Monosynaptic Connections Research Articles

A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling

BackgroundViral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK.MethodsUsing our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties.ResultsH129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer’s disease and control mouse brains.ConclusionsgKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

Glutamate in primary afferents is required for itch transmission

Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3+ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3+ afferents to transmit itch. We further demonstrated that MRGPRA3+ afferents form monosynaptic connections with both NMBR+ and NMBR- neurons and that NMB and glutamate together can enhance the activity of NMBR+ spinal DH neurons. Moreover, Nmb in MRGPRA3+ afferents and NMBR+ DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.

Electrophysiologic Evidence That Directional Deep Brain Stimulation Activates Distinct Neural Circuits in Patients With Parkinson Disease

ObjectivesDeep brain stimulation (DBS) delivered via multicontact leads implanted in the basal ganglia is an established therapy to treat Parkinson disease (PD). However, the different neural circuits that can be modulated through stimulation on different DBS contacts are poorly understood. Evidence shows that electrically stimulating the subthalamic nucleus (STN) causes a therapeutic effect through antidromic activation of the hyperdirect pathway—a monosynaptic connection from the cortex to the STN. Recent studies suggest that stimulating the substantia nigra pars reticulata (SNr) may improve gait. The advent of directional DBS leads now provides a spatially precise means to probe these neural circuits and better understand how DBS affects distinct neural networks. Materials and MethodsWe measured cortical evoked potentials (EPs) using electroencephalography (EEG) in response to low-frequency DBS using the different directional DBS contacts in eight patients with PD. ResultsA short-latency EP at 3 milliseconds originating from the primary motor cortex appeared largest in amplitude when stimulating DBS contacts closest to the dorsolateral STN (p < 0.001). A long-latency EP at 10 milliseconds originating from the premotor cortex appeared strongest for DBS contacts closest to the SNr (p < 0.0001). ConclusionsOur results show that at the individual patient level, electrical stimulation of different nuclei produces distinct EP signatures. Our approach could be used to identify the functional location of each DBS contact and thus help patient-specific DBS programming. Clinical Trial RegistrationThe ClinicalTrials.gov registration number for the study is NCT04658641.

Recent Advances in the Understanding of Specific Efferent Pathways Emerging From the Cerebellum.

The cerebellum has a long history in terms of research on its network structures and motor functions, yet our understanding of them has further advanced in recent years owing to technical developments, such as viral tracers, optogenetic and chemogenetic manipulation, and single cell gene expression analyses. Specifically, it is now widely accepted that the cerebellum is also involved in non-motor functions, such as cognitive and psychological functions, mainly from studies that have clarified neuronal pathways from the cerebellum to other brain regions that are relevant to these functions. The techniques to manipulate specific neuronal pathways were effectively utilized to demonstrate the involvement of the cerebellum and its pathways in specific brain functions, without altering motor activity. In particular, the cerebellar efferent pathways that have recently gained attention are not only monosynaptic connections to other brain regions, including the periaqueductal gray and ventral tegmental area, but also polysynaptic connections to other brain regions, including the non-primary motor cortex and hippocampus. Besides these efferent pathways associated with non-motor functions, recent studies using sophisticated experimental techniques further characterized the historically studied efferent pathways that are primarily associated with motor functions. Nevertheless, to our knowledge, there are no articles that comprehensively describe various cerebellar efferent pathways, although there are many interesting review articles focusing on specific functions or pathways. Here, we summarize the recent findings on neuronal networks projecting from the cerebellum to several brain regions. We also introduce various techniques that have enabled us to advance our understanding of the cerebellar efferent pathways, and further discuss possible directions for future research regarding these efferent pathways and their functions.

