Excitatory Interneurons Research Articles

Selective mGlu1 Potentiation Reverses Cortical Disinhibition and Schizophrenia‐like Social and Cognitive Deficits

There is a critical need to develop novel therapeutic strategies for addressing the negative and cognitive symptom domains in schizophrenia (SCZ). Multiple clinical and preclinical studies support the hypothesis that loss of GABAergic inhibitory transmission in the prefrontal cortex (PFC) and other forebrain regions may play a critical role in the pathophysiological changes underlying cognitive and social behavioral deficits in SCZ patients. Recently, we demonstrated that activation of the mGlu1 subtype of metabotropic glutamate receptor increases inhibitory transmission in the PFC by selective excitation of somatostatin‐expressing GABA interneurons. Genetic studies in humans reveal an association between deleterious non‐synonymous single nucleotide polymorphisms (nsSNPS) in of the human gene encoding mGlu1 (GRM1) and SCZ, raising the possibility that mGlu1 signaling is critical to the function of brain circuits underlying symptoms associated with this disorder.Here, we examine whether novel positive allosteric modulators (PAMs) selective for mGlu1 reverse electrophysiological effects and behavioral deficits in an MK‐801‐induced animal model of SCZ. We used ex vivo whole‐cell patch‐clamp electrophysiology to show that MK‐801 decreased the frequency of spontaneous inhibitory postsynaptic currents onto layer V pyramidal cells of the PFC, and this cortical disinhibition was reversed by mGlu1 activation. Furthermore, acute MK‐801 treatment induces inhibitory deficits onto PFC‐basolateral amygdala (BLA), but not PFC‐nucleus accumbens, projecting layer V pyramidal cells that can be reversed by selective mGlu1 activation. PFC‐BLA projections are known to contribute functionally to social‐approach behavior, while the BLA plays a critical role in object recognition and cognition broadly. We found that the mGlu1 PAM VU6004909 effectively reversed MK801‐induced deficits in social interaction and social novelty preference in a three‐chamber assay. Additionally, VU6004909 restored novel object recognition following MK‐801 treatment. In combination with our previous reports that mGlu1 PAMs reverse deficits in inhibitory transmission in the PFC, the current study provides strong evidence for mGlu1 PAMs as a promising novel approach for the treatment of social and cognitive deficits in SCZ patients.

Chronic, episodic developmental nicotine exposure alters GABAergic synaptic transmission to hypoglossal motor neurons at a critical developmental age in rats

The adverse effects of nicotine exposure on brain structural and functional development is attributed to its action on the nicotinic acetylcholine receptors (nAChRs). nAChRs are expressed post‐synaptically on hypoglossal motor neurons (XIIMNs), the neurons that innervate the tongue. We recently showed that chronic, episodic developmental nicotine exposure alters the intrinsic properties of these neurons, which may be one mechanism to explain impaired tongue muscle function that is commonly observed in nicotine‐ exposed human and animal neonates. Importantly, along with the intrinsic properties of XIIMNs, the balance of excitatory and inhibitory fast‐synaptic inputs to these neurons is important in dictating the strength of motor output to the tongue. nAChRs are also located on the soma, dendrites, and axon end terminals of excitatory and inhibitory interneurons that synapse onto XIIMNs, activation of which increases action potential firing and neurotransmitter release. Here, we tested the hypothesis that chronic, episodic developmental nicotine exposure alters GABAergic transmission, a major source of fast‐synaptic inhibition, to XIIMNs. XIIMNs were recorded in brainstem slices from control and nicotine‐ exposed rats in two age groups: postnatal days (P)1‐5 and P10‐12, which is considered a critical period of brain development. In voltage‐ clamp mode, the frequency of GABAergic spontaneous inhibitory post‐synaptic currents (sIPSCs) was recorded at baseline and in response to activation of nAChRs with acute, bath‐applied nicotine. The baseline frequency of GABAergic sIPSCs was not different between control and nicotine‐exposed neurons at either age. In the P1‐5 age group, acute nicotine increased the frequency of GABAergic sIPSCs in both control and nicotine‐exposed neurons. However, at ages P10‐12, acute nicotine increased the frequency of GABAergic sIPSCs in nicotine‐exposed neurons, but had no effect in control neurons. These findings indicate that chronic, episodic nicotine exposure alters GABAergic synaptic transmission to XIIMNs at a critical developmental age, and may contribute to excitatory/inhibitory imbalance that underlies impaired control of the muscles of the tongue.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Molecular and neural basis of pleasant touch sensation

