classes of excitatory neurons in the main input layer of the piriform cortex. We identify several classes of these currents and show that their properties are similar to those found in better-studied cortical regions. Our detailed quantitative descriptions of these currents will be valuable to computational neuroscientists who aim to build models that explain how the piriform cortex encodes odours. ABSTRACT: The primary olfactory cortex (or piriform cortex, PC) is an anatomically simple palaeocortex that is increasingly used as a model system for investigating cortical sensory processing. However, little information is available on the intrinsic electrical conductances in neurons of the PC, hampering efforts to build realistic computational models of this cortex. In the present study, we used nucleated macropatches and whole-cell recordings to rigorously quantify the biophysical properties of voltage-gated sodium (NaV ), voltage-gated potassium (KV ) and calcium-activated potassium (KCa ) conductances in two major classes of glutamatergic neurons in layer 2 of the PC, semilunar (SL) cells and superficial pyramidal (SP) cells. We found that SL and SP cells both express a fast-inactivating NaV current, two types of KV current (A-type and delayed rectifier-type) and three types of KCa current (fast-, medium- and slow-afterhyperpolarization currents). The kinetic and voltage-dependent properties of the NaV and KV conductances were, with some exceptions, identical in SL and SP cells and similar to those found in neocortical pyramidal neurons. The KCa conductances were also similar across the different types of neurons. Our results are summarized in a series of empirical equations that should prove useful to computational neuroscientists seeking to model the PC. More broadly, our findings indicate that, at the level of single-cell electrical properties, this palaeocortex is not so different from the neocortex, vindicating efforts to use the PC as a model of cortical sensory processing in general.

Pre- and Postsynaptic Activation of GABAB Receptors Modulates Principal Cell Excitation in the Piriform Cortex.

The piriform cortex (PC), like other cortical regions, normally operates in a state of dynamic equilibrium between excitation and inhibition. Here we examined the roles played by pre- and postsynaptic GABAB receptors in maintaining this equilibrium in the PC. Using whole-cell recordings in brain slices from the anterior PC of mice, we found that synaptic activation of postsynaptic GABAB receptors hyperpolarized the two major classes of layer 2 principal neurons and reduced the intrinsic electrical excitability of these neurons. Presynaptic GABAB receptors are expressed on the terminals of associational (intracortical) glutamatergic axons in the PC. Heterosynaptic activation of these receptors reduced excitatory associational inputs onto principal cells. Presynaptic GABAB receptors are also expressed on the axons of GABAergic interneurons in the PC, and blockade of these autoreceptors enhanced inhibitory inputs onto principal cells. Hence, presynaptic GABAB autoreceptors produce disinhibition of principal cells. To study the functional consequences of GABAB activation in vivo, we used 2-photon calcium imaging to simultaneously monitor the activity of ~200 layer 2 neurons. Superfusion of the GABAB agonist baclofen reduced spontaneous random firing but also promoted synchronous epileptiform activity. These findings suggest that, while GABAB activation can dampen excitability by engaging pre- and postsynaptic GABAB heteroreceptors on glutamatergic neurons, it can also promote excitability by disinhibiting principal cells by activating presynaptic GABAB autoreceptors on interneurons. Thus, depending on the dynamic balance of hetero- and autoinhibition, GABAB receptors can function as variable modulators of circuit excitability in the PC.

Spontaneous activity in the piriform cortex extends the dynamic range of cortical odor coding.

Neurons in the neocortex exhibit spontaneous spiking activity in the absence of external stimuli, but the origin and functions of this activity remain uncertain. Here, we show that spontaneous spiking is also prominent in a sensory paleocortex, the primary olfactory (piriform) cortex of mice. In the absence of applied odors, piriform neurons exhibit spontaneous firing at mean rates that vary systematically among neuronal classes. This activity requires the participation of NMDA receptors and is entirely driven by bottom-up spontaneous input from the olfactory bulb. Odor stimulation produces two types of spatially dispersed, odor-distinctive patterns of responses in piriform cortex layer 2 principal cells: Approximately 15% of cells are excited by odor, and another approximately 15% have their spontaneous activity suppressed. Our results show that, by allowing odor-evoked suppression as well as excitation, the responsiveness of piriform neurons is at least twofold less sparse than currently believed. Hence, by enabling bidirectional changes in spiking around an elevated baseline, spontaneous activity in the piriform cortex extends the dynamic range of odor representation and enriches the coding space for the representation of complex olfactory stimuli.

Optogenetic Mapping of Intracortical Circuits Originating from Semilunar Cells in the Piriform Cortex.