Medial prefrontal cortex-basolateral amygdala circuit dysfunction in chronic alcohol-exposed male rats

Alcohol is a commonly used drug that can produce alcohol use disorders (AUDs). Few individuals with AUDs receive treatment and treatment options are complicated by issues with effectiveness and compliance. Alcohol has been shown to differentially affect specific brain regions and an improved understanding of circuit-specific dysregulation caused by alcohol is warranted. Previous work has implicated both the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) in alcohol-associated plasticity, however studies directly examining the impact of alcohol exposure on this circuit are lacking. The current study employed an optogenetic strategy to investigate the prelimbic mPFC to BLA circuit and changes in circuit activity following chronic intragastric ethanol exposure in male Sprague Dawley rats. We observed monosynaptic connections with light-evoked stimulation of mPFC terminals in the BLA with efficacy and short latency. We also found that mPFC-BLA projections are primarily glutamatergic under basal inhibitory control, with a lesser population of GABAergic projections. We examined optically-evoked glutamate currents in the BLA using repeated trains of stimulation that displayed accommodation, or a reduction in evoked current amplitude over repeated stimulations. We found that following chronic ethanol exposure mPFC-BLA glutamatergic connections were dysregulated such that there were decreases in overall function, notably in synaptic strength and accommodation, with no change in probability of evoked glutamate release. The lesser GABAergic component of the mPFC-BLA circuit was not altered by chronic ethanol exposure. Collectively these data indicate that mPFC-BLA circuitry is a significant target of alcohol-associated plasticity, which may contribute to pathological behavior associated with AUDs.This article is part of the special Issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse'.

How does TMS “engage” the human cortex? Insights from structural and functional mapping of the precentral motor hand knob

Abstract Background: TMS of the primary motor hand area (M1-HAND) can probe the function of fast corticomotor connections to the contralateral hand. TMS gives rise to indirect (transsynaptic) excitation of large pyramidal tract (PT) neurons in M1-HAND that make monosynaptic connections with spinal motoneurons in the cervical cord. Other cell types are effectively excited by TMS as well. Yet, it is still unclear which neuronal elements (e.g. axon terminals or axon hillock), which cell types (e.g. PT neurons, intra-telencephalic (IT) neurons, or inhibitory interneurons), and which sites in the precentral hand knob (e.g. crown, lip, or sulcus region) are preferentially depolarized by the TMS-induced electrical field in the precentral gyrus. Objective: To address whether non-invasive neuroimaging in human volunteers can help to pinpoint the primary targets for TMS of M1-HAND. Methods: Structural and functional neuroimaging (MRI and PET) were employed in a series of experiments to target the M1-HAND. Structural MRI was used for shaped-based TMS mapping, electrical field modelling, and mapping of cortical myelin content. Functional MRI and PET were used to localize clusters in the precentral hand knob that are functionally activated by focal TMS of M1-HAND. Results: Inter-individual differences in precentral cortical myelin content account for interindividual differences in rostro-caudal “motor hotspot” location, linking cortical myelination to TMS target engagement. Functional brain mapping reveals different peak target sites in the precentral hand knob and indicates that PT and IT neurons as well as inhibitory interneurons are effectively excited by TMS. Discussion: Structural and functional neuroimaging can help to elucidate how TMS engages its local cortical targets in the precentral gyrus. Causal interpretation of the results needs to consider the potential and pitfalls inherent to the neuroimaging method and the physiological implications of the TMS protocol (e.g. intensity, number of stimuli, online-offline TMS). Keywords: Transcranial magnetic stimulation, Motor cortex, Magnetic resonance imaging, Positron emission tomography

Contralateral Projection of Anterior Cingulate Cortex Contributes to Mirror-Image Pain.

