Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated retrograde tracer that encodes G-CaMP2, a fluorescent calcium sensor protein that is stable at physiological pH and mammalian temperature. This protocol describes the use of PRV369 to express G-CaMP2 in a neuronal circuit and to monitor its activity in a living animal, specifically in the submandibular ganglia (SMG), the peripheral parasympathetic ganglia that innervate the salivary glands. The procedure describes the delivery of PRV369 to the glands and shows how SMG neurons can then be imaged post-inoculation to explore connectivity and activity.
The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.
Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4-11 microm/s to a diameter of approximately 50 microm, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits.
The inferior olive projects climbing fiber axons to cerebellar Purkinje neurons, where they trigger calcium-based dendritic spikes. These responses dynamically shape the immediate spike output of Purkinje cells as well as provide an instructive signal to guide long-term plasticity. Climbing fibers typically fire approximately once a second, and the instructive role is distributed over many such firing events. However, transmission of salient information on an immediate basis needs to occur on a shorter timescale during which a Purkinje cell would typically be activated by a climbing fiber only once. Here we show using in vivo calcium imaging in anesthetized mice and rats that sensory events are rapidly and reliably represented by momentary, simultaneous coactivation of microbands of adjacent Purkinje cells. Microbands were sagittally oriented and spanned up to 100 microm mediolaterally, representing hundreds of Purkinje cells distributed over multiple folia. Spontaneous and sensory-evoked microbands followed boundaries that were close or identical to one another and were desynchronized by olivary injection of the gap junction blocker mefloquine, indicating that excitation to the olive is converted to synchronized firing by electrical coupling. One-time activation of microbands could distinguish a sensory response from spontaneous activity with up to 98% accuracy. Given the anatomy of the olivocerebellar system, microband synchrony may shape the output of neurons in the cerebellar nuclei either via powerful inhibition by Purkinje cells or by direct monosynaptic excitation from the inferior olive.
Like electrical wires, axons carry signals from place to place. However, unlike wires, because of the electrochemical mechanisms for generating and propagating action potentials, the performance of an axon is strongly linked to the costs of its construction and operation. As a consequence, the architecture of brain wiring is biophysically constrained to trade off speed and energetic efficiency against volume. Because the biophysics of axonal conduction is well studied, this tradeoff is amenable to quantitative analysis. In this framework, an examination of axon tract composition can yield insights into neural circuit function in regard to energetics, processing speed, spike timing precision, and average rates of neural activity.
The brains of large mammals have lower rates of metabolism than those of small mammals, but the functional consequences of this scaling are not well understood. An attractive target for analysis is axons, whose size, speed and energy consumption are straightforwardly related. Here we show that from shrews to whales, the composition of white matter shifts from compact, slow-conducting, and energetically expensive unmyelinated axons to large, fast-conducting, and energetically inexpensive myelinated axons. The fastest axons have conduction times of 1-5 ms across the neocortex and <1 ms from the eye to the brain, suggesting that in select sets of communicating fibers, large brains reduce transmission delays and metabolic firing costs at the expense of increased volume. Delays and potential imprecision in cross-brain conduction times are especially great in unmyelinated axons, which may transmit information via firing rate rather than precise spike timing. In neocortex, axon size distributions can account for the scaling of per-volume metabolic rate and suggest a maximum supportable firing rate, averaged across all axons, of 7 +/- 2 Hz. Axon size distributions also account for the scaling of white matter volume with respect to brain size. The heterogeneous white matter composition found in large brains thus reflects a metabolically constrained trade-off that reduces both volume and conduction time.
