Hypothalamic Supramammillary Nucleus Selectively Excites Hippocampal CA3 Interneurons to Suppress CA3 Pyramidal Neuron Activity.

A key mode of neuronal communication between distant brain regions is through excitatory synaptic transmission mediated by long-range glutamatergic projections emitted from principal neurons. The long-range glutamatergic projection normally forms numerous en passant excitatory synapses onto both principal neurons and interneurons along its path. Under physiological conditions, the monosynaptic excitatory drive onto postsynaptic principal neurons outweighs disynaptic feedforward inhibition, with the net effect of depolarizing principal neurons. In contrast with this conventional doctrine, here we report that a glutamatergic projection from the hypothalamic supramammillary nucleus (SuM) largely evades postsynaptic pyramidal neurons (PNs), but preferentially target interneurons in the hippocampal CA3 region to predominantly provide feedforward inhibition. Using viral-based retrograde and anterograde tracing and ChannelRhodopsin2 (ChR2)-assisted patch-clamp recording in mice of either sex, we show that SuM projects sparsely to CA3 and provides minimal excitation onto CA3 PNs. Surprisingly, despite its sparse innervation, the SuM input inhibits all CA3 PNs along the transverse axis. Further, we find that SuM provides strong monosynaptic excitation onto CA3 parvalbumin-expressing interneurons evenly along the transverse axis, which likely mediates the SuM-driven feedforward inhibition. Together, our results demonstrate that a novel long-range glutamatergic pathway largely evades principal neurons, but rather preferentially innervates interneurons in a distant brain region to suppress principal neuron activity. Moreover, our findings reveal a new means by which SuM regulates hippocampal activity through SuM-to-CA3 circuit, independent of the previously focused projections from SuM to CA2 or dentate gyrus.SIGNIFICANCE STATEMENT The dominant mode of neuronal communication between brain regions is the excitatory synaptic transmission mediated by long-range glutamatergic projections, which form en passant excitatory synapses onto both pyramidal neurons and interneurons along its path. Under normal conditions, the excitation onto postsynaptic neurons outweighs feedforward inhibition, with the net effect of depolarization. In contrast with this conventional doctrine, here we report that a glutamatergic input from hypothalamic supramammillary nucleus (SuM) largely evades PNs but selectively targets interneurons to almost exclusively provide disynaptic feedforward inhibition onto hippocampal CA3 PNs. Thus, our findings reveal a novel subcortical-hippocampal circuit that enables SuM to regulate hippocampal activity via SuM-CA3 circuit, independent of its projections to CA2 or dentate gyrus.

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.

Topographic heterogeneity of intrinsic excitability in mouse hippocampal CA3 pyramidal neurons.

Area CA3 in the hippocampus is traditionally thought to act as a homogeneous neural circuit that is vital for spatial navigation and episodic memories. However, recent studies have revealed that CA3 pyramidal neurons in dorsal hippocampus display marked anatomic and functional heterogeneity along the proximodistal (transverse) axis. The hippocampus is also known to be functionally segregated along the dorsoventral (longitudinal) axis, with dorsal hippocampus strongly involved in spatial navigation and ventral hippocampus associated with emotion and anxiety. Surprisingly, however, relatively little is known about CA3 functional heterogeneity along the dorsoventral axis. Here, we carried out mouse-brain-slice patch-clamp recordings and morphological analyses to examine the heterogeneity of CA3 cellular properties along both proximodistal and dorsoventral axes. We find that CA3 pyramidal neurons exhibit considerable heterogeneity of somatodendritic morphology and intrinsic membrane properties, with ventral CA3 (vCA3) displaying more elaborate somatodendritic morphology, lower intrinsic excitability, smaller input resistance, greater cell capacitance, and more prominent hyperpolarization-activated current than dorsal CA3 (dCA3). Furthermore, although both dCA3 and vCA3 exhibit proximal-to-distal gradients in intrinsic properties and neuronal morphology, these proximal-to-distal gradients in vCA3 are more moderate than those in dCA3. Taken together, our results extend previous findings on the proximodistal heterogeneity of dCA3 function and uncover a complex, yet orderly, pattern of topographic organization of CA3 neuronal features that extends to multiple anatomic dimensions and may contribute to its in vivo functional diversity.NEW & NOTEWORTHY Area CA3 is a major hippocampal region that is classically thought to act as a homogeneous neural network vital for spatial navigation and episodic memories. Here, we report that CA3 pyramidal neurons exhibit marked heterogeneity of somatodendritic morphology and cellular electrical properties along both proximodistal and dorsoventral axes. These new results uncover a complex, yet orderly, pattern of topographic organization of CA3 neuronal features that may contribute to its in vivo functional diversity.