Long-term limb nerve injury often leads to mirror-image pain (MIP), an abnormal pain sensation in the limb contralateral to the injury. Although it is clear that MIP is mediated in part by central nociception processing, the underlying mechanisms remain poorly understood. The anterior cingulate cortex (ACC) is a key brain region that receives relayed peripheral nociceptive information from the contralateral limb. In this study, we induced MIP in male mice, in which a unilateral chronic constrictive injury of the sciatic nerve (CCI) induced a decreased nociceptive threshold in both hind limbs and an increased number of c-Fos-expressing neurons in the ACC both contralateral and ipsilateral to the injured limb. Using viral-mediated projection mapping, we observed that a portion of ACC neurons formed monosynaptic connections with contralateral ACC neurons. Furthermore, the number of cross-callosal projection ACC neurons that exhibited c-Fos signal was increased in MIP-expressing mice, suggesting enhanced transmission between ACC neurons of the two hemispheres. Moreover, selective inhibition of the cross-callosal projection ACC neurons contralateral to the injured limb normalized the nociceptive sensation of the uninjured limb without affecting the increased nociceptive sensation of the injured limb in CCI mice. In contrast, inhibition of the non-cross-callosal projection ACC neurons contralateral to the injury normalized the nociceptive sensation of the injured limb without affecting the MIP exhibited in the uninjured limb. These results reveal a circuit mechanism, namely, the cross-callosal projection of ACC between two hemispheres, that contributes to MIP and possibly other forms of contralateral migration of pain sensation.SIGNIFICANCE STATEMENT Mirror-image pain (MIP) refers to the increased pain sensitivity of the contralateral body part in patients with chronic pain. This pathology requires central processing, yet the mechanisms are less known. Here, we demonstrate that the cross-callosal projection neurons in the anterior cingulate cortex (ACC) contralateral to the injury contribute to MIP exhibited in the uninjured limb, but do not affect nociceptive sensation of the injured limb. In contrast, the non-cross-callosal projection neurons in the ACC contralateral to the injury contribute to nociceptive sensation of the injured limb, but do not affect MIP exhibited in the uninjured limb. Our study depicts a novel cross-callosal projection of ACC that contributes to MIP, providing a central mechanism for MIP in chronic pain state.

Hippocampus-Prefrontal Coupling Regulates Recognition Memory for Novelty Discrimination.

Recognition memory provides the ability to distinguish familiar from novel objects and places, and is important for recording and updating events to guide appropriate behavior. The hippocampus (HPC) and medial prefrontal cortex (mPFC) have both been implicated in recognition memory, but the nature of HPC-mPFC interactions, and its impact on local circuits in mediating this process is not known. Here we show that novelty discrimination is accompanied with higher theta activity (4-10 Hz) and increased c-Fos expression in both these regions. Moreover, theta oscillations were highly coupled between the HPC and mPFC during recognition memory retrieval for novelty discrimination, with the HPC leading the mPFC, but not during initial learning. Principal neurons and interneurons in the mPFC responded more strongly during recognition memory retrieval compared with learning. Optogenetic silencing of HPC input to the mPFC disrupted coupled theta activity between these two structures, as well as the animals' (male Sprague Dawley rats) ability to differentiate novel from familiar objects. These results reveal a key role of monosynaptic connections between the HPC and mPFC in novelty discrimination via theta coupling and identify neural populations that underlie this recognition memory-guided behavior.SIGNIFICANCE STATEMENT Many memory processes are highly dependent on the interregional communication between the HPC and mPFC via neural oscillations. However, how these two brain regions coordinate their oscillatory activity to engage local neural populations to mediate recognition memory for novelty discrimination is poorly understood. This study revealed that the HPC and mPFC theta oscillations and their temporal coupling is correlated with recognition memory-guided behavior. During novel object recognition, the HPC drives mPFC interneurons to effectively reduce the activity of principal neurons. This study provides the first evidence for the requirement of the HPC-mPFC pathway to mediate recognition memory for novelty discrimination and describes a mechanism for how this memory is regulated.

CellExplorer: A framework for visualizing and characterizing single neurons

The large diversity of neuron types provides the means by which cortical circuits perform complex operations. Neuron can be described by biophysical and molecular characteristics, afferent inputs, and neuron targets. To quantify, visualize, and standardize those features, we developed the open-source, MATLAB-based framework CellExplorer. It consists of three components: a processing module, a flexible data structure, and a powerful graphical interface. The processing module calculates standardized physiological metrics, performs neuron-type classification, finds putative monosynaptic connections, and saves them to a standardized, yet flexible, machine-readable format. The graphical interface makes it possible to explore the computed features at the speed of a mouse click. The framework allows users to process, curate, and relate their data to a growing public collection of neurons. CellExplorer can link genetically identified cell types to physiological properties of neurons collected across laboratories and potentially lead to interlaboratory standards of single-cell metrics.

