Whole-brain mapping of afferent projections to the suprachiasmatic nucleus of the tree shrew.

The suprachiasmatic nucleus (SCN) is essential for the neural control of mammalian circadian timing system. The circadian activity of the SCN is modulated by its afferent projections. In the present study, we examine neuroanatomical characteristics and afferent projections of the SCN in the tree shrew (Tupaia belangeri chinensis) using immunocytochemistry and retrograde tracer Fluoro-Gold (FG). Distribution of the vasoactive intestinal peptide was present in the SCN from rostral to caudal, especially concentrated in its ventral part. FG-labeled neurons were observed in the lateral septal nucleus, septofimbrial nucleus, paraventricular thalamic nucleus, posterior hypothalamic nucleus, posterior complex of the thalamus, ventral subiculum, rostral linear nucleus of the raphe, periaqueductal gray, mesencephalic reticular formation, dorsal raphe nucleus, pedunculopontine tegmental nucleus, medial parabrachial nucleus, locus coeruleus, parvicellular reticular nucleus, intermediate reticular nucleus, and ventrolateral reticular nucleus. In summary, the morphology of the SCN in tree shrews is described from rostral to caudal. In addition, our data demonstrate for the first time that the SCN in tree shrews receives inputs from numerous brain regions in the telencephalon, diencephalon, mesencephalon, metencephalon, and myelencephalon. This comprehensive knowledge of the afferent projections of the SCN in tree shrews provides further insights into the neural organization and physiological processes of circadian rhythms.

Phototoxicities Caused by Continuous Light Exposure Were Not Induced in Retinal Ganglion Cells Transduced by an Optogenetic Gene.

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Hyperactivation of proprioceptors induces microglia-mediated long-lasting pain in a rat model of chronic fatigue syndrome.

BACKGROUND: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. METHODS: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. RESULTS: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. CONCLUSION: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.

Cell-type specific parallel circuits in the bed nucleus of the stria terminalis and the central nucleus of the amygdala of the mouse.

The central extended amygdala (EAc) is a forebrain macrosystem which has been widely implicated in reward, fear, anxiety, and pain. Its two key structures, the lateral bed nucleus of the stria terminalis (BSTL) and the central nucleus of the amygdala (CeA), share similar mesoscale connectivity. However, it is not known whether they also share similar cell-specific neuronal circuits. We addressed this question using tract-tracing and immunofluorescence to reveal the EAc microcircuits involving two neuronal populations expressing either protein kinase C delta (PKCδ) or somatostatin (SOM). PKCδ and SOM are expressed predominantly in the dorsal BSTL (BSTLD) and in the lateral/capsular parts of CeA (CeL/C). We found that, in both BSTLD and CeL/C, PKCδ+ cells are the main recipient of extra-EAc inputs from the lateral parabrachial nucleus (LPB), while SOM+ cells constitute the main source of long-range projections to extra-EAc targets, including LPB and periaqueductal gray. PKCδ+ cells can also integrate inputs from the basolateral nucleus of the amygdala or insular cortex. Within EAc, PKCδ+, but not SOM+ neurons, serve as the major source of inputs to the ventral BSTL and to the medial part of CeA. However, both cell types can be involved in mutual connections between BSTLD and CeL/C. These results unveil the pivotal positions of PKCδ+ and SOM+ neurons in organizing parallel cell-specific neuronal circuits within CeA and BSTL, but also between them, which further reinforce the notion of EAc as a structural and functional macrosystem.

Targeting the Nav1.8 ion channel engenders sex-specific responses in lysophosphatidic acid-induced joint neuropathy.

Joint neuropathic pain occurs in a subset of arthritis patients, and lysophosphatidic acid (LPA) has been implicated as a mediator of joint neuropathy. The mechanism by which LPA promotes neuropathic pain is unknown but may be related to altered signalling of the voltage-gated sodium channel Nav1.8 located on nociceptors. Because arthritis and neuropathic pain are more prevalent in females, this study aimed to explore potential sex differences in the development of LPA-induced joint neuropathy and whether Nav1.8 played a role in the associated neuropathic pain. Joint neuropathy was induced in male and female Wistar rats (179-284 g) by intra-articular injection of 50-µg LPA. Pain behaviour was assessed over 21 days using von Frey hair algesiometry. On day 21, electrophysiological recordings of joint primary afferents were conducted to measure peripheral sensitisation. Saphenous nerve morphology and expression of the nerve-damage marker ATF3 and Nav1.8 in ipsilateral dorsal root ganglions were compared on the basis of sex. The analgesic properties of the selective Nav1.8 antagonist A-803467 was determined in pain behaviour and electrophysiology experiments. Females developed more severe mechanical allodynia than males after LPA treatment. Lysophosphatidic acid caused more pronounced demyelination of the saphenous nerve in females, but no sex differences were observed in the expression of ATF3 or Nav1.8 in dorsal root ganglion neurones. Blockade of Nav1.8 channels with A-803467 resulted in a decrease in joint mechanosensitivity and secondary allodynia with females exhibiting a greater response. These findings suggest that LPA has sex-specific effects on joint neuropathy and Nav1.8 gating, which should be considered when treating neuropathic arthritis patients.

