MATLAB-based automated patch-clamp system for awake behaving mice.

Automation has been an important part of biomedical research for decades, and the use of automated and robotic systems is now standard for such tasks as DNA sequencing, microfluidics, and high-throughput screening. Recently, Kodandaramaiah and colleagues (Nat Methods 9: 585-587, 2012) demonstrated, using anesthetized animals, the feasibility of automating blind patch-clamp recordings in vivo. Blind patch is a good target for automation because it is a complex yet highly stereotyped process that revolves around analysis of a single signal (electrode impedance) and movement along a single axis. Here, we introduce an automated system for blind patch-clamp recordings from awake, head-fixed mice running on a wheel. In its design, we were guided by 3 requirements: easy-to-use and easy-to-modify software; seamless integration of behavioral equipment; and efficient use of time. The resulting system employs equipment that is standard for patch recording rigs, moderately priced, or simple to make. It is written entirely in MATLAB, a programming environment that has an enormous user base in the neuroscience community and many available resources for analysis and instrument control. Using this system, we obtained 19 whole cell patch recordings from neurons in the prefrontal cortex of awake mice, aged 8-9 wk. Successful recordings had series resistances that averaged 52 ± 4 MΩ and required 5.7 ± 0.6 attempts to obtain. These numbers are comparable with those of experienced electrophysiologists working manually, and this system, written in a simple and familiar language, will be useful to many cellular electrophysiologists who wish to study awake behaving mice.

A simple MATLAB toolbox for analyzing calcium imaging data in vitro and in vivo.

BACKGROUND: Fluorescence imaging of calcium dynamics in neuronal populations is powerful because it offers a way of relating the activity of individual cells to the broader population of nearby cells. The method's growth across neuroscience has particularly been driven by the introduction of sophisticated mathematical techniques related to motion correction, image registration, cell detection, spike estimation, and population characterization. However, for many researchers, making good use of these techniques has been difficult because they have been devised by different workers and impose differing - and sometimes stringent - technical requirements on those who seek to use them. NEW METHOD: We have built a simple toolbox of analysis routines that encompass the complete workflow for analyzing calcium imaging data. The workflow begins with preprocessing of data, includes motion correction and longitudinal image registration, detects active cells using constrained non-negative matrix factorization, and offers multiple options for estimating spike times and characterizing population activity. The routines can be navigated through a simple graphical user interface. Although written in MATLAB, a standalone version for researchers who do not have access to MATLAB is included. RESULTS: We have used the toolbox on two very different preparations: spontaneously active brain slices and microendoscopic imaging from deep structures in awake behaving mice. In both cases, the toolbox offered a seamless flow from raw data all the way through to prepared graphs. CONCLUSION: The field of calcium imaging has benefited from the development of numerous innovative mathematical techniques. Here we offer a simple toolbox that allows ordinary researchers to fully exploit these techniques.

Distinct subpopulations of ventral pallidal cholinergic projection neurons encode valence of olfactory stimuli.

To better understand the function of cholinergic projection neurons in the ventral pallidum (VP), we examined behavioral responses to appetitive (APP) and aversive (AV) odors that elicited approach or avoidance, respectively. Exposure to each odor increased cFos expression and calcium signaling in VP cholinergic neurons. Activity and Cre-dependent viral vectors selectively labeled VP cholinergic neurons that were activated and reactivated in response to either APP or AV odors, but not both, identifying two non-overlapping populations of VP cholinergic neurons differentially activated by the valence of olfactory stimuli. These two subpopulations showed differences in electrophysiological properties, morphology, and projections to the basolateral amygdala. Although VP neurons are engaged in both approach and avoidance behavioral responses, cholinergic signaling is only required for approach behavior. Thus, two distinct subpopulations of VP cholinergic neurons differentially encode valence of olfactory stimuli and play distinct roles in approach and avoidance behaviors.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically-encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can "learn" the association between a naïve tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24h later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can 'learn' the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

Distinct subpopulations of ventral pallidal cholinergic projection neurons encode valence of olfactory stimuli.