Chemogenetic inactivation reveals the inhibitory control function of the prefronto-striatal pathway in the macaque brain

The lateral prefrontal cortex (LPFC) has a strong monosynaptic connection with the caudate nucleus (CdN) of the striatum. Previous human MRI studies have suggested that this LPFC-CdN pathway plays an important role in inhibitory control and working memory. We aimed to validate the function of this pathway at a causal level by pathway-selective manipulation of neural activity in non-human primates. To this end, we trained macaque monkeys on a delayed oculomotor response task with reward asymmetry and expressed an inhibitory type of chemogenetic receptors selectively to LPFC neurons that project to the CdN. Ligand administration reduced the inhibitory control of impulsive behavior, as well as the task-related neuronal responses observed in the local field potentials from the LPFC and CdN. These results show that we successfully suppressed pathway-selective neural activity in the macaque brain, and the resulting behavioral changes suggest that the LPFC-CdN pathway is involved in inhibitory control.

Temporal Course of Transient Direct Corticomotoneuronal Connections during Development in Rodents

We previously observed in rodents that during the 2nd postnatal week corticospinal axons make monosynaptic connections with motoneurons. Prior to that finding, it had been believed that such contacts only occur in higher primates. Although an in vitro electrophysiological study is prerequisite for studying the developmental time course of synaptic connections, the technical difficulty of reliably recording synaptic responses from spinal motoneurons in animals over 2 weeks old hampered the study. Instead, we used retrograde transsynaptic labeling with a genetically modified rabies virus to confirm the presence of direct corticomotoneuronal connections at an early developmental stage and to show that these connections were subsequently eliminated. However, determination of an accurate elimination time course and quantitative evaluation of synaptic connectivity cannot be achieved through viral-tracing experiments.For the present study, we improved the slice preparation procedure and maintenance of slice viability, which enabled us to record postsynaptic responses using the whole cell patch-clamp technique from retrogradely labeled forearm motoneurons up until postnatal week 7. We examined the extent of corticomotoneuronal monosynaptic connections and studied the time course of their accumulation and loss. Positive ratios of monosynaptic corticomotoneuronal EPSCs increased from P6 to P8 and then plateaued (P8–P13: 65%). Thereafter, the monosynaptic connections declined until P21, at which time they were no longer detected. The time course of the falling phase and elimination was confirmed by experiments using optogenetic stimulation. The timing of the elimination fell within the same range (P18-22) estimated in our earlier study using retrograde transsynaptic labeling.

Frequency-Dependent Synaptic Dynamics Differentially Tune CA1 and CA2 Pyramidal Neuron Responses to Cortical Input.

Entorhinal cortex neurons make monosynaptic connections onto distal apical dendrites of CA1 and CA2 pyramidal neurons through the perforant path (PP) projection. Previous studies show that differences in dendritic properties and synaptic input density enable the PP inputs to produce a much stronger excitation of CA2 compared with CA1 pyramidal neurons. Here, using mice of both sexes, we report that the difference in PP efficacy varies substantially as a function of presynaptic firing rate. Although a single PP stimulus evokes a 5- to 6-fold greater EPSP in CA2 compared with CA1, a brief high-frequency train of PP stimuli evokes a strongly facilitating postsynaptic response in CA1, with relatively little change in CA2. Furthermore, we demonstrate that blockade of NMDARs significantly reduces strong temporal summation in CA1 but has little impact on that in CA2. As a result of the differences in the frequency- and NMDAR-dependent temporal summation, naturalistic patterns of presynaptic activity evoke CA1 and CA2 responses with distinct dynamics, differentially tuning CA1 and CA2 responses to bursts of presynaptic firing versus single presynaptic spikes, respectively.SIGNIFICANCE STATEMENT Recent studies have demonstrated that abundant entorhinal cortical innervation and efficient dendritic propagation enable hippocampal CA2 pyramidal neurons to produce robust excitation evoked by single cortical stimuli, compared with CA1. Here we uncovered, unexpectedly, that the difference in efficacy of cortical excitation varies substantially as a function of presynaptic firing rate. A burst of stimuli evokes a strongly facilitating response in CA1, but not in CA2. As a result, the postsynaptic response of CA1 and CA2 to presynaptic naturalistic firing displays contrasting temporal dynamics, which depends on the activation of NMDARs. Thus, whereas CA2 responds to single stimuli, CA1 is selectively recruited by bursts of cortical input.