No latency to dentate granule cell epileptogenesis in experimental temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: To determine when spontaneous granule cell epileptiform discharges first occur after hippocampal injury, and to identify the postinjury "latent" period as either a "silent" gestational state of epileptogenesis or a subtle epileptic state in gradual transition to a more obvious epileptic state. METHODS: Nonconvulsive status epilepticus evoked by perforant path stimulation in urethane-sedated rats produced selective and extensive hippocampal injury and a "latent" period that preceded the onset of the first clinically obvious epileptic seizures. Continuous granule cell layer depth recording and video monitoring assessed the time course of granule cell hyperexcitability and the onset/offset times of spontaneous epileptiform discharges and behavioral seizures. RESULTS: One day postinjury, granule cells in awake rats were hyperexcitable to afferent input, and continuously generated spontaneous population spikes. During the ~2-4 week "latent" period, granule cell epileptiform discharges lasting ~30 seconds caused subtle focal seizures characterized by immobilization and facial automatisms that were undetected by behavioral assessment alone but identified post hoc. Granule cell layer epileptiform discharge duration eventually tripled, which caused the first clinically obvious seizure, ending the "latent" period. Behavioral seizure duration was linked tightly to spontaneous granule cell layer events. Granule cell epileptiform discharges preceded all behavioral seizure onsets, and clonic behaviors ended abruptly within seconds of the termination of each granule cell epileptiform discharge. Noninjurious hippocampal excitation produced no evidence of granule cell hyperexcitability or epileptogenesis. SIGNIFICANCE: The latent period in this model is a subtle epileptic state in transition to a more clinically obvious epileptic state, not a seizure-free "gestational" state when an unidentified epileptogenic mechanism gradually develops. Based on the onset/offset times of electrographic and behavioral events, granule cell behavior may be the prime determinant of seizure onset, phenotype, duration, and offset in this model of hippocampal-onset epilepsy. Extensive hippocampal neuron loss could be the primary epileptogenic mechanism.

Orexinergic neurons are involved in the chemosensory control of breathing during the dark phase in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra compacta (SNpc) and the only risk factor is aging. We showed that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the neuronal profile within the brainstem ventral respiratory column with a decrease in the hypercapnic ventilatory response. Here we tested the involvement of orexin cells from the lateral hypothalamus/perifornical area (LH/PeF) on breathing in a 6-OHDA PD model. In this model of PD, there is a reduction in the total number of orexinergic neurons and in the number of orexinergic neurons that project to the RTN, without changing the number of CO2-activated orexinergic neurons during the dark phase. The ventilation at rest and in response to hypercapnia (7% CO2) was assessed in animals that received 6-OHDA or vehicle injections into the striatum and saporin anti-Orexin-B or IgG saporin into the LH/PeF during the sleep and awake states. The experiments showed a reduction of respiratory frequency (fR) at rest during the light phase in PD animals only during sleep. During the dark phase, there was an impaired fR response to hypercapnia in PD animals with depletion of orexinergic neurons in awake and sleeping rats. In conclusion, the degeneration of orexinergic neurons in this model of PD can be related to impaired chemoreceptor function in the dark phase.

Optic nerve as a source of activated retinal microglia post-injury.

Using mice expressing green fluorescent protein (GFP) from a transgenic CD11c promoter we found that a controlled optic nerve crush (ONC) injury attracted GFPhi retinal myeloid cells to the dying retinal ganglion cells and their axons. However, the origin of these retinal myeloid cells was uncertain. In this study we use transgenic mice in conjunction with ONC, partial and full optic nerve transection (ONT), and parabiosis to determine the origin of injury induced retinal myeloid cells. Analysis of parabiotic mice and fate mapping showed that responding retinal myeloid cells were not derived from circulating macrophages and that GFPhi myeloid cells could be derived from GFPlo microglia. Comparison of optic nerve to retina following an ONC showed a much greater concentration of GFPhi cells and GFPlo microglia in the optic nerve. Optic nerve injury also induced Ki67+ cells in the optic nerve but not in the retina. Comparison of the retinal myeloid cell response after full versus partial ONT revealed fewer GFPhi cells and GFPlo microglia in the retina following a full ONT despite it being a more severe injury, suggesting that full transection of the optic nerve can block the migration of responding myeloid cells to the retina. Our results suggest that the optic nerve can be a reservoir for activated microglia and other retinal myeloid cells in the retina following optic nerve injury.