Neuronal activity and microglial activation support corticospinal tract and proprioceptive afferent sprouting in spinal circuits after a corticospinal system lesion.

Spared corticospinal tract (CST) and proprioceptive afferent (PA) axons sprout after injury and contribute to rewiring spinal circuits, affecting motor recovery. Loss of CST connections post-injury results in corticospinal signal loss and associated reduction in spinal activity. We investigated the role of activity loss and injury on CST and PA sprouting. To understand activity-dependence after injury, we compared CST and PA sprouting after motor cortex (MCX) inactivation, produced by chronic MCX muscimol microinfusion, with sprouting after a CST lesion produced by pyramidal tract section (PTx). Activity suppression, which does not produce a lesion, is sufficient to trigger CST axon outgrowth from the active side to cross the midline and to enter the inactivated side of the spinal cord, to the same extent as PTx. Activity loss was insufficient to drive significant CST gray matter axon elongation, an effect of PTx. Activity suppression triggered presynaptic site formation, but less than PTx. Activity loss triggered PA sprouting, as PTx. To understand injury-dependent sprouting further, we blocked microglial activation and associated inflammation after PTX by chronic minocycline administration after PTx. Minocycline inhibited myelin debris phagocytosis contralateral to PTx and abolished CST axon elongation, formation of presynaptic sites, and PA sprouting, but not CST axon outgrowth from the active side to cross the midline. Our findings suggest sprouting after injury has a strong activity dependence and that microglial activation after injury supports axonal elongation and presynaptic site formation. Combining spinal activity support and inflammation control is potentially more effective in promoting functional restoration than either alone.

Transneuronal Downregulation of the Premotor Cholinergic System After Corticospinal Tract Loss.

Injury to the supraspinal motor systems, especially the corticospinal tract, leads to movement impairments. In addition to direct disruption of descending motor pathways, spinal motor circuits that are distant to and not directly damaged by the lesion undergo remodeling that contributes significantly to the impairments. Knowing which spinal circuits are remodeled and the underlying mechanisms are critical for understanding the functional changes in the motor pathway and for developing repair strategies. Here, we target spinal premotor cholinergic interneurons (IN) that directly modulate motoneuron excitability via their cholinergic C-bouton terminals. Using a model of unilateral medullary corticospinal tract lesion in male rats, we found transneuronal downregulation of the premotor cholinergic pathway. Phagocytic microglial cells were upregulated in parallel with cholinergic pathway downregulation and both were blocked by minocycline, a microglia activation inhibitor. Additionally, we found a transient increase in interneuronal complement protein C1q expression that preceded cell loss. 3D reconstructions showed ongoing phagocytosis of C1q-expressing cholinergic INs by microglia 3 d after injury, which was complete by 10 d after injury. Unilateral motor cortex inactivation using the GABAA receptor agonist muscimol replicated the changes detected at 3 d after lesion, indicating activity dependence. The neuronal loss after the lesion was rescued by increasing spinal activity using cathodal trans-spinal direct current stimulation. Our finding of activity-dependent modulation of cholinergic premotor INs after CST injury provides the mechanistic insight that maintaining activity, possibly during a critical period, helps to protect distant motor circuits from further damage and, as a result, may improve motor functional recovery and rehabilitation.SIGNIFICANCE STATEMENT Supraspinal injury to the motor system disrupts descending motor pathways, leading to movement impairments. Whether and how intrinsic spinal circuits are remodeled after a brain injury is unclear. Using a rat model of unilateral corticospinal tract lesion in the medulla, we show activity-dependent, transneuronal downregulation of the spinal premotor cholinergic system, which is mediated by microglial phagocytosis, possibly involving a rapid and transient increase in neuronal C1q before neuronal loss. Spinal cord neuromodulation after injury to augment spinal activity rescued the premotor cholinergic system. Our findings provide the mechanistic insight that maintaining activity, possibly during an early critical period, could protect distant motor circuits from further damage mediated by microglia and interneuronal complement protein and improve motor functional outcomes.