The ventral pallidum (VP) mediates motivated behaviors largely via the action of VP GABA and glutamatergic neurons. In addition to these neuronal subtypes, there is a population of cholinergic projection neurons in the VP, whose functional significance remains unclear. To understand the functional role of VP cholinergic neurons, we first examined behavioral responses to an appetitive (APP) odor that elicited approach, and an aversive (AV) odor that led to avoidance. To examine how VP cholinergic neurons were engaged in APP vs. AV responses, we used an immediate early gene marker and in-vivo fiber photometry, examining the activation profile of VP cholinergic neurons in response to each odor. Exposure to each odor led to an increase in the number of cFos counts and increased calcium signaling of VP cholinergic neurons. Activity and cre-dependent viral vectors were designed to label engaged VP cholinergic neurons in two distinct contexts: (1) exposure to the APP odor, (2) followed by subsequent exposure to the AV odor, and vice versa. These studies revealed two distinct, non-overlapping subpopulations of VP cholinergic neurons: one activated in response to the APP odor, and a second distinct population activated in response to the AV odor. These two subpopulations of VP cholinergic neurons are spatially intermingled within the VP, but show differences in electrophysiological properties, neuronal morphology, and projections to the basolateral amygdala. Although VP cholinergic neurons are engaged in behavioral responses to each odor, VP cholinergic signaling is only required for approach behavior. Indeed, inhibition of VP cholinergic neurons not only blocks approach to the APP odor, but reverses the behavior, leading to active avoidance. Our results highlight the functional heterogeneity of cholinergic projection neurons within the VP. These two subpopulations of VP cholinergic neurons differentially encode valence of olfactory stimuli and play unique roles in approach and avoidance behaviors.

Synaptic and intrinsic balancing during postnatal development in rat pups exposed to valproic acid in utero.

Valproic acid (VPA) is among the most teratogenic of commonly prescribed anticonvulsants, increasing the risk in humans of major malformations and impaired cognitive development. Likewise, rats exposed prenatally to VPA exhibit a variety of neuroanatomical and behavioral abnormalities. Previous work has shown that pyramidal neuron physiology in young VPA-exposed animals is marked by two strong abnormalities: an impairment in intrinsic neuronal excitability and an increase in NMDA synaptic currents. In this study, we investigated these abnormalities across postnatal development using whole-cell patch recordings from layer 2/3 neurons of medial prefrontal cortex. We found that both abnormalities were at a peak soon after birth but were gradually corrected as animals matured, to the extent that normal excitability and NMDA currents had been restored by early adolescence. The manner in which this correction happened suggested coordination between the two processes. Using computational models fitted to the physiological data, we argue that the two abnormalities trade off against each other, with the effects on network activity of the one balancing the effects of the other. This may constitute part of the nervous system's homeostatic response to teratogenic insult: an attempt to maintain stability despite a strong challenge.

Pyramidal neuron conductance state gates spike-timing-dependent plasticity.

Neocortical neurons in vivo process each of their individual inputs in the context of ongoing synaptic background activity, produced by the thousands of presynaptic partners a typical neuron has. Previous work has shown that background activity affects multiple aspects of neuronal and network function. However, its effect on the induction of spike-timing dependent plasticity (STDP) is not clear. Here we report that injections of simulated background conductances (produced by a dynamic-clamp system) into pyramidal cells in rat brain slices selectively reduced the magnitude of timing-dependent synaptic potentiation while leaving the magnitude of timing-dependent synaptic depression unchanged. The conductance-dependent suppression also sharpened the STDP curve, with reliable synaptic potentiation induced only when EPSPs and action potentials (APs) were paired within 8 ms of each other. Dual somatic and dendritic patch recordings suggested that the deficit in synaptic potentiation arose from shunting of dendritic EPSPs and APs. Using a biophysically detailed computational model, we were not only able to replicate the conductance-dependent shunting of dendritic potentials, but show that synaptic background can truncate calcium dynamics within dendritic spines in a way that affects potentiation more strongly than depression. This conductance-dependent regulation of synaptic plasticity may constitute a novel homeostatic mechanism that can prevent the runaway synaptic potentiation to which Hebbian networks are vulnerable.

Critical periods for experience-dependent synaptic scaling in visual cortex.