In vivo multiphoton fluorescence microscopy allows imaging of cellular structures in brain tissue to depths of hundreds of micrometers and, when combined with the use of activity-dependent indicator dyes, opens the possibility of observing intact, functioning neural circuitry. We have developed tools for analyzing in vivo multiphoton data sets to identify responding structures and events in single cells as well as patterns of activity within the neural ensemble. Data were analyzed from populations of cerebellar Purkinje cell dendrites, which generate calcium-based complex action potentials. For image segmentation, active dendrites were identified using a correlation-based method to group covarying pixels. Firing events were extracted from dendritic fluorescence signals with a 95% detection rate and an 8% false-positive rate. Because an event that begins in one movie frame is sometimes not detected until the next frame, detection delays were compensated using a likelihood-based correction procedure. To identify groups of dendrites that tended to fire synchronously, a k-means-based procedure was developed to analyze pairwise correlations across the population. Because repeated runs of k-means often generated dissimilar clusterings, the runs were combined to determine a consensus cluster number and composition. This procedure, termed meta-k-means, gave clusterings as good as individual runs of k-means, was independent of random initial seeding, and allowed the exclusion of outliers. Our methods should be generally useful for analyzing multicellular activity recordings in a variety of brain structures.
Associative long-term depression (LTD) at cerebellar parallel fiber-Purkinje cell synapses is sensitive to the temporal order in which the parallel fiber is coactivated with the climbing fiber input, but how order sensitivity is achieved is unknown. Here we show that the cerebellar inositol-1,4,5-trisphosphate (IP3) receptor, whose activation is required for LTD induction, is sensitive in situ to the order of presentation of its coagonists, IP3 and cytoplasmic calcium. By focally photolyzing a novel caged IP3 compound in dendritic spines, we find that pairing IP3 with climbing fiber-mediated calcium entry leads to a large calcium release transient if the climbing fiber is activated up to 100 ms before or up to 500 ms after IP3 uncaging. This asymmetric timing window for coactivation follows the kinetics of calcium removal and IP3 unbinding from the receptor and is not limited by IP3 metabolism. IP3 receptor binding thus acts as an eligibility trace that can drive temporal order-dependent calcium release and LTD induction in Purkinje cells and event order-dependent sensory plasticity in the whole animal.
Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP3) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP3 than for conventional single-caged IP3, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP3 led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP2), a poorly metabolizable IP3 analog, led to less well localized release. Thus, IP3 breakdown is necessary for spine-specificity. IP3- and gPIP2-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP3 degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP3-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP3 to act as a synapse-specific second messenger. Unexpectedly, single-caged IP3 led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP2 was inhibited by the addition of single-caged IP3, suggesting that single-caged IP3 is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.
At individual synapses, post-synaptic responses include a mixture of "successes" and "failures" in which transmitter is released or not released, respectively. Previously we measured synaptic strength at CA3-CA1 synapses averaged over all trials, including both successes and failures, using an induction protocol that allowed us to observe potentiation and depression events as step-like changes. Here we report quantal properties of 15 of the earlier experiments, including 14 potentiation events and eight depression events. In five experiments both potentiation events and depression events were evoked at the same synapse. During potentiation, success rate increased from 0.56 +/- 0.14 (mean +/- SD) to 0.69 +/- 0.12, and during depression, success rate decreased from 0.70 +/- 0.09 to 0.51 +/- 0.10. During potentiation potency increased from 10 +/- 5 to 19 +/- 9 pA, and during depression, potency decreased from 18 +/- 12 to 12 +/- 7 pA. On average, changes in potency accounted for 76% of the change in response size in potentiation events and 60% of the change in depression events. A reduced-assumption spectral analysis method showed evidence for multiple quantal peaks in distributions of post-synaptic current amplitudes. Consistent with the observed changes in potency, estimated quantal size (Q) increased with potentiation and decreased with depression. A change in potency, which is thought to reflect post-synaptic expression mechanisms, was followed within seconds to minutes by a change in success rate, which is thought to reflect pre-synaptic expression mechanisms. Synaptic plasticity events may therefore consist of changes that occur on both sides of a synapse in a temporally coordinated fashion.