Spinal protein kinase C/extracellular signal-regulated kinase signal pathway mediates hyperalgesia priming.

Chronic pain can be initiated by one or more acute stimulations to sensitize neurons into the primed state. In the primed state, the basal nociceptive thresholds of the animal are normal, but, in response to another hyperalgesic stimulus, the animal develops enhanced and prolonged hyperalgesia. The exact mechanism of how primed state is formed is not completely understood. Here, we showed that spinal protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signal pathway is required for neuronal plasticity change, hyperalgesic priming formation, and the development of chronic hyperalgesia using acid-induced muscle pain model in mice. We discovered that phosphorylated extracellular signal-regulated kinase-positive neurons in the amygdala, spinal cord, and dorsal root ganglion were significantly increased after first acid injection. Inhibition of the phosphorylated extracellular signal-regulated kinase activity intrathecally, but not intracerebroventricularly or intramuscularly before first acid injection, prevented the development of chronic pain induced by second acid injection, which suggests that hyperalgesic priming signal is stored at spinal cord level. Furthermore, intrathecal injection of PKC but not protein kinase A blocker prevented the development of chronic pain, and PKC agonist was sufficient to induce prolonged hyperalgesia response after acid injection. We also found that mammalian target of rapamycin-dependent protein synthesis was required for the priming establishment. To test whether hyperalgesic priming leads to synaptic plasticity change, we recorded field excitatory postsynaptic potentials from spinal cord slices and found enhanced long-term potentiation in mice that received one acid injection. This long-term potentiation enhancement was prevented by inhibition of extracellular signal-regulated kinase. These findings show that the activation of PKC/ERK signal pathway and downstream protein synthesis is required for hyperalgesic priming and the consolidation of pain singling.

Direct and indirect nigrofugal projections to the nucleus reticularis pontis caudalis mediate in the motor execution of the acoustic startle reflex.

The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.

Neuronal PTEN deletion in adult cortical neurons triggers progressive growth of cell bodies, dendrites, and axons.

Deletion of the phosphatase and tensin (PTEN) gene in neonatal mice leads to enlargement of the cell bodies of cortical motoneurons (CMNs) in adulthood (Gutilla et al., 2016). Here, we assessed whether PTEN deletion in adult mice would trigger growth of mature neurons. PTEN was deleted by injecting AAV-Cre into the sensorimotor cortex of adult transgenic mice with a lox-P flanked exon 5 of the PTEN gene and Cre-dependent reporter gene tdTomato. PTEN-deleted CMN's identified by tdT expression and retrograde labeling with fluorogold (FG) were significantly enlarged four months following PTEN deletion, and continued to increase in size through the latest time intervals examined (12-15 months post-deletion). Sholl analyses of tdT-positive pyramidal neurons revealed increases in dendritic branches at 6 months following adult PTEN deletion, and greater increases at 12 months. 12 months after adult PTEN deletion, axons in the medullary pyramids were significantly larger and G-ratios were higher. Mice with PTEN deletion exhibited no overt neurological symptoms and no seizures. Assessment of motor function on the rotarod and cylinder test revealed slight impairment of coordination with unilateral deletion; however, mice with bilateral PTEN deletion in the motor cortex performed better than controls on the rotarod at 8 and 10 months post-deletion. Our findings demonstrate that robust neuronal growth can be induced in fully mature cortical neurons long after the developmental period has ended and that this continuous growth occurs without obvious functional impairments.

Behavioural and neural sequelae of stressor exposure are not modulated by controllability in females.