The mechanisms underlying experience-dependent plasticity and refinement of central circuits are not yet fully understood. A non-Hebbian form of synaptic plasticity, which scales synaptic strengths up or down to stabilize firing rates, has recently been discovered in cultured neuronal networks. Here we demonstrate the existence of a similar mechanism in the intact rodent visual cortex. The frequency of miniature excitatory postsynaptic currents (mEPSCs) in principal neurons increased steeply between post-natal days 12 and 23. There was a concomitant decrease in mEPSC amplitude, which was prevented by rearing rats in complete darkness from 12 days of age. In addition, as little as two days of monocular deprivation scaled up mEPSC amplitude in a layer- and age-dependent manner. These data indicate that mEPSC amplitudes can be globally scaled up or down as a function of development and sensory experience, and suggest that synaptic scaling may be involved in the activity-dependent refinement of cortical connectivity.

Synaptic bombardment modulates muscarinic effects in forelimb motor cortex.

Neocortical neurons in vivo exist in an environment of continuous synaptic bombardment, receiving a complex barrage of excitatory and inhibitory inputs. This background activity (by depolarizing neurons, increasing membrane conductance, and introducing fluctuations) strongly alters many aspects of neuronal responsiveness. In this study, we asked how it shapes neuromodulation of postsynaptic responses. Specifically, we examined muscarinic modulation of forelimb motor cortex, a brain area in which cholinergic stimulation is known to be necessary for modifications during motor skill learning. Using a dynamic clamp system to inject simulated conductances into pyramidal neurons in motor cortical slices, we mimicked in vivo-like activity by introducing a random background of excitatory and inhibitory inputs. When muscarinic receptors were stimulated with the agonist oxotremorine-M, several previously described currents were modified, and excitability was increased. However, the presence of the background conductances strongly attenuated most muscarinic agonist effects, with the notable exception that sustained firing responses to trains of inputs were well preserved. This may be important for promoting plasticity in vivo.

A Dynamic Clamp on Every Rig.

The dynamic clamp should be a standard part of every cellular electrophysiologist's toolbox. That it is not, even 25 years after its introduction, comes down to three issues: money, the disruption that adding dynamic clamp to an existing electrophysiology rig entails, and the technical prowess required of experimenters. These have been valid and limiting issues in the past, but no longer. Technological advances associated with the so-called maker movement render them moot. We demonstrate this by implementing a fast (∼100 kHz) dynamic clamp system using an inexpensive microcontroller (Teensy 3.6). The overall cost of the system is less than USD$100, and assembling it requires no prior electronics experience. Modifying it-for example, to add Hodgkin-Huxley-style conductances-requires no prior programming experience. The system works together with existing electrophysiology data acquisition systems (for Macintosh, Windows, and Linux); it does not attempt to supplant them. Moreover, the process of assembling, modifying, and using the system constitutes a useful pedagogical exercise for students and researchers with no background but an interest in electronics and programming. We demonstrate the system's utility by implementing conductances as fast as a transient sodium conductance and as complex as the Ornstein-Uhlenbeck conductances of the "point conductance" model of synaptic background activity.

Bursts as a unit of neural information: selective communication via resonance.

What is the functional significance of generating a burst of spikes, as opposed to a single spike? A dominant point of view is that bursts are needed to increase the reliability of communication between neurons. Here, we discuss the alternative, but complementary, hypothesis: bursts with specific resonant interspike frequencies are more likely to cause a postsynaptic cell to fire than are bursts with higher or lower frequencies. Such a frequency preference might occur at the level of individual synapses because of the interplay between short-term synaptic depression and facilitation, or at the postsynaptic cell level because of subthreshold membrane potential oscillations and resonance. As a result, the same burst could resonate for some synapses or cells and not resonate for others, depending on their natural resonance frequencies. This observation suggests that, in addition to increasing reliability of synaptic transmission, bursts of action potentials might provide effective mechanisms for selective communication between neurons.

