Subthreshold Responses Research Articles

In vivo characterization of colorectal and cutaneous inputs to lumbosacral dorsal horn neurons in the mouse spinal cord

Chronic abdominal pain is a common symptom of inflammatory bowel disease and often persists in the absence of gut inflammation. Although the mechanisms responsible for ongoing pain are unknown, clinical and preclinical evidence suggests lumbosacral spinal cord dorsal horn neurons contribute to these symptoms. At present, we know little about the intrinsic and synaptic properties of this population of neurons in either normal or inflammed conditions. Therefore, we developed an in vivo preparation to make patch-clamp recordings from superficial dorsal horn (SDH) neurons receiving colonic inputs in naïve male mice. Recordings were made in the lumbosacral spinal cord (L6–S1) under isoflurane anesthesia. Noxious colorectal distension (CRD) was used to determine whether SDH neurons received inputs from mechanical stimulation/distension of the colon. Responses to hind paw/tail cutaneous stimulation and intrinsic and synaptic properties were also assessed, as well as action potential discharge properties. Approximately 11% of lumbosacral SDH neurons in the cohort of neurons sampled responded to CRD and a majority of these responses were subthreshold. Most CRD-responsive neurons (80%) also responded to cutaneous stimuli, compared with <50% of CRD-non-responsive neurons. Furthermore, CRD-responsive neurons had more hyperpolarized resting membrane potentials, larger rheobase currents, and reduced levels of excitatory drive, compared to CRD-non-responsive neurons. Our results demonstrate that CRD-responsive neurons can be distinguished from CRD-non-responsive neurons by several differences in their membrane properties and excitatory synaptic inputs. We also demonstrate that SDH neurons with colonic inputs show predominately subthreshold responses to CRD and exhibit a high degree of viscerosomatic convergence.

Specific Early and Late Oddball-Evoked Responses in Excitatory and Inhibitory Neurons of Mouse Auditory Cortex.

A major challenge for sensory processing in the brain is considering stimulus context, such as stimulus probability, which may be relevant for survival. Excitatory neurons in auditory cortex, for example, adapt to repetitive tones in a stimulus-specific manner without fully generalizing to a low-probability deviant tone ("oddball") that breaks the preceding regularity. Whether such stimulus-specific adaptation (SSA) also prevails in inhibitory neurons and how it might relate to deviance detection remains elusive. We obtained whole-cell recordings from excitatory neurons and somatostatin- and parvalbumin-positive GABAergic interneurons in layer 2/3 of mouse auditory cortex and measured tone-evoked membrane potential responses. All cell types displayed SSA of fast ("early") subthreshold and suprathreshold responses with oddball tones of a deviant frequency eliciting enlarged responses compared with adapted standards. SSA was especially strong when oddball frequency matched neuronal preference. In addition, we identified a slower "late" response component (200-400 ms after tone onset), most clearly in excitatory and parvalbumin-positive neurons, which also displayed SSA. For excitatory neurons, this late component reflected genuine deviance detection. Moreover, intracellular blockade of NMDA receptors reduced early and late responses in excitatory but not parvalbumin-positive neurons. The late component in excitatory neurons thus shares time course, deviance detection, and pharmacological features with the deviant-evoked event-related potential known as mismatch negativity (MMN) and provides a potential link between neuronal SSA and MMN. In summary, our results suggest a two-phase cortical activation upon oddball stimulation, with oddball tones first reactivating the adapted auditory cortex circuitry and subsequently triggering delayed reverberating network activity. Significance statement: Understanding how the brain encodes sensory context in addition to stimulus feature has been a main focus in neuroscience. Using in vivo targeted whole-cell recordings from excitatory and inhibitory neurons of mouse primary auditory cortex, we report two temporally distinct components of membrane potential responses encoding oddball tones that break stimulus regularity. Both components display stimulus-specific adaptation upon oddball paradigm stimulation in the three recorded cell types. The late response component, in particular, carries signatures of genuine deviance detection. In excitatory but not parvalbumin-positive inhibitory neurons, both early and late components depend on NMDA receptor-signaling. Our work proposes a potential neuronal substrate of a known deviant-evoked event-related potential, which is of fundamental significance in basic and clinical neuroscience.