Pleasant touch provides emotional and psychological support that helps mitigate social isolation and stress. However, the underlying mechanisms remain poorly understood. Using a pleasant touch-conditioned place preference (PT-CPP) test, we show that genetic ablation of spinal excitatory interneurons expressing prokineticin receptor 2 (PROKR2), or its ligand PROK2 in sensory neurons, abolishes PT-CPP without impairing pain and itch behaviors in mice. Mutant mice display profound impairments in stress response and prosocial behaviors. Moreover, PROKR2 neurons respond most vigorously to gentle stroking and encode reward value. Collectively, we identify PROK2 as a long-sought neuropeptide that encodes and transmits pleasant touch to spinal PROKR2 neurons. These findings may have important implications for elucidating mechanisms by which pleasant touch deprivation contributes to social avoidance behavior and mental disorders.

Time‐limited alterations in cortical activity of a knock‐in mouse model of KCNQ2‐related developmental and epileptic encephalopathy

De novo missense variants in the KCNQ2 gene encoding the Kv7.2 subunit of voltage-gated potassium Kv7/M channels are the main cause of developmental and epileptic encephalopathy with neonatal onset. Although seizures usually resolve during development, cognitive/motor deficits persist. To gain a better understanding of the cellular mechanisms underlying network dysfunction and their progression over time, we investigated in vivo, using local field potential recordings of freely moving animals, and ex vivo in layers II/III and V of motor cortical slices, using patch-clamp recordings, the electrophysiological properties of pyramidal cells from a heterozygous knock-in mouse model carrying the Kv7.2 p.T274M pathogenic variant during neonatal, postweaning and juvenile developmental stages. We found that knock-in mice displayed spontaneous seizures preferentially at postweaning rather than at juvenile stages. At the cellular level, the variant led to a reduction in M ​​current density/conductance and to neuronal hyperexcitability. These alterations were observed during the neonatal period in pyramidal cells of layers II/III and during the postweaning stage in pyramidal cells of layer V. Moreover, there was an increase in the frequency of spontaneous network-driven events mediated by GABA receptors, suggesting that the excitability of interneurons was also increased. However, all these alterations were no longer observed in layers II/III and V of juvenile mice. Thus, our data indicate that the action of the variant is regulated developmentally. This raises the possibility that the age-related seizure remission observed in KCNQ2-related developmental and epileptic encephalopathy patients results from a time-limited alteration of Kv7 channel activity and neuronal excitability. KEY POINTS: The electrophysiological impact of the pathogenic c.821C>T mutation of the KCNQ2 gene (p.T274M variant in Kv7.2 subunit) related to developmental and epileptic encephalopathy has been analysed both in vivo and ex vivo in layers II/III and V of motor cortical slices from a knock-in mouse model during development at neonatal, postweaning and juvenile stages. Mcurrent density and conductance are decreased and the excitability of layer II/III pyramidal cells is increased in slices from neonatal and postweaning knock-in mice but not from juvenile knock-in mice. Mcurrent and excitability of layer V pyramidal cells are impacted in knock-in mice only at the postweaning stage. Spontaneous GABAergic network-driven events can be recorded until the postweaning stage, and their frequency is increased in layers II/III of the knock-in mice. Knock-in mice display spontaneous seizures preferentially at postweaning rather than at juvenile stages.

Locomotor Pattern and Force Generation Modulated by Ionic Channels: A Computational Study of Spinal Networks Underlying Locomotion.