Trace Eyeblink Conditioning in Mice Is Dependent upon the Dorsal Medial Prefrontal Cortex, Cerebellum, and Amygdala: Behavioral Characterization and Functional Circuitry

Trace eyeblink conditioning is useful for studying the interaction of multiple brain areas in learning and memory. The goal of the current work was to determine whether trace eyeblink conditioning could be established in a mouse model in the absence of elicited startle responses and the brain circuitry that supports this learning. We show here that mice can acquire trace conditioned responses (tCRs) devoid of startle while head-restrained and permitted to freely run on a wheel. Most mice (75%) could learn with a trace interval of 250 ms. Because tCRs were not contaminated with startle-associated components, we were able to document the development and timing of tCRs in mice, as well as their long-term retention (at 7 and 14 d) and flexible expression (extinction and reacquisition). To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented. Furthermore, inactivation of the cerebellum with muscimol completely abolished tCRs, demonstrating that learned responses were driven by the cerebellum. Finally, inactivation of the mPFC and amygdala in trained animals nearly abolished tCRs. Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons. The data provide the first report of trace eyeblink conditioning in mice in which tCRs were driven by the cerebellum and required a localized region of mPFC for acquisition. The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

Homeostatic plasticity in the CNS: synaptic and intrinsic forms.

The study of experience-dependent plasticity has been dominated by questions of how Hebbian plasticity mechanisms act during learning and development. This is unsurprising as Hebbian plasticity constitutes the most fully developed and influential model of how information is stored in neural circuits and how neural circuitry can develop without extensive genetic instructions. Yet Hebbian plasticity may not be sufficient for understanding either learning or development: the dramatic changes in synapse number and strength that can be produced by this kind of plasticity tend to threaten the stability of neural circuits. Recent work has suggested that, in addition to Hebbian plasticity, homeostatic regulatory mechanisms are active in a variety of preparations. These mechanisms alter both the synaptic connections between neurons and the intrinsic electrical properties of individual neurons, in such a way as to maintain some constancy in neuronal properties despite the changes wrought by Hebbian mechanisms. Here we review the evidence for homeostatic plasticity in the central nervous system, with special emphasis on results from cortical preparations.

Homeostatic Plasticity and STDP: Keeping a Neuron's Cool in a Fluctuating World.

Spike-timing-dependent plasticity (STDP) offers a powerful means of forming and modifying neural circuits. Experimental and theoretical studies have demonstrated its potential usefulness for functions as varied as cortical map development, sharpening of sensory receptive fields, working memory, and associative learning. Even so, it is unlikely that STDP works alone. Unless changes in synaptic strength are coordinated across multiple synapses and with other neuronal properties, it is difficult to maintain the stability and functionality of neural circuits. Moreover, there are certain features of early postnatal development (e.g., rapid changes in sensory input) that threaten neural circuit stability in ways that STDP may not be well placed to counter. These considerations have led researchers to investigate additional types of plasticity, complementary to STDP, that may serve to constrain synaptic weights and/or neuronal firing. These are collectively known as "homeostatic plasticity" and include schemes that control the total synaptic strength of a neuron, that modulate its intrinsic excitability as a function of average activity, or that make the ability of synapses to undergo Hebbian modification depend upon their history of use. In this article, we will review the experimental evidence for homeostatic forms of plasticity and consider how they might interact with STDP during development, and learning and memory.

Early postnatal plasticity in neocortex of Fmr1 knockout mice.

Fragile X syndrome is produced by a defect in a single X-linked gene, called Fmr1, and is characterized by abnormal dendritic spine morphologies with spines that are longer and thinner in neocortex than those from age-matched controls. Studies using Fmr1 knockout mice indicate that spine abnormalities are especially pronounced in the first month of life, suggesting that altered developmental plasticity underlies some of the behavioral phenotypes associated with the syndrome. To address this issue, we used intracellular recordings in neocortical slices from early postnatal mice to examine the effects of Fmr1 disruption on two forms of plasticity active during development. One of these, long-term potentiation of intrinsic excitability, is intrinsic in expression and requires mGluR5 activation. The other, spike timing-dependent plasticity, is synaptic in expression and requires N-methyl-d-aspartate receptor activation. While intrinsic plasticity was normal in the knockout mice, synaptic plasticity was altered in an unusual and striking way: long-term depression was robust but long-term potentiation was entirely absent. These findings underscore the ideas that Fmr1 has highly selective effects on plasticity and that abnormal postnatal development is an important component of the disorder.

Relating STDP to BCM.

We demonstrate that the BCM learning rule follows directly from STDP when pre- and postsynaptic neurons fire uncorrelated or weakly correlated Poisson spike trains, and only nearest-neighbor spike interactions are taken into account.