Synaptic Basis for Differential Orientation Selectivity between Complex and Simple Cells in Mouse Visual Cortex.

Simple and complex cells, two classes of principal neurons in the primary visual cortex (V1), are generally thought to be equally selective for orientation. In mouse V1, we report that complex cells, identified by their overlapping on/off subfields, has significantly weaker orientation selectivity (OS) than simple cells. This can be primarily attributed to the differential tuning selectivity of inhibitory synaptic input: inhibition in complex cells is more narrowly tuned than excitation, whereas in simple cells inhibition is more broadly tuned than excitation. In addition, there is a good correlation between inhibitory tuning selectivity and the spatial organization of inhibitory inputs. These complex and simple cells with differential degree of OS may provide functionally distinct signals to different downstream targets.

T Cells Integrate Local and Global Cues to Discriminate between Structurally Similar Antigens.

T lymphocytes’ ability to discriminate between structurally related antigens has been attributed to the unique signaling properties of the T cell receptor. However, recent studies have suggested that the output of this discrimination process is conditioned by environmental cues. Here, we demonstrate how the IL-2 cytokine, collectively generated by strongly activated T cell clones, can induce weaker T cell clones to proliferate. We identify the PI3K pathway as being critical for integrating the antigen and cytokine responses and for controlling cell-cycle entry. We build a hybrid stochastic/deterministic computational model that accounts for such signal synergism and demonstrates quantitatively how T cells tune their cell-cycle entry according to environmental cytokine cues. Our findings indicate that antigen discrimination by T cells is not solely an intrinsic cellular property but rather a product of integration of multiple cues, including local cues such as antigen quality and quantity, to global ones like the extracellular concentration of inflammatory cytokines.

Changes in the electrophysiological properties of Bötzinger Complex post‐inspiratory neurons during post‐natal development in rats

Bötzinger Complex (BötC) post-inspiratory (post-I) neurons are part of ventral respiratory group and are involved in the control of respiratory phase duration and upper airway resistance. The post-I neurons are immature at birth and the changes in their electrophysiological properties during the post-natal development were not yet investigated. We investigated the respiratory pattern, the electrophysiological properties and phenotype of BötC post-I neurons on different post-natal days [neonatal, P1-5 (n=10); intermediate, P6-15 (n=19); juvenile, P15-21 (n=29); adult, P22-40 (n=35)] in the in situ preparation of rats. Age-dependent increase in both duration of expiration and post-inspiration over the 14 days of life was observed. Similarly, the post-I increase in the upper airway resistance recorded from subglottal pressure also changed from a small with a short duration, to a pattern of sharp rise and longer duration (> 14 days). In the glicinergic and glutamatergic BötC post-I neurons, burst activity, low threshold spike and low-voltage calcium current also increased during the development. Changes in subthreshold responses were also observed, including an increase in input resistance and excitability. Rheobase current, spike frequency adaptation and action potential duration decreased during post-natal development. We conclude that the post-natal maturation of the respiratory pattern and respiratory control of upper airway resistance in rats are associated to major changes in the electrophysiological properties of BötC post-I neurons. Support: FAPESP

Subthreshold membrane responses underlying sparse spiking to natural vocal signals in auditory cortex.