Motor system plasticity after unilateral injury in the developing brain.

In maturity, motor skills depend on the corticospinal tract (CST) and brainstem pathways that together synapse on interneurons and motoneurons in the spinal cord. Descending signals to spinal neurons that mediate voluntary control can be distinguished from peripheral sensory signals, primarily for feedback control. These motor system circuits depend initially on developmental genetic mechanisms to establish their connections and neural activity- and use-dependent synaptic refinement during the early postnatal period to enable motor skills to develop. In this review we consider four key activity-dependent developmental mechanisms that provide insights into how the motor systems establish the proper connections for skilled movement control and how the same mechanisms also inform the mechanisms of motor impairments and developmental plasticity after corticospinal system injury: (1) synaptic competition between the CSTs from each hemisphere; (2) interactions between the CST and spinal cord neurons; (3) synaptic competition between the CST and proprioceptive sensory fibres; and (4) interactions between the developing corticospinal motor system and the rubrospinal tract. Our findings suggest that the corticospinal motor system effectively 'oversees' development of its subcortical targets through synaptic competition and trophic-like interactions and this has important implications for motor impairments after perinatal cortical stroke. WHAT THIS PAPER ADDS: Neural activity-dependent processes inform the brain and spinal cord response to injury. The corticospinal motor system may 'oversee' development of its downstream subcortical targets through activity, trophic-like interactions, and synaptic competition.

Competition with Primary Sensory Afferents Drives Remodeling of Corticospinal Axons in Mature Spinal Motor Circuits.

Injury to the mature motor system drives significant spontaneous axonal sprouting instead of axon regeneration. Knowing the circuit-level determinants of axonal sprouting is important for repairing motor circuits after injury to achieve functional rehabilitation. Competitive interactions are known to shape corticospinal tract axon outgrowth and withdrawal during development. Whether and how competition contributes to reorganization of mature spinal motor circuits is unclear. To study this question, we examined plastic changes in corticospinal axons in response to two complementary proprioceptive afferent manipulations: (1) enhancing proprioceptive afferents activity by electrical stimulation; or (2) diminishing their input by dorsal rootlet rhizotomy. Experiments were conducted in adult rats. Electrical stimulation produced proprioceptive afferent sprouting that was accompanied by significant corticospinal axon withdrawal and a decrease in corticospinal connections on cholinergic interneurons in the medial intermediate zone and C boutons on motoneurons. In contrast, dorsal rootlet rhizotomy led to a significant increase in corticospinal connections, including those on cholinergic interneurons; C bouton density increased correspondingly. Motor cortex-evoked muscle potentials showed parallel changes to those of corticospinal axons, suggesting that reciprocal corticospinal axon changes are functional. Using the two complementary models, we showed that competitive interactions between proprioceptive and corticospinal axons are an important determinant in the organization of mature corticospinal axons and spinal motor circuits. The activity- and synaptic space-dependent properties of the competition enables prediction of the remodeling of spared corticospinal connection and spinal motor circuits after injury and informs the target-specific control of corticospinal connections to promote functional recovery. SIGNIFICANCE STATEMENT: Neuroplasticity is limited in maturity, but it is promoted after injury. Axons of the major descending motor pathway for motor skills, the corticospinal tract (CST), sprout after brain or spinal cord injury. This contributes to spontaneous spinal motor circuit repair and partial motor recovery. Knowing the determinants that enhance this plasticity is critical for functional rehabilitation. Here we examine the remodeling of CST axons directed by sensory fibers. We found that the CST projection is regulated dynamically in maturity by the competitive, activity-dependent actions of sensory fibers. Knowledge of the properties of this competition enables prediction of the remodeling of CST connections and spinal circuits after injury and informs ways to engineer target-specific control of CST connections to promote recovery.