Despite its comparatively simple trilaminar architecture, the primary olfactory (piriform) cortex of mammals is capable of performing sophisticated sensory processing, an ability that is thought to depend critically on its extensive associational (intracortical) excitatory circuits. Here, we used a novel transgenic mouse model and optogenetics to measure the connectivity of associational circuits that originate in semilunar (SL) cells in layer 2a of the anterior piriform cortex (aPC). We generated a mouse line (48L) in which channelrhodopsin-2 (ChR) could be selectively expressed in a subset of SL cells. Light-evoked excitatory postsynaptic currents (EPSCs) could be evoked in superficial pyramidal cells (17.4% of n = 86 neurons) and deep pyramidal cells (33.3%, n = 9) in the aPC, but never in ChR- SL cells (0%, n = 34). Thus, SL cells monosynaptically excite pyramidal cells, but not other SL cells. Light-evoked EPSCs were also selectively elicited in 3 classes of GABAergic interneurons in layer 3 of the aPC. Our results show that SL cells are specialized for providing feedforward excitation of specific classes of neurons in the aPC, confirming that SL cells comprise a functionally distinctive input layer.

Persistent barrage firing in cortical interneurons can be induced in vivo and may be important for the suppression of epileptiform activity.

Neural circuits are typically maintained in a state of dynamic equilibrium by balanced synaptic excitation and inhibition. However, brain regions that are particularly susceptible to epilepsy may have evolved additional specialized mechanisms for inhibiting over-excitation. Here we identify one such possible mechanism in the cerebral cortex and hippocampus of mice. Recently it was reported that some types of GABAergic interneurons can slowly integrate excitatory inputs until eventually they fire persistently in the absence of the original stimulus. This property, called persistent firing or retroaxonal barrage firing (BF), is of unknown physiological importance. We show that two common types of interneurons in cortical regions, neurogliaform (NG) cells and fast-spiking (FS) cells, are unique in exhibiting BF in acute slices (~85 and ~23% success rate for induction, respectively). BF can also be induced in vivo, although the success rate for induction is lower (~60% in NG cells). In slices, BF could reliably be triggered by trains of excitatory synaptic input, as well as by exposure to proconvulsant bath solutions (elevated extracellular K(+), blockade of GABAA receptors). Using pair recordings in slices, we confirmed that barrage-firing NG cells can produce synaptic inhibition of nearby pyramidal neurons, and that this inhibition outlasts the original excitation. The ubiquity of NG and FS cells, together with their ability to fire persistently following excessive excitation, suggests that these interneurons may function as cortical sentinels, imposing an activity-dependent brake on undesirable neuronal hyperexcitability.

Neurons and circuits for odor processing in the piriform cortex.

Increased understanding of the early stages of olfaction has lead to a renewed interest in the higher brain regions responsible for forming unified 'odor images' from the chemical components detected by the nose. The piriform cortex, which is one of the first cortical destinations of olfactory information in mammals, is a primitive paleocortex that is critical for the synthetic perception of odors. Here we review recent work that examines the cellular neurophysiology of the piriform cortex. Exciting new findings have revealed how the neurons and circuits of the piriform cortex process odor information, demonstrating that, despite its superficial simplicity, the piriform cortex is a remarkably subtle and intricate neural circuit.

Microcircuits mediating feedforward and feedback synaptic inhibition in the piriform cortex.

Local inhibition by GABA-releasing neurons is important for the operation of sensory cortices, but the details of these inhibitory circuits remain unclear. We addressed this question in the olfactory system by making targeted recordings from identified classes of inhibitory and glutamatergic neurons in the piriform cortex (PC) of mice. First, we looked for feedforward synaptic inhibition provided by interneurons located in the outermost layer of the PC, layer Ia, which is the unique recipient of afferent fibers from the olfactory bulb. We found two types of feedforward inhibition: a fast-rising, spatially restricted kind that was generated by horizontal cells, and a slow-rising, more diffuse kind generated by neurogliaform cells. Both cell types targeted the distal apical dendrites of layer II principal neurons. Next, we studied feedback synaptic inhibition in isolation by making a tissue cut across layer I to selectively remove feedforward inhibitory connections. We identified a powerful type of feedback inhibition of layer II neurons, mostly generated by soma-targeting fast-spiking multipolar cells in layer III, which in turn were driven by feedforward excitation from layer II semilunar cells. Dynamic clamp simulation of feedback inhibition revealed differential effects of this inhibition on the two main types of layer II principal neurons. Thus, our results articulate the connectivity and functions of two important classes of inhibitory microcircuits in the PC. Feedforward and feedback inhibition generated by these circuits is likely to be required for the operation of this sensory paleocortex during the processing of olfactory information.