Natural acoustic communication signals, such as speech, are typically high-dimensional with a wide range of co-varying spectro-temporal features at multiple timescales. The synaptic and network mechanisms for encoding these complex signals are largely unknown. We are investigating these mechanisms in high-level sensory regions of the songbird auditory forebrain, where single neurons show sparse, object-selective spiking responses to conspecific songs. Using whole-cell in vivo patch clamp techniques in the caudal mesopallium and the caudal nidopallium of starlings, we examine song-driven subthreshold and spiking activity. We find that both the subthreshold and the spiking activity are reliable (i.e. the same song drives a similar response each time it is presented) and specific (i.e. responses to different songs are distinct). Surprisingly, however, the reliability and specificity of the subthreshold response was uniformly high regardless of when the cell spiked, even for song stimuli that drove no spikes. We conclude that despite a selective and sparse spiking response, high-level auditory cortical neurons are under continuous, non-selective, stimulus-specific synaptic control. To investigate the role of local network inhibition in this synaptic control, we then recorded extracellularly while pharmacologically blocking local GABAergic transmission. This manipulation modulated the strength and the reliability of stimulus-driven spiking, consistent with a role for local inhibition in regulating the reliability of network activity and the stimulus specificity of the subthreshold response in single cells. We discuss these results in the context of underlying computations that could generate sparse, stimulus-selective spiking responses, and models for hierarchical pooling.

Cortical fosGFP Expression Reveals Broad Receptive Field Excitatory Neurons Targeted by POm

Neighboring cortical excitatory neurons show considerable heterogeneity in their responses to sensory stimulation. We hypothesized that a subset of layer 2 excitatory neurons in the juvenile (P18 to 27) mouse whisker somatosensory cortex, distinguished by expression of the activity-dependent fosGFP reporter gene, would be preferentially activated by whisker stimulation. In fact, two-photon targeted, dual whole-cell recordings showed that principal whisker stimulation elicits similar amplitude synaptic responses in fosGFP-expressing and fosGFP(-) neurons. FosGFP(+) neurons instead displayed shorter latency and larger amplitude subthreshold responses to surround whisker stimulation. Using optogenetic stimulation, we determined that these neurons are targeted by axons from the posteromedial nucleus (POm), a paralemniscal thalamic nucleus associated with broad receptive fields and widespread cortical projections. We conclude that fosGFP expression discriminates between single- and multi-whisker receptive field layer 2 pyramidal neurons.

Density of trap states in a polymer field-effect transistor

We report a more accurate method to determine the density of trap states in a polymer field-effect transistor. In the approach, we describe in this letter, we take into consideration the sub-threshold behavior in the calculation of the density of trap states. This is very important since the sub-threshold regime of operation extends to fairly large gate voltages in these disordered semiconductor based transistors. We employ the sub-threshold drift-limited mobility model (for sub-threshold response) and the conventional linear mobility model for above threshold response. The combined use of these two models allows us to extract the density of states from charge transport data much more accurately. We demonstrate our approach by analyzing data from diketopyrrolopyrrole based co-polymer transistors with high mobility. This approach will also work well for other disordered semiconductors in which sub-threshold conduction is important.

Spiking in auditory cortex following thalamic stimulation is dominated by cortical network activity.

The state of the sensory cortical network can have a profound impact on neural responses and perception. In rodent auditory cortex, sensory responses are reported to occur in the context of network events, similar to brief UP states, that produce “packets” of spikes and are associated with synchronized synaptic input (Bathellier et al., 2012; Hromadka et al., 2013; Luczak et al., 2013). However, traditional models based on data from visual and somatosensory cortex predict that ascending sensory thalamocortical (TC) pathways sequentially activate cells in layers 4 (L4), L2/3, and L5. The relationship between these two spatio-temporal activity patterns is unclear. Here, we used calcium imaging and electrophysiological recordings in murine auditory TC brain slices to investigate the laminar response pattern to stimulation of TC afferents. We show that although monosynaptically driven spiking in response to TC afferents occurs, the vast majority of spikes fired following TC stimulation occurs during brief UP states and outside the context of the L4>L2/3>L5 activation sequence. Specifically, monosynaptic subthreshold TC responses with similar latencies were observed throughout layers 2–6, presumably via synapses onto dendritic processes located in L3 and L4. However, monosynaptic spiking was rare, and occurred primarily in L4 and L5 non-pyramidal cells. By contrast, during brief, TC-induced UP states, spiking was dense and occurred primarily in pyramidal cells. These network events always involved infragranular layers, whereas involvement of supragranular layers was variable. During UP states, spike latencies were comparable between infragranular and supragranular cells. These data are consistent with a model in which activation of auditory cortex, especially supragranular layers, depends on internally generated network events that represent a non-linear amplification process, are initiated by infragranular cells and tightly regulated by feed-forward inhibitory cells.