Photolysis of caged compounds is a powerful tool for studying subcellular physiological functions. Here we describe protocols for the alignment and calibration of a focal uncaging system. We also report procedures for convenient quantitative calibration of uncaging. Using these methods, we can achieve submicron lateral resolution of photolysis and probe biological function in spines, the smallest signaling compartments of neurons. Initially, the entire alignment procedure takes 4-6 h to perform; periodic fine-tuning of the system takes 1-2 h.
The magnitude and direction of synaptic plasticity can be determined by the precise timing of presynaptic and postsynaptic action potentials on a millisecond timescale. In vivo, however, neural activity has structure on longer timescales. Here we show that plasticity at the CA3-CA1 synapse depends strongly on parameters other than millisecond spike timing. As a result, the notion that a single spike-timing-dependent plasticity (STDP) rule alone can fully describe the mapping between neural activity and synapse strength is invalid. We have begun to explore the influence of additional behaviorally relevant activity parameters on STDP and found conditions under which underlying spike-timing-dependent rules for potentiation and depression can be separated from one another. Potentiation requires postsynaptic burst firing at 5 Hz or higher, a firing pattern that occurs during the theta rhythm. Potentiation is measurable after only tens of presynaptic-before-postsynaptic pairings. Depression requires hundreds of pairings but has less stringent long timescale requirements and broad timing dependence. By varying these parameters, we obtain STDP curves that are long-term potentiation only, bidirectional, or long-term depression only. This expanded description of the CA3-CA1 learning rule reconciles apparent contradictions between spike-timing-dependent plasticity and previous work at CA3-CA1 synapses.
In populations of synapses, overall synaptic strength can undergo either a net strengthening (long-term potentiation) or weakening (long-term depression). These phenomena have distinct induction pathways, but the functional outcome is usually measured as a single lumped quantity. In hippocampal CA3-CA1 synapses, we took two approaches to study the activity dependence of each phenomenon in isolation. First, we selectively blocked one process by applying kinase or phosphatase inhibitors known, respectively, to block potentiation or depression. Second, we saturated depression or potentiation and examined the activity dependence of the converse process. The resulting unidirectional learning rules could be recombined to give a well-known bidirectional frequency-dependent learning rule under the assumption that when both pathways are activated kinases dominate, resulting in potentiation. Saturation experiments revealed an additional process in which potentiated synapses can be locked at high strength. Saturability of the components of plasticity implies that the amount of plasticity contributed by each pathway depends on the initial level of strength of the synapses. Variation in the distribution of initial synaptic strengths predicts a form of metaplasticity and can account for differences in learning rules observed under several physiological and genetic manipulations.
Biological information storage events are often rapid transitions between discrete states. In neural systems, the initiation of bidirectional plasticity by all-or-none events may help confer robustness on memory storage. Here, we report that at CA3-CA1 hippocampal synapses, individual potentiation and depression plasticity events are discrete and heterogeneous in nature. Individual synapses began from extreme high and low strength states. Unitary plasticity events were all-or-none and drove synaptic strength between extremes in <1 min. Under naïve conditions, approximately three-fourths of synapses began in a low-strength state. The timing of these unitary events can account for the time course of macroscopic synaptic plasticity.
In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-d-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.
Light-sensitive 'caged' molecules provide a means of rapidly and noninvasively manipulating biochemical signals with submicron spatial resolution. Here we describe a new optical system for rapid uncaging in arbitrary patterns to emulate complex neural activity. This system uses TeO(2) acousto-optical deflectors to steer an ultraviolet beam rapidly and can uncage at over 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing us to combine patterned uncaging with imaging and electrophysiology. By photolyzing caged neurotransmitter in brain slices we can generate precise, complex activity patterns for dendritic integration. The method can also be used to activate many presynaptic neurons at once. Patterned uncaging opens new vistas in the study of signal integration and plasticity in neuronal circuits and other biological systems.