Changes in dendritic axial resistance alter synaptic integration in cerebellar Purkinje cells.

The ability of neurons to process synaptic inputs depends critically on their passive electrical properties. The intracellular resistivity, R(i), is one of the parameters that determine passive properties, yet few experiments have explored how changes in R(i) might affect synaptic integration. In this work, I addressed this issue by using targeted dendritic occlusion to locally increase R(i) in cerebellar Purkinje cells and examining the consequences of this manipulation for the summation of synaptic inputs. To achieve dendritic occlusion, I used two glass micropipettes to gently pinch the dendritic trunk close to the soma. This pinching produced stereotypical changes in the responses to test pulses applied at the soma under voltage and current clamp. A simple model confirmed that these changes were due to increases in R(i) in the dendritic trunk. These localized increases in R(i) produced striking alterations in the shapes of postsynaptic potentials at the soma, increasing their amplitude and accelerating their decay kinetics. As a consequence, dendritic occlusion sharpened temporal precision during the summation of synaptic inputs. These findings highlight the importance of local changes in intracellular resistivity for the passive electrical properties of neurons, with implications for their ability to process synaptic information.

Two layers of synaptic processing by principal neurons in piriform cortex.

The primary olfactory (or piriform) cortex is a trilaminar paleocortex that is thought to construct unified "odor images" from the odor components identified by the olfactory bulb. How the piriform cortex (PC) accomplishes this sophisticated synthetic task, despite its relatively simple architecture, is unknown. Here we used in vitro patch-clamp recordings from acute slices of the anterior PC of mice to identify microcircuits involved in excitatory synaptic processing. Cluster analysis confirmed the presence of two prominent classes of glutamatergic principal cells in the main input layer (layer II) of the PC: semilunar (SL) cells and superficial pyramidal (SP) cells. SL cells received stronger afferent excitatory input from the olfactory bulb, on average, than did SP cells. This was due to the larger mean strength of single-fiber afferents onto SL cells. In contrast, SP cells received stronger associational (intracortical) excitatory inputs, most likely due to their more extensive dendritic trees within the associational layers. Tissue-cut experiments and dual recordings from SL and SP cells in disinhibited slices were consistent with the distinctive patterns of connectivity of these two cell classes. Our findings suggest that the anterior PC employs at least two layers of excitatory synaptic processing: one involving strong afferent inputs onto SL cells, and another involving strong intracortical inputs onto SP cells. This architecture may allow the PC to sequentially process olfactory information within segregated subcircuits.

Distinctive classes of GABAergic interneurons provide layer-specific phasic inhibition in the anterior piriform cortex.

The primary olfactory (or piriform) cortex is a trilaminar paleocortex that is seen increasingly as an attractive model system for the study of cortical sensory processing. Recent findings highlight the importance of γ-amino butyric acid (GABA)-releasing interneurons for the function of the piriform cortex (PC), yet little is known about the different types of interneurons in the PC. Here, we provide the first detailed functional characterization of the major classes of GABAergic interneurons in the anterior piriform cortex (aPC) and show how these classes differentially engage in phasic synaptic inhibition. By measuring the electrical properties of interneurons and combining this with information about their morphology, laminar location, and expression of molecular markers, we have identified 5 major classes in the aPC of the mouse. Each layer contains at least one class of interneuron that is tuned to fire either earlier or later in a train of stimuli resembling the input received by the PC in vivo during olfaction. This suggests that the different subtypes of interneuron are specialized for providing synaptic inhibition at different phases of the sniff cycle. Thus, our results suggest mechanisms by which classes of interneurons play specific roles in the processing performed by the PC in order to recognize odors.

Inhibitory neurons in the anterior piriform cortex of the mouse: classification using molecular markers.

The primary olfactory cortex (or piriform cortex, PC) is attracting increasing attention as a model system for the study of cortical sensory processing, yet little is known about inhibitory neurons in the PC. Here we provide the first systematic classification of GABA-releasing interneurons in the anterior PC of mice, based on the expression of molecular markers. Our experiments used GAD67-GFP transgenic mice, in which gamma-aminobutyric acid (GABA)-containing cells are labeled with green fluorescent protein (GFP). We first confirmed, using paired whole-cell recordings, that GFP(+) neurons in the anterior PC of GAD67-GFP mice are functionally GABAergic. Next, we performed immunolabeling of GFP(+) cells to quantify their expression of every possible pairwise combination of seven molecular markers: calbindin, calretinin, parvalbumin, cholecystokinin, neuropeptide Y, somatostatin, and vasoactive intestinal peptide. We found that six main categories of interneurons could be clearly distinguished in the anterior PC, based on the size and laminar location of their somata, intensity of GFP fluorescence, patterns of axonal projections, and expression of one or more of the seven markers. A number of rarer categories of interneurons could also be identified. These data provide a road map for further work that examines the functional properties of the six main classes of interneurons. Together, this information elucidates the cellular architecture of the PC and provides clues about the roles of GABAergic interneurons in olfactory processing.