Sound-induced hyperpolarization of hippocampal neurons

The hippocampus is involved in episodic memory, which is composed of subjective experiences in the multisensory world; however, little is known about the subthreshold membrane potential responses of individual hippocampal neurons to sensory stimuli. Using in-vivo whole-cell patch-clamp recordings from hippocampal CA1 neurons in awake mice, we found that almost all hippocampal neurons exhibited a hyperpolarization of 1-2 mV immediately after the onset of a sound. This large-scale hyperpolarization was unaffected by the duration or pitch of the tone. The response was abolished by general anesthesia and a surgical fimbria-fornix lesion.

Controlling Brain States

Neurons in mouse V1 increase their response to visual stimulation during locomotion. In this issue of Neuron, Lee et al. (2014) show that subthreshold optogenetic stimulation of a brainstem locomotion area can mimic the effect of locomotion on sensory processing.

Subthreshold delta‐frequency resonance in thalamic reticular neurons

The thalamic reticular nucleus (nRt) is an assembly of GABAergic projection neurons that participate in the generation of brain rhythms during synchronous sleep and absence epilepsy. NRt cells receive inhibitory and excitatory synaptic inputs, and are endowed with an intricate set of intrinsic conductances. However, little is known about how intrinsic and synaptic properties interact to generate rhythmic discharges in these neurons. In order to better understand this interaction, I studied the subthreshold responses of nRt cells to time-varying inputs. Patch-clamp recordings were performed in acute slices of rat thalamus (postnatal days 12-21). Sinusoidal current waveforms of linearly changing frequencies were injected into the soma, and the resulting voltage oscillations were recorded. At the resting membrane potential, the impedance profile showed a characteristic resonance at 1.7Hz. The relative strength of the resonance was 1.2, and increased with membrane hyperpolarization. Small suprathreshold current injections led to preferred spike generation at the resonance frequency. Bath application of ZD7288 or Cs(+) , inhibitors of the hyperpolarization-activated cation current (Ih ), transformed the resonance into low-pass behaviour, whereas the T-channel blockers mibefradil and Ni(2+) decreased the strength of the resonance. It is concluded that nRt cells have an Ih -mediated intrinsic frequency preference in the subthreshold voltage range that favours action potential generation in the delta-frequency band.

Plastic corollary discharge predicts sensory consequences of movements in a cerebellum-like circuit.

The capacity to predict the sensory consequences of movements is critical for sensory, motor, and cognitive function. Though it is hypothesized that internal signals related to motor commands, known as corollary discharge, serve to generate such predictions, this process remains poorly understood at the neural circuit level. Here we demonstrate that neurons in the electrosensory lobe (ELL) of weakly electric mormyrid fish generate negative images of the sensory consequences of the fish's own movements based on ascending spinal corollary discharge signals. These results generalize previous findings describing mechanisms for generating negative images of the effects of the fish's specialized electric organ discharge (EOD) and suggest that a cerebellum-like circuit endowed with associative synaptic plasticity acting on corollary discharge can solve the complex and ubiquitous problem of predicting sensory consequences of movements.

Calretinin and calbindin distribution patterns specify subpopulations of type I and type II spiral ganglion neurons in postnatal murine cochlea