Rapid and persistent impairments of the forelimb motor representations following cervical deafferentation in rats.

Skilled motor control is regulated by the convergence of somatic sensory and motor signals in brain and spinal motor circuits. Cervical deafferentation is known to diminish forelimb somatic sensory representations rapidly and to impair forelimb movements. Our focus was to determine what effect deafferentation has on the motor representations in motor cortex, knowledge of which could provide new insights into the locus of impairment following somatic sensory loss, such as after spinal cord injury or stroke. We hypothesized that somatic sensory information is important for cortical motor map topography. To investigate this we unilaterally transected the dorsal rootlets in adult rats from C4 to C8 and mapped the forelimb motor representations using intracortical microstimulation, immediately after rhizotomy and following a 2-week recovery period. Immediately after deafferentation we found that the size of the distal representation was reduced. However, despite this loss of input there were no changes in motor threshold. Two weeks after deafferentation, animals showed a further distal representation reduction, an expansion of the elbow representation, and a small elevation in distal movement threshold. These changes were specific to the forelimb map in the hemisphere contralateral to deafferentation; there were no changes in the hindlimb or intact-side forelimb representations. Degradation of the contralateral distal forelimb representation probably contributes to the motor control deficits after deafferentation. We propose that somatic sensory inputs are essential for the maintenance of the forelimb motor map in motor cortex and should be considered when rehabilitating patients with peripheral or spinal cord injuries or after stroke.

Spinal morphine but not ziconotide or gabapentin analgesia is affected by alternative splicing of voltage-gated calcium channel CaV2.2 pre-mRNA.

Presynaptic voltage-gated calcium Ca(V)2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of Ca(V)2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. Ca(V)2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the Ca(V)2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to Ca(V)2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing.

Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances µ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

Characteristics of HCN channels and their participation in neuropathic pain.

Neuropathic pain is induced by the injury to nervous systems and characterized by hyperalgesia, allodynia and spontaneous pain. The underlying mechanisms include peripheral and central sensitization resulted from neuronal hyperexcitability. A number of ion channels are considered to contribute to the neuronal hyperexcitability. Here, we particularly concentrate on an interesting ion channel, hyperpolarization-activated cyclic nucleotide gated (HCN) channels. We overview its biophysical properties, physiological functions, followed by focusing on the current progress in the study of its role in the development of neuropathic pain. We attempt to provide a comprehensive review of the potential valuable target, HCN channels, in the treatment of neuropathic pain.

Axonal accumulation of hyperpolarization-activated cyclic nucleotide-gated cation channels contributes to mechanical allodynia after peripheral nerve injury in rat.

Peripheral nerve injury causes neuropathic pain including mechanical allodynia and thermal hyperalgesia due to central and peripheral sensitization. Spontaneous ectopic discharges derived from dorsal root ganglion (DRG) neurons and from the sites of injury are a key factor in the initiation of this sensitization. Numerous studies have focused primarily on DRG neurons; however, the injured axons themselves likely play an equally important role. Previous studies of neuropathic pain rats with spinal nerve ligation (SNL) showed that the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel in DRG neuronal bodies is important for the development of neuropathic pain. Here, we investigate the role of the axonal HCN channel in neuropathic pain rats. Using the chronic constriction injury (CCI) model, we found abundant axonal accumulation of HCN channel protein at the injured sites accompanied by a slight decrease in DRG neuronal bodies. The function of these accumulated channels was verified by local application of ZD7288, a specific HCN blocker, which significantly suppressed the ectopic discharges from injured nerve fibers with no effect on impulse conduction. Moreover, mechanical allodynia, but not thermal hyperalgesia, was relieved significantly by ZD7288. These results suggest that axonal HCN channel accumulation plays an important role in ectopic discharges from injured spinal nerves and contributes to the development of mechanical allodynia in neuropathic pain rats.