Cerebellar parallel fibers are among the thinnest known vertebrate axons and represent an extreme anatomical adaptation. Until now a systematic examination of their properties across species has not been carried out. We used transmission electron microscopy and light microscopy to compare parallel fibers in mammals of different brain sizes. From mouse to macaque, the average unmyelinated parallel fiber diameter was 0.2-0.3 microm, consistent with the idea that they are evolutionarily selected for compactness. Average unmyelinated parallel fiber diameter scaled up slightly with brain size, and across species the estimated total conduction time is 5-10 ms. However, these conduction times can vary by milliseconds, and unmyelinated PFs consume large amounts of energy per action potential. These functional disadvantages are overcome in myelinated parallel fibers, which we found in the deep regions nearest the Purkinje cell layer in marmoset, cat and macaque. These axons were 0.4-1.1 microm wide, have expected conduction times of 0.5-1.0 ms, and may convey fast feedforward inhibition via basket cells to Purkinje cells.
Vertebrate brains vary tremendously in size, but differences in form are more subtle. To bring out functional contrasts that are independent of absolute size, we have normalized brain component sizes to whole brain volume. The set of such volume fractions is the cerebrotype of a species. Using this approach in mammals we previously identified specific associations between cerebrotype and behavioral specializations. Among primates, cerebrotypes are linked principally to enlargement of the cerebral cortex and are associated with increases in the complexity of social structure. Here we extend this analysis to include a second major vertebrate group, the birds. In birds the telencephalic volume fraction is strongly correlated with social complexity. This correlation accounts for almost half of the observed variation in telencephalic size, more than any other behavioral specialization examined, including the ability to learn song. A prominent exception to this pattern is owls, which are not social but still have very large forebrains. Interpolating the overall correlation for Archaeopteryx, an ancient bird, suggests that its social complexity was likely to have been on a par with modern domesticated chickens. Telencephalic volume fraction outperforms residuals-based measures of brain size at separating birds by social structure. Telencephalic volume fraction may be an anatomical substrate for social complexity, and perhaps cognitive ability, that can be generalized across a range of vertebrate brains, including dinosaurs.
Although descriptions of form have been a mainstay of comparative neuroanatomy, less well explored is the use of quantitative approaches, especially at the cellular level. In the neocortex, many gross and cellular anatomical measures show striking regularities over a wide range of brain sizes. Here we review our recent efforts to accurately characterize these scaling trends and explain them in functional terms. We focus on the expansion of white matter volume with increasing brain size and the formation of surface folds, in addition to principles of processing speed and energetics that may explain these phenomena. We also consider exceptional cases of neocortical morphology as a means of testing putative functional principles and developmental mechanisms. We illustrate this point by describing several morphological specializations at the cellular level that may constitute functional adaptations. Taken together, these approaches illustrate the benefits of a synthesis between comparative neuroanatomy and biophysics.
Comparison of mammalian brain parts has often focused on differences in absolute size, revealing only a general tendency for all parts to grow together. Attempts to find size-independent effects using body weight as a reference variable obscure size relationships owing to independent variation of body size and give phylogenies of questionable significance. Here we use the brain itself as a size reference to define the cerebrotype, a species-by-species measure of brain composition. With this measure, across many mammalian taxa the cerebellum occupies a constant fraction of the total brain volume (0.13 +/- 0.02), arguing against the hypothesis that the cerebellum acts as a computational engine principally serving the neocortex. Mammalian taxa can be well separated by cerebrotype, thus allowing the use of quantitative neuroanatomical data to test evolutionary relationships. Primate cerebrotypes have progressively shifted and neocortical volume fractions have become successively larger in lemurs and lorises, New World monkeys, Old World monkeys, and hominoids, lending support to the idea that primate brain architecture has been driven by directed selection pressure. At the same time, absolute brain size can vary over 100-fold within a taxon, while maintaining a relatively uniform cerebrotype. Brains therefore constitute a scalable architecture.