Locomotion is a fundamental movement in vertebrates produced by spinal networks known as central pattern generators (CPG). During fictive locomotion cat lumbar motoneurons (MNs) exhibit changes in membrane properties, including hyperpolarization of voltage threshold, reduction of afterhyperpolarization and input resistance, and amplification of nonlinear membrane properties. Both modeling and electrophysiological studies suggest that these changes can be produced by upregulating voltage-gated sodium channel (VGSC), persistent sodium (NaP), or L-type calcium channel (LTCC) or downregulating delayed-rectifier potassium (K(DR)) or calcium-dependent potassium channel (KCa) in spinal MNs. Further studies implicate that these channel modulations increase motor output and facilitate MN recruitment. However, it remains unknown how the channel modulation of CPG networks or MN pools affects the rhythmic generation of locomotion and force production of skeletal muscle during locomotion. In order to investigate this issue, we built a two-level CPG model composed of excitatory interneuron pools (Exc-INs), coupled reciprocally with inhibitory interneuron pools (Inh-INs), and projected to the flexor-extensor MN pools innervating skeletal muscles. Each pool consisted of 100 neurons with membrane properties based on cat spinal neurons. VGSC, K(DR), NaP, KCa, LTCC, and H-current channels were included in the model. Simulation results showed that (1) upregulating VGSC, NaP, or LTCC or downregulating KCa in MNs increased discharge rate and recruitment of MNs, thus facilitating locomotor pattern formation, increased amplitude of electroneurogram (ENG) bursting, and enhanced force generation of skeletal muscles. (2) The same channel modulation in Exc-INs increased the firing frequency of the Exc-INs, facilitated rhythmic generation, and increased flexor-extensor durations of step cycles. (3) Contrarily, downregulation of NaP or LTCC in MNs or Exc-INs or both CPG (Exc-INs and Inh-INs) and MNs disrupted locomotor pattern and reduced or even blocked the ENG bursting of MNs and force generation of skeletal muscles. (4) Pharmacological experiments showed that bath application of 25 μM nimodipine or 2 μM riluzole completely blocked fictive locomotion in isolated rat spinal cord, consistent with simulation results. We concluded that upregulation of VGSC, NaP, or LTCC or downregulation of KCa facilitated rhythmic generation and force production during walking, with NaP and LTCC playing an essential role.

A new transgenic reporter line reveals expression of protocadherin 9 at a cellular level within the zebrafish central nervous system

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.

The T-Type Calcium Channel Cav3.2 in Somatostatin Interneurons in Spinal Dorsal Horn Participates in Mechanosensation and Mechanical Allodynia in Mice.

Somatostatin-positive (SOM+) neurons have been proposed as one of the key populations of excitatory interneurons in the spinal dorsal horn involved in mechanical pain. However, the molecular mechanism for their role in pain modulation remains unknown. Here, we showed that the T-type calcium channel Cav3.2 was highly expressed in spinal SOM+ interneurons. Colocalization of Cacna1h (which codes for Cav3.2) and SOMtdTomato was observed in the in situ hybridization studies. Fluorescence-activated cell sorting of SOMtdTomato cells in spinal dorsal horn also proved a high expression of Cacna1h in SOM+ neurons. Behaviorally, virus-mediated knockdown of Cacna1h in spinal SOM+ neurons reduced the sensitivity to light touch and responsiveness to noxious mechanical stimuli in naïve mice. Furthermore, knockdown of Cacna1h in spinal SOM+ neurons attenuated thermal hyperalgesia and dynamic allodynia in the complete Freund’s adjuvant-induced inflammatory pain model, and reduced both dynamic and static allodynia in a neuropathic pain model of spared nerve injury. Mechanistically, a decrease in the percentage of neurons with Aβ-eEPSCs and Aβ-eAPs in superficial dorsal horn was observed after Cacna1h knockdown in spinal SOM+ neurons. Altogether, our results proved a crucial role of Cav3.2 in spinal SOM+ neurons in mechanosensation under basal conditions and in mechanical allodynia under pathological pain conditions. This work reveals a molecular basis for SOM+ neurons in transmitting mechanical pain and shows a functional role of Cav3.2 in tactile and pain processing at the level of spinal cord in addition to its well-established peripheral role.

Voltage imaging and spinal circuits get along swimmingly

Voltage imaging promises to unite optical and electrical approaches to accelerate circuit discovery. In this issue of Neuron, Böhm etal. (2022) use voltage imaging to explore the structure and functional dynamics of spinal excitatory interneurons in larval zebrafish and reveal the role of V3 neurons in adaptive locomotor control.