HoxD transcription factors define monosynaptic sensory-motor specificity in the developing spinal cord.

The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. Here, we show that members of the HoxD family of transcription factors play a crucial role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10 and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.

Macaque monkey trigeminal blink reflex circuits targeting levator palpebrae superioris motoneurons.

For normal viewing, the eyes are held open by the tonic actions of the levator palpebrae superioris (levator) muscle raising the upper eyelid. This activity is interrupted during blinks, when the eyelid sweeps down to spread the tear film or protect the cornea. We examined the circuit connecting the principal trigeminal nucleus to the levator motoneurons by use of both anterograde and retrograde tracers in macaque monkeys. Injections of anterograde tracer were made into the principal trigeminal nucleus using either a stereotaxic approach or localization following physiological characterization of trigeminal second order neurons. Anterogradely labeled axonal arbors were located both within the caudal central subdivision, which contains levator motoneurons, and in the adjacent supraoculomotor area. Labeled boutons made synaptic contacts on retrogradely labeled levator motoneurons indicating a monosynaptic connection. As the eye is also retracted through the actions of the rectus muscles during a blink, we examined whether these trigeminal injections labeled boutons contacting rectus motoneurons within the oculomotor nucleus. These were not found when the injection sites were confined to the principal trigeminal nucleus region. To identify the source of the projection to the levator motoneurons, we injected retrograde tracer into the oculomotor complex. Retrogradely labeled cells were confined to a narrow, dorsoventrally oriented cell population that lined the rostral edge of the principal trigeminal nucleus. Presumably these cells inhibit levator motoneurons, while other parts of the trigeminal sensory complex are activating orbicularis oculi motoneurons, when a blink is initiated by sensory stimuli contacting the face.

The subiculum sensitizes retrosplenial cortex layer 2/3 pyramidal neurons

Neurons in the retrosplenial cortex (RSC), a cerebral region that connects synaptically with various brain regions, are known to increase neuronal activity in accordance with hippocampal sharp wave-ripples. Pyramidal cells in granular RSC (gRSC) layer 2/3, but not layer 5, exhibit slowly ramping depolarization and considerably delayed spikes in response to a step-pulse current injection. The latencies of delayed spikes in RSC layer 2/3 pyramidal neurons were shortened by a preceding current injection. This effect was mimicked by activation of axonal afferents from the subiculum, but not of neocortical afferents. The subiculum is likely to facilitate information processing and flow in the RSC. The retrosplenial cortex (RSC), a cerebral region involved in diverse cognitive functions, is an anatomical hub that forms monosynaptic connections with various brain areas. Here, we report a unique form of short-term intrinsic plasticity in mouse granular RSC layer 2/3 pyramidal cells. These cells exhibited delayed spikes in response to somatic current injection, but the spike latencies were shortened by a preceding brief depolarization (priming). This priming-induced sensitization is distinct from desensitization, which is commonly observed in other cortical neurons. The facilitatory priming effect lasted for more than 3s, providing a time window for increased sensitivity to RSC inputs. Based on in vitro and in vivo patch-clamp recordings following optogenetic stimulation of axonal fibres, we found that preactivation of subicular afferents replicated the facilitatory priming effect. The results suggest that subicular inputs to RSC layer 2/3 neurons may modulate subsequent information integration in the RSC layer 2/3 circuits.

Inferring thalamocortical monosynaptic connectivity in vivo.