The degree of behavioural control that an organism has over a stressor is a potent modulator of the stressor's impact; controllable stressors produce none of the neurochemical and behavioural sequelae that occur if the stressor is uncontrollable. Research demonstrating the importance of control and the neural mechanisms responsible has been conducted almost entirely with male rats. It is unknown if behavioural control is stress blunting in females, and whether or not a similar resilience circuitry is engaged. Female rats were exposed to controllable, yoked uncontrollable or no tailshock. In separate experiments, behavioural (juvenile social exploration, fear and shuttle box escape) and neurochemical (activation of dorsal raphe serotonin and dorsal raphe-projecting prelimbic neurons) outcomes, which are sensitive to the dimension of control in males, were assessed. Despite successful acquisition of the controlling response, behavioural control did not mitigate dorsal raphe serotonergic activation and behavioural outcomes induced by tailshock, as it does in males. Moreover, behavioural control failed to selectively engage prelimbic cells that project to the dorsal raphe as in males. Pharmacological activation of the prelimbic cortex restored the stress-buffering effects of control. Collectively, the data demonstrate stressor controllability phenomena are absent in females and that the protective prelimbic circuitry is present but not engaged. Reduced benefit from coping responses may represent a novel approach for understanding differential sex prevalence in stress-related psychiatric disorders.

Early sensory experience influences the development of multisensory thalamocortical and intracortical connections of primary sensory cortices.

The nervous system integrates information from multiple senses. This multisensory integration already occurs in primary sensory cortices via direct thalamocortical and corticocortical connections across modalities. In humans, sensory loss from birth results in functional recruitment of the deprived cortical territory by the spared senses but the underlying circuit changes are not well known. Using tracer injections into primary auditory, somatosensory, and visual cortex within the first postnatal month of life in a rodent model (Mongolian gerbil) we show that multisensory thalamocortical connections emerge before corticocortical connections but mostly disappear during development. Early auditory, somatosensory, or visual deprivation increases multisensory connections via axonal reorganization processes mediated by non-lemniscal thalamic nuclei and the primary areas themselves. Functional single-photon emission computed tomography of regional cerebral blood flow reveals altered stimulus-induced activity and higher functional connectivity specifically between primary areas in deprived animals. Together, we show that intracortical multisensory connections are formed as a consequence of sensory-driven multisensory thalamocortical activity and that spared senses functionally recruit deprived cortical areas by an altered development of sensory thalamocortical and corticocortical connections. The functional-anatomical changes after early sensory deprivation have translational implications for the therapy of developmental hearing loss, blindness, and sensory paralysis and might also underlie developmental synesthesia.

Quantitative Analysis of Mouse Dural Afferent Neurons Expressing TRPM8, VGLUT3, and NF200.

OBJECTIVE: To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice. BACKGROUND: With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice. METHODS: We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca2+ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs). RESULTS: The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca2+ influx in less than 7% VGLUT3-expressing PANs in adult mice. CONCLUSIONS: TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Generating level-dependent models of cervical and thoracic spinal cord injury: Exploring the interplay of neuroanatomy, physiology, and function.

The majority of spinal cord injuries (SCI) occur at the cervical level, which results in significant impairment. Neurologic level and severity of injury are primary endpoints in clinical trials; however, how level-specific damages relate to behavioural performance in cervical injury is incompletely understood. We hypothesized that ascending level of injury leads to worsening forelimb performance, and correlates with loss of neural tissue and muscle-specific neuron pools. A direct comparison of multiple models was made with injury realized at the C5, C6, C7 and T7 vertebral levels using clip compression with sham-operated controls. Animals were assessed for 10weeks post-injury with numerous (40) outcome measures, including: classic behavioural tests, CatWalk, non-invasive MRI, electrophysiology, histologic lesion morphometry, neuron counts, and motor compartment quantification, and multivariate statistics on the total dataset. Histologic staining and T1-weighted MR imaging revealed similar structural changes and distinct tissue loss with cystic cavitation across all injuries. Forelimb tests, including grip strength, F-WARP motor scale, Inclined Plane, and forelimb ladder walk, exhibited stratification between all groups and marked impairment with C5 and C6 injuries. Classic hindlimb tests including BBB, hindlimb ladder walk, bladder recovery, and mortality were not different between cervical and thoracic injuries. CatWalk multivariate gait analysis showed reciprocal and progressive changes forelimb and hindlimb function with ascending level of injury. Electrophysiology revealed poor forelimb axonal conduction in cervical C5 and C6 groups alone. The cervical enlargement (C5-T2) showed progressive ventral horn atrophy and loss of specific motor neuron populations with ascending injury. Multivariate statistics revealed a robust dataset, rank-order contribution of outcomes, and allowed prediction of injury level with single-level discrimination using forelimb performance and neuron counts. Level-dependent models were generated using clip-compression SCI, with marked and reliable differences in forelimb performance and specific neuron pool loss.

Intracortical signal processing of periodontal ligament sensations in rat.