Optimization of stimulation waveforms for regulating spike-wave discharges in a thalamocortical model

Based on our modified thalamocortical model considering excitatory interneuron, this study incorporates deep brain stimulation (DBS) applied to pyramidal cluster and focuses on optimizing stimulus waveforms of DBS. The epileptic activities can be reproduced and modulated by inhibitory and excitatory interneurons, which are closely related to the generation of absence seizures. Under the bifurcation mechanism analysis, fold limit cycle and Hopf bifurcations well explain the termination and transition of various discharge states. To offer a potential way to improve control effects and energy savings, we investigate a range of waveforms covering rectangular, linear increase and decrease, triangular, exponential increase and decrease, Gaussian and sinusoidal pulse in detail. These single pulses all can effectively remove spike-wave discharges (SWD) with appropriate stimulus parameters, except for exponential decrease pulse. Additionally, stimulation outcomes are uncovered by adjusting stimulus timing and pulse width, and the optimal pulse width of each waveform has been suggested. By comprehensively analyzing the control rate, charge injected ability and energy requirement, the sinusoidal pulse achieves a better impact on eliminating SWD. From the perspective of rectangular and sinusoidal stimulations, the single pulse is of great advantage comparing to the traditional DBS, which may give some enlightenment for the clinical treatment, especially extend the application of DBS in neurological disorders.

Cerebellar noninvasive neuromodulation influences the reactivity of the contralateral primary motor cortex and surrounding areas: a TMS-EMG-EEG study.

Understanding cerebellar-cortical physiological interactions is of fundamental importance to advance the efficacy of neurorehabilitation strategies for patients with cerebellar damage. Previous works have aimed to modulate this pathway by applying transcranial electrical or magnetic stimulation (TMS) over the cerebellum and probing the resulting changes in the primary motor cortex (M1) excitability with motor-evoked potentials (MEPs). While these protocols produce changes in cerebellar excitability, their ability to modulate MEPs has produced inconsistent results, mainly due to the MEP being a highly variable outcome measure that is susceptible to fluctuations in the excitability of M1 neurons and spinal interneurons. To overcome this limitation, we combined TMS with electroencephalography (EEG) to directly record TMS-evoked potentials (TEPs) and oscillations from the scalp. In three sessions, we applied intermittent theta-burst stimulation (iTBS), cathodal direct current stimulation (c-DC) or sham stimulation to modulate cerebellar activity. To assess the effects on M1 and nearby cortex, we recorded TMS-EEG and MEPs before, immediately after (T1) and 15min (T2) following cerebellar neuromodulation. We found that cerebellar iTBS immediately increased TMS-induced alpha oscillations and produced lasting facilitatory effects on TEPs, whereas c-DC immediately decreased TMS-induced alpha oscillations and reduced TEPs. We also found increased MEP following iTBS but not after c-DC. All of the TMS-EEG measures showed high test-retest repeatability. Overall, this work importantly shows that cerebellar neuromodulation influences both cortical and corticospinal physiological measures; however, they are more pronounced and detailed when utilizing TMS-EEG outcome measures. These findings highlight the advantage of using TMS-EEG over MEPs when assessing the effects of neuromodulation.

KNa1.1 gain-of-function preferentially dampens excitability of murine parvalbumin-positive interneurons

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

V3 Interneurons Are Active and Recruit Spinal Motor Neurons during In Vivo Fictive Swimming in Larval Zebrafish.

Survival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude of motor output combine in a multiplicative manner to determine features of locomotion such as distance traveled, speed, force (thrust), and vigor. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is a less thorough mechanistic understanding for how locomotor amplitude is established. Recent evidence indicates that locomotor amplitude is regulated in part by a subset of functionally and morphologically distinct V2a excitatory spinal interneurons (Type II, nonbursting) in larval and adult zebrafish. Here, we provide direct evidence that most V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. We also show that elimination of the spinal V3-IN population reduces the proportion of active motor neurons (MNs) during fictive swimming but does not alter the range of locomotor frequencies produced. These data are consistent with V3-INs providing excitatory drive to spinal MNs during swimming in larval zebrafish and may contribute to the production of locomotor amplitude independently of locomotor frequency.

126 Regulator of G Protein Signaling 4 (RGS4) Gene Expression is Upregulated in the Superficial Spinal Dorsal Horn and in Dorsal Root Ganglion Neurons in a Murine Model of Neuropathic Pain

INTRODUCTION: Regulator of G protein signaling 4 (RGS4), a potent negative regulator of G-protein coupled receptor signaling, is a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and is upregulated in response to peripheral inflammation and nerve injury. METHODS: To understand the dynamics of RGS4 gene expression after nerve injury, we perform the spared-nerve injury (SNI) model of neuropathic pain in C57BL/6. At 21 days (DRG) or 2.5 months (spinal cord) post-surgery (SNI vs sham), animals were sacrificed and specimens were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RESULTS: RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (p = 0.0384). In sham-treated mice, there were no differences in cell-type-specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p = 0.4170). Cell-type-specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; p = 0.0015; one-way ANOVA) following SNI. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p = 0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p = 0.0012). In addition, neuronal-specific RGS4 expression was significantly increased in the DRG in SNI vs Sham-injured mice (p = 0.0050). CONCLUSION: Our data suggest that upregulation of RGS4 expression in the superficial dorsal horn and DRG may play a crucial role in maintenance of chronic pain following nerve injury.