As the tools to simultaneously record electrophysiological signals from large numbers of neurons within and across brain regions become increasingly available, this opens up for the first time the possibility of establishing the details of causal relationships between monosynaptically connected neurons and the patterns of neural activation that underlie perception and behavior. Although recorded activity across synaptically connected neurons has served as the cornerstone for much of what we know about synaptic transmission and plasticity, this has largely been relegated to ex vivo preparations that enable precise targeting under relatively well-controlled conditions. Analogous studies in vivo, where image-guided targeting is often not yet possible, rely on indirect, data-driven measures, and as a result such studies have been sparse and the dependence upon important experimental parameters has not been well studied. Here, using in vivo extracellular single-unit recordings in the topographically aligned rodent thalamocortical pathway, we sought to establish a general experimental and computational framework for inferring synaptic connectivity. Specifically, attacking this problem within a statistical signal detection framework utilizing experimentally recorded data in the ventral-posterior medial (VPm) region of the thalamus and the homologous region in layer 4 of primary somatosensory cortex (S1) revealed a trade-off between network activity levels needed for the data-driven inference and synchronization of nearby neurons within the population that results in masking of synaptic relationships. Here, we provide a framework for establishing connectivity in multisite, multielectrode recordings based on statistical inference, setting the stage for large-scale assessment of synaptic connectivity within and across brain structures.NEW & NOTEWORTHY Despite the fact that all brain function relies on the long-range transfer of information across different regions, the tools enabling us to measure connectivity across brain structures are lacking. Here, we provide a statistical framework for identifying and assessing potential monosynaptic connectivity across neuronal circuits from population spiking activity that generalizes to large-scale recording technologies that will help us to better understand the signaling within networks that underlies perception and behavior.

Respiratory axon regeneration in the chronically injured spinal cord

Promoting the combination of robust regeneration of damaged axons and synaptic reconnection of these growing axon populations with appropriate neuronal targets represents a major therapeutic goal following spinal cord injury (SCI). A key impediment to achieving this important aim includes an intrinsic inability of neurons to extend axons in adult CNS, particularly in the context of the chronically-injured spinal cord. We tested whether an inhibitory peptide directed against phosphatase and tensin homolog (PTEN: a central inhibitor of neuron-intrinsic axon growth potential) could restore inspiratory diaphragm function by reconnecting critical respiratory neural circuitry in a rat model of chronic cervical level 2 (C2) hemisection SCI. We found that systemic delivery of PTEN antagonist peptide 4 (PAP4) starting at 8 weeks after C2 hemisection promoted substantial, long-distance regeneration of injured bulbospinal rostral Ventral Respiratory Group (rVRG) axons into and through the lesion and back toward phrenic motor neurons (PhMNs) located in intact caudal C3-C5 spinal cord. Despite this robust rVRG axon regeneration, PAP4 stimulated only minimal recovery of diaphragm function. Furthermore, re-lesion through the hemisection site completely removed PAP4-induced functional improvement, demonstrating that axon regeneration through the lesion was responsible for this partial functional recovery. Interestingly, there was minimal formation of putative excitatory monosynaptic connections between regrowing rVRG axons and PhMN targets, suggesting that (1) limited rVRG-PhMN synaptic reconnectivity was responsible at least in part for the lack of a significant functional effect, (2) chronically-injured spinal cord presents an obstacle to achieving synaptogenesis between regenerating axons and post-synaptic targets, and (3) addressing this challenge is a potentially-powerful strategy to enhance therapeutic efficacy in the chronic SCI setting. In conclusion, our study demonstrates a non-invasive and transient pharmacological approach in chronic SCI to repair the critically-important neural circuitry controlling diaphragmatic respiratory function, but also sheds light on obstacles to circuit plasticity presented by the chronically-injured spinal cord.

074 Basal Forebrain GABAergic Neurons Promote Arousal by Disinhibiting the Orexin Neurons via Local GABAergic Interneurons