The somatosensory information from the orofacial region, including the periodontal ligament (PDL), is processed in a manner that differs from that used for other body somatosensory information in the related cortices. It was reported that electrical stimulation to rat PDL elicited activation of the insular oral region (IOR) and the primary (S1) and secondary (S2) somatosensory cortices. However, the physiological relationship between S1 and S2/IOR is not well understood. To address this issue, we performed in vivo optical imaging using a voltage-sensitive dye. Our results demonstrated that the electrical stimulation to the PDL of the mandibular incisor evoked the simultaneous activation of S1 and the S2/IOR. The stimulation to the initial response area of the S1 evoked responses in the S2/IOR, and vice versa. An injection of tetrodotoxin (TTX) to the cortical region between S1 and S2/IOR attenuated such elicited responses only in the non-stimulated cortical partner site. The cortico-cortical interaction between S1 and S2/IOR was suppressed by the application of TTX, indicating that these two cortical regions bi-directionally communicate the signal processing of PDL sensations. An injection of FluoroGold™ (FG) to the initial response area in S1 or the S2/IOR showed that FG-positive cells were scattered in the non-injected cortical counterpart. This morphological result demonstrated the presence of a bi-directional intracortical connection between the initial response areas in S1 and the S2/IOR. These findings suggest the presence of a mutual connection between S1 and the S2/IOR as an intracortical signal processing network for orofacial nociception.

Afferent and efferent projections of the anterior cortical amygdaloid nucleus in the mouse.

The anterior cortical amygdaloid nucleus (ACo) is a chemosensory area of the cortical amygdala that receives afferent projections from both the main and accessory olfactory bulbs. The role of this structure is unknown, partially due to a lack of knowledge of its connectivity. In this work, we describe the pattern of afferent and efferent projections of the ACo by using fluorogold and biotinylated dextranamines as retrograde and anterograde tracers, respectively. The results show that the ACo is reciprocally connected with the olfactory system and basal forebrain, as well as with the chemosensory and basomedial amygdala. In addition, it receives dense projections from the midline and posterior intralaminar thalamus, and moderate projections from the posterior bed nucleus of the stria terminalis, mesocortical structures and the hippocampal formation. Remarkably, the ACo projects moderately to the central nuclei of the amygdala and anterior bed nucleus of the stria terminalis, and densely to the lateral hypothalamus. Finally, minor connections are present with some midbrain and brainstem structures. The afferent projections of the ACo indicate that this nucleus might play a role in emotional learning involving chemosensory stimuli, such as olfactory fear conditioning. The efferent projections confirm this view and, given its direct output to the medial part of the central amygdala and the hypothalamic 'aggression area', suggest that the ACo can initiate defensive and aggressive responses elicited by olfactory or, to a lesser extent, vomeronasal stimuli.

Ascending projections of nociceptive neurons from trigeminal subnucleus caudalis: A population approach.

Second-order neurons in trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1) are critical for craniofacial pain processing and project rostrally to terminate in: ventral posteromedial thalamic nucleus (VPM), medial thalamic nuclei (MTN) and parabrachial nuclei (PBN). The contribution of each region to trigeminal nociception was assessed by the number of phosphorylated extracellular signal-regulated kinase-immunoreactive (pERK-IR) neurons co-labeled with fluorogold (FG). The phenotype of pERK-IR neurons was further defined by the expression of neurokinin 1 receptor (NK1). The retrograde tracer FG was injected into VPM, MTN or PBN of the right hemisphere and after seven days, capsaicin was injected into the left upper lip in male rats. Nearly all pERK-IR neurons were found in superficial laminae of Vc-C1 ipsilateral to the capsaicin injection. Nearly all VPM and MTN FG-labeled neurons in Vc-C1 were found contralateral to the injection site, whereas FG-labeled neurons were found bilaterally after PBN injection. The percentage of FG-pERK-NK1-IR neurons was significantly greater (>10%) for PBN projection neurons than for VPM and MTN projection neurons (<3%). pERK-NK1-IR VPM projection neurons were found mainly in the middle-Vc, while pERK-NK1-immunoreactive MTN or PBN projection neurons were found in the middle-Vc and caudal Vc-C1. These results suggest that a significant percentage of capsaicin-responsive neurons in superficial laminae of Vc-C1 project directly to PBN, while neurons that project to VPM and MTN are subject to greater modulation by pERK-IR local interneurons. Furthermore, the rostrocaudal distribution differences of FG-pERK-NK1-IR neurons in Vc-C1 may reflect functional differences between these projection areas regarding craniofacial pain.