As the first neural element in the auditory pathway, neurons in the spiral ganglion shape the initial coding of sound stimuli for subsequent processing. Within the ganglion, type I and type II neurons form divergent and convergent innervation patterns, respectively, with their hair cell sensory receptors, indicating that very different information is gathered and conveyed. Layered onto these basic innervation patterns are structural and electrophysiological features that provide additional levels of processing multifaceted sound stimuli. To understand the nature of this additional complexity of signal coding, we characterized the distribution of calretinin and calbindin, two regulators of intracellular calcium that serve as markers for neuronal subpopulations. We showed in acute preparations and in vitro that calretinin and calbindin staining levels were heterogeneous. Immunocytochemical analysis of colocalization further showed that high levels of staining for the two molecules rarely overlapped. Although varied amounts of calbindin and calretinin were found within each tonotopic location and neuronal type, some distinct subdistributions were noted. For example, calretinin levels were highest in neurons innervating the midcochlea region, whereas calbindin levels were similar across the entire ganglion. Furthermore, we noted that apical type II neurons, identified by antiperipherin labeling, had significantly lower levels of calretinin and higher levels of calbindin. We also established that the endogenous firing feature of onset tau of the subthreshold response showed a pattern related to quantified calretinin and calbindin staining levels. Taken together, our results suggest an additional dimension of complexity within the spiral ganglion beyond that currently categorized.

Mossy fiber-evoked subthreshold responses induce timing-dependent plasticity at hippocampal CA3 recurrent synapses

Dentate granule cells exhibit exceptionally low levels of activity and rarely elicit action potentials in targeted CA3 pyramidal cells. It is thus unclear how such weak input from the granule cells sustains adequate levels of synaptic plasticity in the targeted CA3 network. We report that subthreshold potentials evoked by mossy fibers are sufficient to induce synaptic plasticity between CA3 pyramidal cells, thereby complementing the sparse action potential discharge. Repetitive pairing of a CA3-CA3 recurrent synaptic response with a subsequent subthreshold mossy fiber response induced long-term potentiation at CA3 recurrent synapses in rat hippocampus in vitro. Reversing the timing of the inputs induced long-term depression. The underlying mechanism depends on a passively conducted giant excitatory postsynaptic potential evoked by a mossy fiber that enhances NMDA receptor-mediated current at active CA3 recurrent synapses by relieving magnesium block. The resulting NMDA spike generates a supralinear depolarization that contributes to synaptic plasticity in hippocampal neuronal ensembles implicated in memory.

Differential Receptive Field Properties of Parvalbumin and Somatostatin Inhibitory Neurons in Mouse Auditory Cortex

Cortical inhibitory circuits play important roles in shaping sensory processing. In auditory cortex, however, functional properties of genetically identified inhibitory neurons are poorly characterized. By two-photon imaging-guided recordings, we specifically targeted 2 major types of cortical inhibitory neuron, parvalbumin (PV) and somatostatin (SOM) expressing neurons, in superficial layers of mouse auditory cortex. We found that PV cells exhibited broader tonal receptive fields with lower intensity thresholds and stronger tone-evoked spike responses compared with SOM neurons. The latter exhibited similar frequency selectivity as excitatory neurons. The broader/weaker frequency tuning of PV neurons was attributed to a broader range of synaptic inputs and stronger subthreshold responses elicited, which resulted in a higher efficiency in the conversion of input to output. In addition, onsets of both the input and spike responses of SOM neurons were significantly delayed compared with PV and excitatory cells. Our results suggest that PV and SOM neurons engage in auditory cortical circuits in different manners: while PV neurons may provide broadly tuned feedforward inhibition for a rapid control of ascending inputs to excitatory neurons, the delayed and more selective inhibition from SOM neurons may provide a specific modulation of feedback inputs on their distal dendrites.

Distributional reaction time properties in the Eriksen task: marked differences or hidden similarities with the Simon task?