Abstract Introduction Optogenetic and chemogenetic studies have shown that activation of basal forebrain (BF) GABAergic neurons rapidly wakes up mice from non-REM (NREM) sleep. These wake-promoting responses have been attributed to BF GABAergic neurons projecting to the cerebral cortex and more specifically to the inhibition of cortical fast-spiking interneurons. Tracing studies have however found that BF GABAergic neurons also densely innervate the lateral hypothalamus (LH) perifornical area, although the role of this pathway in behavioral state control remains mostly unexplored. Methods We conducted in vivo and in vitro optogenetic studies. We selectively expressed channelrhodopsin-2 (ChR2) in BF GABAergic neurons by injecting a cre-dependent viral vector encoding for ChR2 into the BF of VGAT-cre mice. We photostimulated the BF GABAergic input to the LH with optical fibers placed into the LH of EEG instrumented mice. For in vitro recordings we expressed ChR2 in BF GABAergic neurons and we fluorescently labeled orexin or LH GABAergic neurons. We recorded in brain slices from identified orexin neurons or GABA neurons while photostimulating the BF GABAergic input. Results Optogenetic stimulation of the BF GABAergic fibers in the LH produced rapid arousals from NREM sleep. The same stimulation however did not wake up the mice if they were in REM sleep. We conducted additional studies in brain slices to identify the postsynaptic neurons in the LH targeted by the BF GABAergic input. We found that while optogenetic stimulation of the BF GABAergic input did not produce opto-evoked synaptic responses in the orexin neurons, it produced short-latency opto-evoked inhibitory postsynaptic currents (IPSCs) in LH GABAergic neurons. These opto-evoked IPSCs were GABAA receptor-mediated and were maintained in tetrodotoxin (TTX) indicating monosynaptic connectivity. We have previously found that orexin neurons are inhibited by local LH GABAergic neurons. Our hypothesis is that these local GABAergic interneurons are the target of the BF GABAergic arousal input. Conclusion BF GABAergic neurons drive arousal through projections to the LH. We propose that this arousal response is due to the inhibition of local GABAergic interneurons which in turn disinhibit the LH wake-promoting neurons including the orexin neurons. Support (if any) NS091126 and HL149630

Subthreshold membrane potential dynamics of posterior parietal cortical neurons coupled with hippocampal ripples.

During behavioral states of immobility, sleep, and anesthesia, the hippocampus generates high-frequency oscillations called ripples. Ripples occur simultaneously with synchronous neuronal activity in the neocortex, known as slow waves, and contribute to memory consolidation. During these ripples, various neocortical regions exhibit modulations in spike rates and local field activity irrespective of whether they receive direct synaptic inputs from the hippocampus. However, little is known about the subthreshold dynamics of the membrane potentials of neocortical neurons during ripples. We patch-clamped layer 2/3 pyramidal cells in the posterior parietal cortex (PPC), a neocortical region that is involved in allocentric spatial representation of behavioral exploration and sequential series of relevant action potentials during ripples. We simultaneously monitored the membrane potentials of post hoc-identified PPC neurons and the local field potentials of the hippocampus in anesthetized mice. More than 50% of the recorded PPC neurons exhibited significant depolarizations and/or hyperpolarizations during ripples. Histological inspections of the recorded neurons revealed that the ripple-modulated PPC neurons were distributed in the PPC in a spatially non-biased fashion. These results suggest that hippocampal ripples are widely but selectively associated with the subthreshold dynamics of the membrane potentials of PPC neurons even though there is no monosynaptic connectivity between the hippocampus and the PPC.

Monosynaptic connection from the subiculum to medial mammillary nucleus neurons projecting to the anterior thalamus and Gudden’s ventral tegmental nucleus

As a major hippocampal output structure, the subiculum projects to diverse cortical and subcortical areas, and its projection to the medial mammillary nucleus (MM) has been implicated in memory. Major efferent targets of the MM are the anteroventral and anteromedial thalamic nuclei and Gudden’s ventral tegmental nucleus. These projections may play a key role in distributing subicular information. However, it remains unknown whether neurons in the MM that receive monosynaptic input from the subiculum project to these target regions. We addressed this issue with anterograde transsynaptic tracing mediated using adeno-associated virus serotype 1 (AAV1). Injection of AAV1-Cre and a Cre-dependent AAV encoding enhanced yellow fluorescent protein (EYFP) into the rat dorsal subiculum and MM, respectively, labeled the soma of the MM and axons in the anteroventral / anteromedial thalamic nuclei and Gudden’s ventral tegmental nucleus with EYFP. The EYFP-positive neurons in the MM were immunoreactive for glutamate and leu-enkephalin and received perisomatic GABAergic inputs. These results revealed monosynaptic projections from the subiculum to MM neurons projecting to the anteroventral / anteromedial thalamic nuclei and Gudden’s ventral tegmental nucleus. This monosynaptic connection may support a fast and robust signal flow through the hippocampal–mammillothalamic and hippocampal–mammillotegmental pathways.