Individual arcuate nucleus proopiomelanocortin neurons project to select target sites

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its afferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their afferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct afferent projections may serve as a way for the POMC population to organize its many functions.

A retrograde mechanism coordinates memory allocation across brain regions

Memories engage ensembles of neurons across different brain regions within a memory system. However, it is unclear whether the allocation of a memory to these ensembles is coordinated across brain regions. To address this question, we used CREB expression to bias memory allocation in one brain region, and rabies retrograde tracing to test memory allocation in connected presynaptic neurons in the other brain regions. We find that biasing allocation of CTA memory in the basolateral amygdala (BLA) also biases memory allocation in presynaptic neurons of the insular cortex (IC). By manipulating the allocation of CTA memory to specific neurons in both BLA and IC, we found that we increased their connectivity and enhanced CTA memory performance. These results, which are corroborated by mathematical simulations and by studies with auditory fear conditioning, demonstrate that a retrograde mechanism coordinates the allocation of memories across different brain regions.

Efferent and Afferent Connections of Neuropeptide Y Neurons in the Nucleus Accumbens of Mice

Neuropeptide Y (NPY) is a neural peptide distributed widely in the brain and has various functions in each region. We previously reported that NPY neurons in the nucleus accumbens (NAc) are involved in the regulation of anxiety behavior. Anterograde and retrograde tracing studies suggest that neurons in the NAc project to several areas, such as the lateral hypothalamus (LH) and ventral pallidum (VP), and receive afferent projections from the cortex, thalamus, and amygdala. However, the neural connections between accumbal NPY neurons and other brain areas in mice remain unclear. In this study, we sought to clarify these anatomical connections of NPY neurons in the NAc by investigating their neural outputs and inputs. To selectively map NPY neuronal efferents from the NAc, we injected Cre-dependent adeno-associated viruses (AAVs) into the NAc of NPY-Cre mice. This revealed that NAc NPY neurons exclusively projected to the LH. We confirmed this by injecting cholera toxin b subunit (CTb), a retrograde tracer, into the LH and found that approximately 7–10% of NPY neurons in the NAc were double-labeled for mCherry and CTb. Moreover, retrograde tracing using recombinant rabies virus (rRABV) also identified NAc NPY projections to the LH. Finally, we investigated monosynaptic input to the NPY neurons in the NAc using rRABV. We found that NPY neurons in the NAc received direct synaptic connections from the midline thalamic nuclei and posterior basomedial amygdala. These findings provide new insight into the neural networks of accumbal NPY neurons and should assist in elucidating their functional roles.

The role of V3 neurons in speed-dependent interlimb coordination during locomotion in mice

Speed-dependent interlimb coordination allows animals to maintain stable locomotion under different circumstances. We have previously demonstrated that a subset of spinal V3 neurons contributes to stable locomotion by mediating mutual excitation between left and right lumbar rhythm generators (RGs). Here, we expanded our investigation to the V3 neurons involved in ascending long propriospinal interactions (aLPNs). Using retrograde tracing, we revealed a subpopulation of lumbar V3 aLPNs with contralateral cervical projections. V3OFF mice, in which all V3 neurons were silenced, had a significantly reduced maximal locomotor speed, were unable to move using stable trot, gallop, or bound, and predominantly used lateral-sequence walk. To understand the functional roles of V3 aLPNs, we adapted our previous model of spinal circuitry controlling quadrupedal locomotion (Danner et al., 2017), by incorporating diagonal V3 aLPNs mediating inputs from each lumbar RG to the contralateral cervical RG. The updated model reproduces our experimental results and suggests that locally projecting V3 neurons, mediating left–right interactions within lumbar and cervical cords, promote left–right synchronization necessary for gallop and bound, whereas the V3 aLPNs promote synchronization between diagonal fore and hind RGs necessary for trot. The model proposes the organization of spinal circuits available for future experimental testing.

Spatially patterned excitatory neuron subtypes and projections of the claustrum

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

An Approach to Maximize Retrograde Transport Based on the Spatial Distribution of Motor Endplates in Mouse Hindlimb Muscles