Synaptic transmission: excitatory autapses find a function?

An autapse is a synapse between a neuron and itself, a peculiar structure with an unclear function. A new study suggests that excitatory autapses contribute to a positive-feedback loop that maintains persistent electrical activity in neurons.

Counting the number of releasable synaptic vesicles in a presynaptic terminal.

Synaptic transmission depends on the continued availability of neurotransmitter-filled synaptic vesicles (SVs) for triggered release from presynaptic boutons. Surprisingly, small boutons in the brain, that already contain comparatively few SVs, are thought to retain the majority of these SVs in a "reserve" pool that is not mobilized under physiological conditions. Why such a scarce synaptic resource is normally inaccessible has been a matter of debate. Here, we readdress this issue by developing an electrophysiological approach for counting SVs released from boutons formed by a single, isolated neuron on itself ("autapses"). We show that, after treatment with Bafilomycin A1 to prevent reloading of discharged SVs with glutamate, each SV is counted only once on first-time release. Hence, by integrating all autaptic currents as they run down over time, we can estimate the total number of SVs released by a single neuron. This total can be normalized to the number of boutons on the neuron, giving the mean number of SVs released per bouton. We estimate that up to approximately 130 vesicles can be released per bouton over approximately 10 min of stimulation at 0.2 Hz. This number of vesicles represents a substantial proportion of the total number of SVs (100-200) that have been counted in these boutons by using electron microscopy. Thus, mild electrical stimulation, when maintained for sufficient time, causes the eventual release of many of the SVs in a bouton, including those in the putative reserve pool. This result suggests that SVs are functionally homogeneous in that the majority can contribute to basal synaptic transmission.

Distinctive quantal properties of neurotransmission at excitatory and inhibitory autapses revealed using variance-mean analysis.

Normal brain function depends on an interplay between glutamatergic and GABAergic synaptic transmission, yet questions remain about the biophysical differences between these two classes of synapse. By taking advantage of a simple culture system, we present here a detailed comparison of excitatory and inhibitory neurotransmission under identical conditions using the variance-mean (V-M) method of quantal analysis. First, we validate V-M analysis for glutamatergic autapses formed by isolated hippocampal pyramidal neurons in culture, confirming that the analysis accurately predicts the quantal amplitude (Q). We also show that V-M analysis is only weakly sensitive to intersite and intrasite quantal variance and to the known inhomogeneities in release probability (P(r)). Next, by repeating the experiments with GABAergic autapses, we confirm that V-M analysis provides an accurate account of inhibitory neurotransmission in this system. Mean P(r), provided by V-M analysis, shows a dependence on extracellular Ca(2+) concentration that is nearly identical for both excitatory and inhibitory autapses. Finally, the V-M method allows us to compare the locus of short-term synaptic plasticity at these connections. Glutamatergic autapses exhibit paired-pulse depression that depends mainly on changes in P(r), whereas depression at GABAergic autapses appears to depend primarily on changes in the number of release sites. We conclude that, apart from differences in the mechanisms of short-term plasticity, the basic quantal properties of excitatory and inhibitory connections in this hippocampal system are remarkably similar.

Inhibitory interneurons in the piriform cortex.

1. The piriform cortex (PC) is the largest subdivision of the olfactory cortex and the first cortical destination of olfactory information. Despite the relatively simple anatomy of the PC and its obvious appeal as a model system for the study of cortical sensory processing, there are many outstanding questions about its basic cell physiology. In the present article, we review what is known about GABAergic inhibitory interneurons in the PC. 2. The GABA-containing neurons in the PC are morphologically diverse, ranging from small neurogliaform cells to large multipolar forms. Some of these classes are distributed across all three main layers of the PC, whereas others have a more restricted laminar expression. 3. Distinct and overlapping populations of GABAergic basket cells in Layers II and III of the PC express different combinations of calcium-binding proteins and neuropeptides. Few Layer I interneurons express any of the molecular markers so far examined. 4. The intrinsic firing properties of one or two types of putative PC interneurons have been measured and inhibitory post-synaptic responses have been recorded in PC pyramidal cells following extracellular stimulation. However, little is known about the physiology of the subtypes of interneurons identified. 5. In view of the likely importance of PC interneurons in olfactory learning, olfactory coding and epileptogenesis, further investigation of their properties is likely to be highly informative.