Mechanism analysis for excitatory interneurons dominating poly-spike wave and optimization of electrical stimulation.

In addition to inhibitory interneurons, there exist excitatory interneurons (EINs) in the cortex, which mainly have excitatory projections to pyramidal neurons. In this study, we improve a thalamocortical model by introducing EIN, investigate the dominant role of EIN in generating spike and slow wave discharges (SWDs), and consider a non-rectangular pulse to control absence seizures. First, we display here that the improved model can reproduce typical SWDs of absence seizures. Moreover, we focus on the function of EIN by means of bifurcation analysis and find that EIN can induce transition behaviors under Hopf-type and fold limit cycle bifurcations. Specifically, the system has three stable solutions composing a tri-stable region. In this region, there are three attraction basins, which hints that external stimulation can drive the system trajectory from one basin to another, thereby eliminating abnormal oscillations. Furthermore, we compare the increasing ramp with rectangular pulse and optimize stimulation waveforms from the perspective of electrical charges input. The controlling role of the single increasing ramp to absence seizures is remarkable and the optimal stimulus parameters have been found theoretically. This work provides a computational model containing EIN and a theoretical basis for future physiological experiments and clinical research studies.

Diverse roles and modulations of IA in spinal cord pain circuits.

This review highlights recent findings of different amplitude ranges, roles, and modulations of A-type K+ currents (IA) in excitatory (GAD67-GFP-) and inhibitory (GAD67-GFP+) interneurons in mouse spinal cord pain pathways. Endogenous neuropeptides, such as TAFA4, oxytocin, and dynorphin in particular, have been reported to modulate IA in these pain pathways, but only TAFA4 has been shown to fully reverse the opposing modulations that occur selectively in LIIo GAD67-GFP- and LIIi GAD67-GFP+ interneurons following bothneuropathic and inflammatory pain. If, as hypothesized here, Kv4 subunits underlie IA in both GAD67-GFP- and GAD67-GFP+ interneurons, then IA diversity in spinal cord pain pathways may depend on the interneuron-subtype-selective expression of Kv4 auxiliary subunits with functionally different N-terminal variants. Thus, IA emerges as a good candidate for explaining the mechanisms underlying injury-induced mechanical hypersensitivity.

Recurrent Excitatory Feedback From Mossy Cells Enhances Sparsity and Pattern Separation in the Dentate Gyrus via Indirect Feedback Inhibition.

It is generally appreciated that storing memories of specific events in the mammalian brain, and associating features of the environment with behavioral outcomes requires fine-tuning of the strengths of connections between neurons through synaptic plasticity. It is less understood whether the organization of neuronal circuits comprised of multiple distinct neuronal cell types provides an architectural prior that facilitates learning and memory by generating unique patterns of neuronal activity in response to different stimuli in the environment, even before plasticity and learning occur. Here we simulated a neuronal network responding to sensory stimuli, and systematically determined the effects of specific neuronal cell types and connections on three key metrics of neuronal sensory representations: sparsity, selectivity, and discriminability. We found that when the total amount of input varied considerably across stimuli, standard feedforward and feedback inhibitory circuit motifs failed to discriminate all stimuli without sacrificing sparsity or selectivity. Interestingly, networks that included dedicated excitatory feedback interneurons based on the mossy cells of the hippocampal dentate gyrus exhibited improved pattern separation, a result that depended on the indirect recruitment of feedback inhibition. These results elucidate the roles of cellular diversity and neural circuit architecture on generating neuronal representations with properties advantageous for memory storage and recall.

Synaptic connectivity to L2/3 of primary visual cortex measured by two-photon optogenetic stimulation.