Knowledge regarding the relationship between muscles and the corresponding motor neurons would allow therapeutic genes to transport into specific spinal cord segments. Retrograde tracing technique by targeting the motor endplate (MEP), a highly specialized structure that offers direct access to the spinal motor neurons, has been used to elucidate the connectivity between skeletal muscles and the innervating motor neuron pools. However, current injection strategies mainly based on blind injection or the local MEP region might lead to an underestimation of the motor neuron number due to the uneven distribution of MEP in skeletal muscles. In this work, we proposed a novel intramuscular injection strategy based on the 3D distribution of the MEPs in skeletal muscles, applied the 3D intramuscular injection to the gastrocnemius and tibialis anterior for retrograde tracing of the corresponding motor neurons, and compared this with the existing injection strategy. The intramuscular diffusion of the tracer demonstrated that 3D injection could maximize the retrograde transport by ensuring a greater uptake of the tracer by the MEP region. In combination with optical clearing and imaging, we performed 3D mapping and quantification of the labeled motor neurons and confirmed that 3D injection could label more motor neurons than the current injection method. It is expected that 3D intramuscular injection strategy will help elucidate the connective relationship between muscles and motor neurons faithfully and becomes a promising tool in the development of gene therapy strategies for motor neuron diseases.

Single-Cell Transcriptional Profiling of the Adult Corticospinal Tract Reveals Forelimb and Hindlimb Molecular Specialization

The corticospinal tract (CST) is refractory to repair after CNS trauma, resulting in chronic debilitating functional motor deficits after spinal cord injury. While novel pro-axon growth activators have stimulated plasticity and regeneration of corticospinal neurons (CSNs) after injury, robust functional recovery remains elusive. These repair strategies are sub-optimal in part due to underexplored molecular heterogeneity within the developing and adult CST. In this study, we combine retrograde CST tracing with single-cell RNA sequencing to build a comprehensive atlas of CSN subtypes. By comparing CSNs to non-spinally projecting neurons in layer Vb, we identify pan-CSN markers including Wnt7b. By leveraging retrograde tracing, we are able to compare forelimb and hindlimb projecting CSNs, identifying subtype-specific markers, including Cacng7 and Slc16a2 respectively. These markers are expressed in embryonic and neonatal CSNs and can be used to study early postnatal patterning of the CST. Our results provide molecular insight into the differences between anatomically distinct CSN subtypes and provide a resource for future screening and exploitation of these subtypes to repair the damaged CST after injury and disease.

Decreased Resting-State Functional Connectivity of Periaqueductal Gray in Temporal Lobe Epilepsy Comorbid With Migraine

Objective: Patients with temporal lobe epilepsy (TLE) are at high risk for having a comorbid condition of migraine, and these two common diseases are proposed to have some shared pathophysiological mechanisms. Our recent study indicated the dysfunction of periaqueductal gray (PAG), a key pain-modulating structure, contributes to the development of pain hypersensitivity and epileptogenesis in epilepsy. This study is to investigate the functional connectivity of PAG network in epilepsy comorbid with migraine.Methods: Thirty-two patients with TLE, including 16 epilepsy patients without migraine (EwoM) and 16 epilepsy patients with comorbid migraine (EwM), and 14 matched healthy controls (HCs) were recruited and underwent resting functional magnetic resonance imaging (fMRI) scans to measure the resting-state functional connectivity (RsFC) of PAG network. The frequency and severity of migraine attacks were assessed using the Migraine Disability Assessment Questionnaire (MIDAS) and Visual Analog Scale/Score (VAS). In animal experiments, FluoroGold (FG), a retrograde tracing agent, was injected into PPN and its fluorescence detected in vlPAG to trace the neuronal projection from vlPAG to PPN. FG traced neuron number was used to evaluate the neural transmission activity of vlPAG-PPN pathway. The data were processed and analyzed using DPARSF and SPSS17.0 software. Based on the RsFC finding, the excitatory transmission of PAG and the associated brain structure was studied via retrograde tracing in combination with immunohistochemical labeling of excitatory neurons.Results: Compared to HCs group, the RsFC between PAG and the left pedunculopontine nucleus (PPN), between PAG and the corpus callosum (CC), was decreased both in EwoM and EwM group, while the RsFC between PAG and the right PPN was increased only in EwoM group but not in EwM group. Compared to EwoM group, the RsFC between PAG and the right PPN was decreased in EwM group. Furthermore, the RsFC between PAG and PPN was negatively correlated with the frequency and severity of migraine attacks. In animal study, a seizure stimulation induced excitatory transmission from PAG to PPN was decreased in rats with chronic epilepsy as compared to that in normal control rats.Conclusion: The comorbidity of epilepsy and migraine is associated with the decreased RsFC between PAG and PPN.

Spatially patterned excitatory neuron subtypes and circuits within the claustrum

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow region that primarily projects to the neocortex, whereas circuit-based approaches suggest a broader region embedding neocortical and other neural circuits. Here, we took a bottom up, cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum is comprised of two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential projection targets. Thus, the claustrum is comprised of excitatory neuron subtypes with distinct molecular and circuit properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.