In conflict tasks, the irrelevant stimulus attribute needs to be suppressed for the correct response to be produced. In the Simon task, earlier researchers have proposed that this suppression is the reason that, after an initial increase, the interference effect decreases for longer RTs, as reflected by late, negative-going delta plots. This view has been challenged by observations of positive-going delta plots, even for long RTs, in other conflict tasks, despite a similar necessity for suppression. For late negative-going delta plots to be interpreted as reflecting suppression, a necessary, although maybe not sufficient, condition is that similar patterns should be observed for other conflict tasks. We reasoned that a similar suppression could be present, but hidden, in the Eriksen flanker task. By recording and analyzing electromyograms of the muscles involved in response execution, we could compute delta plots separately for trials that elicited a subthreshold incorrect response activation (partial error). Late negative-going delta plots were observable on partial-error trials, although they were weaker than for the Simon task, reducing the impact of this inversion on the overall distribution. We further showed that this pattern is modulated by time pressure. Those results indicate that mechanisms leading to negative-going delta plots, similar to those observed in the Simon task, are also at play in the Eriksen task. The link between negative-going delta plots and executive online control is discussed.

An ephaptic transmission model of CA3 pyramidal cells: an investigation into electric field effects.

Extracellular electric fields existing throughout the living brain affect the neural coding and information processing via ephaptic transmission, independent of synapses. A two-compartment whole field effect model (WFEM) of pyramidal neurons embedded within a resistive array which simulates the extracellular medium i.e. ephapse is developed to study the effects of electric field on neuronal behaviors. We derive the two linearized filed effect models (LFEM-1 and LFEM-2) from WFEM at the stable resting state. Through matching these simplified models to the subthreshold membrane response in experiments of the resting pyramidal cells exposed to applied electric fields, we not only verify our proposed model's validity but also found the key parameters which dominate subthreshold frequency response characteristic. Moreover, we find and give its underlying biophysical mechanism that the unsymmetrical properties of active ion channels results in the very different low-frequency response of somatic and dendritic compartments. Following, WFEM is used to investigate both direct-current (DC) and alternating-current field effect on the neural firing patterns by bifurcation analyses. We present that DC electric field could modulate neuronal excitability, with the positive field improving the excitability, the modest negative field suppressing the excitability, but interestingly, the larger negative field re-exciting the neuron back into spiking behavior. The neuron exposed to the sinusoidal electric field exhibits abundant firing patterns sensitive to the input frequency and intensity. In addition, the electrical properties of ephapse can modulate the efficacy of field effect. Our simulated results are qualitatively in line with the relevant experimental results and can explain some experimental phenomena. Furthermore, they are helpful to provide the predictions which can be tested in future experiments.

Subthreshold Mechanisms Underlying State-Dependent Modulation of Visual Responses

The processing of sensory information varies widely across behavioral states. However, little is known about how behavioral states modulate the intracellular activity of cortical neurons to effect changes in sensory responses. Here, we performed whole-cell recordings from neurons in upper-layer primary visual cortex of awake mice during locomotion and quiet wakefulness. We found that the signal-to-noise ratio for sensory responses was improved during locomotion by two mechanisms: (1) a decrease in membrane potential variability leading to a reduction in background firing rates and (2) an enhancement in the amplitude and reliability of visually evoked subthreshold responses mediated by an increase in total conductance and a depolarization of the stimulus-evoked reversal potential. Consistent with the enhanced signal-to-noise ratio for visual responses during locomotion, we demonstrate that performance is improved in a visual detection task during this behavioral state.

Improved detection of electrical activity with a voltage probe based on a voltage‐sensing phosphatase

One of the most awaited techniques in modern physiology is the sensitive detection of spatiotemporal electrical activity in a complex network of excitable cells. The use of genetically encoded voltage probes has been expected to enable such analysis. However, in spite of recent progress, existing probes still suffer from low signal amplitude and/or kinetics too slow to detect fast electrical activity. Here, we have developed an improved voltage probe named Mermaid2, which is based on the voltage-sensor domain of the voltage-sensing phosphatase from Ciona intestinalis and Förster energy transfer between a pair of fluorescent proteins. In mammalian cells, Mermaid2 permits ratiometric readouts of fractional changes of more than 50% over a physiologically relevant voltage range with fast kinetics, and it was used to follow a train of action potentials at frequencies of up to 150 Hz. Mermaid2 was also able to detect single action potentials and subthreshold voltage responses in hippocampal neurons in vitro, in addition to cortical electrical activity evoked by sound stimuli in single trials in living mice.