Understanding cortical microcircuits requires thorough measurement of physiological properties of synaptic connections formed within and between diverse subclasses of neurons. Towards this goal, we combined spatially precise optogenetic stimulation with multicellular recording to deeply characterize intralaminar and translaminar monosynaptic connections to supragranular (L2/3) neurons in the mouse visual cortex. The reliability and specificity of multiphoton optogenetic stimulation were measured across multiple Cre lines, and measurements of connectivity were verified by comparison to paired recordings and targeted patching of optically identified presynaptic cells. With a focus on translaminar pathways, excitatory and inhibitory synaptic connections from genetically defined presynaptic populations were characterized by their relative abundance, spatial profiles, strength, and short-term dynamics. Consistent with the canonical cortical microcircuit, layer 4 excitatory neurons and interneurons within L2/3 represented the most common sources of input to L2/3 pyramidal cells. More surprisingly, we also observed strong excitatory connections from layer 5 intratelencephalic neurons and potent translaminar inhibition from multiple interneuron subclasses. The hybrid approach revealed convergence to and divergence from excitatory and inhibitory neurons within and across cortical layers. Divergent excitatory connections often spanned hundreds of microns of horizontal space. In contrast, divergent inhibitory connections were more frequently measured from postsynaptic targets near each other.

Are Neurophysiological Biomarkers Able to Discriminate Multiple Sclerosis Clinical Subtypes?

Secondary progressive multiple sclerosis (SPMS) subtype is retrospectively diagnosed, and biomarkers of the SPMS are not available. We aimed to identify possible neurophysiological markers exploring grey matter structures that could be used in clinical practice to better identify SPMS. Fifty-five people with MS and 31 healthy controls underwent a transcranial magnetic stimulation protocol to test intracortical interneuron excitability in the primary motor cortex and somatosensory temporal discrimination threshold (STDT) to test sensory function encoded in cortical and deep grey matter nuclei. A logistic regression model was used to identify a combined neurophysiological index associated with the SP subtype. We observed that short intracortical inhibition (SICI) and STDT were the only variables that differentiated the RR from the SP subtype. The logistic regression model provided a formula to compute the probability of a subject being assigned to an SP subtype based on age and combined SICI and STDT values. While only STDT correlated with disability level at baseline evaluation, both SICI and STDT were associated with disability at follow-up. SICI and STDT abnormalities reflect age-dependent grey matter neurodegenerative processes that likely play a role in SPMS pathophysiology and may represent easily accessible neurophysiological biomarkers for the SPMS subtype.

Excitatory and Inhibitory Neurons of the Spinal Cord Superficial Dorsal Horn Diverge in Their Somatosensory Responses and Plasticity in Vivo.

The superficial dorsal horn (SDH) of the spinal cord represents the first site of integration between innocuous and noxious somatosensory stimuli. According to gate control theory, diverse populations of excitatory and inhibitory interneurons within the SDH are activated by distinct sensory afferents, and their interplay determines the net nociceptive output projecting to higher pain centers. Although specific SDH cell types are ill defined, numerous classifications schemes find that excitatory and inhibitory neurons fundamentally differ in their morphology, electrophysiology, neuropeptides, and pain-associated plasticity; yet little is known about how these neurons respond over a range of natural innocuous and noxious stimuli. To address this question, we applied an in vivo imaging approach in male mice where the genetically encoded calcium indicator GCaMP6s was expressed either in vGluT2-positive excitatory or vIAAT-positive inhibitory neurons. We found that inhibitory neurons were markedly more sensitive to innocuous touch than excitatory neurons but still responded dynamically over a wide range of noxious mechanical stimuli. Inhibitory neurons were also less sensitive to thermal stimuli than their excitatory counterparts. In a capsaicin model of acute pain sensitization, the responses of excitatory neurons were significantly potentiated to innocuous and noxious mechanical stimuli, whereas inhibitory neural responses were only depressed to noxious stimuli. These in vivo findings show that excitatory and inhibitory SDH neurons diverge considerably in their somatosensory responses and plasticity, as postulated by gate control theory.SIGNIFICANCE STATEMENT Gate control theory posits that opposing spinal excitatory and inhibitory neurons, differently tuned across somatosensory modalities, determine the net nociceptive output to higher pain centers. Little is known about how natural stimuli activate these two neural populations. This study applied an in vivo calcium imaging approach to genetically target these neurons and contrast their responses over a range of innocuous and noxious mechanical and thermal stimuli. Compared with excitatory neurons, we found that inhibitory neurons are more sensitive to innocuous touch and far less sensitive to thermal stimuli. An acute model of pain also revealed that these subtypes undergo divergent mechanosensory plasticity. Our data provide important and novel insights for gate-control inspired models of pain processing.