Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Proprioceptive thalamus receiving forelimb and neck muscle spindle inputs via the external cuneate nucleus in the rat.

Proprioceptive signals from body muscles have historically been considered to project to the rostrodorsal shell of the ventrobasal thalamic complex [the ventral posterolateral nucleus (VPL) and ventral posteromedial nucleus (VPM)]. However, we have recently found that proprioception from rat jaw-closing muscle spindles (JCMSs) is conveyed via the supratrigeminal nucleus to the caudo-ventromedial edge of the VPM, but not to the rostrodorsal shell of the VPM. Therefore, proprioception from other body muscles may also project to thalamic regions other than the rostrodorsal shell of the VPL. We thus examined the thalamic projection from the rat external cuneate nucleus (ECu), which receives proprioceptive inputs from forelimb and neck muscles. After injection of anterograde tracer into the ECu, axon terminals were contralaterally labeled in the ventromedial part (VPLvm) of the VPL, but not in the rostrodorsal shell of the VPL. After anterograde tracer injection into the cuneate nucleus (Cu), axon terminals were widely labeled in the contralateral VPL including the VPLvm. In the VPLvm, we electrophysiologically confirmed the proprioceptive inputs responsive to electrical stimulation of the ECu or median nerve and to the pressure of forelimb/neck muscles or wrist flexion. After retrograde tracer injection into the VPLvm, neurons were contralaterally labeled in the ECu and Cu. After retrograde tracer injection into the VPL where no such proprioceptive inputs were recorded, no ECu neurons were labeled. These findings indicate that proprioception from forelimb/neck muscle spindles and JCMSs is somatotopically transmitted to the ventromedial floor of the ventrobasal thalamic complex, but not to its rostrodorsal shell.

Transcortical descending pathways through granular insular cortex conveying orofacial proprioception.

Our motor behavior can be affected by proprioceptive information. However, little is known about which brain circuits contribute to this process. We have recently revealed that the proprioceptive information arising from jaw-closing muscle spindles (JCMSs) is conveyed to the supratrigeminal nucleus (Su5) by neurons in the trigeminal mesencephalic nucleus (Me5), then to the caudo-ventromedial edge of ventral posteromedial thalamic nucleus (VPMcvm), and finally to the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of secondary somatosensory cortex (dGIrvs2). Our next question is which brain areas receive the information from the dGIrvs2 for the jaw-movements. To test this issue, we injected an anterograde tracer, biotinylated dextranamine, into the dGIrvs2, and analyzed the resultant distribution profiles of the labeled axon terminals. Anterogradely labeled axons were distributed in the pontomedullary areas (including the Su5) which are known to receive JCMS proprioceptive inputs conveyed directly by the Me5 neurons and to contain premotoneurons projecting to the jaw-closing motoneurons in the trigeminal motor nucleus (Mo5). They were also found in and around the VPMcvm. In contrast, no labeled axonal terminals were detected on the cell bodies of Me5 neurons and motoneurons in the Mo5. These data suggest that jaw-movements, which are evoked by the classically defined jaw-reflex arc originating from the peripheral JCMS proprioceptive information, could also be modulated by the transcortical feedback connections from the dGIrvs2 to the VPMcvm and Su5.

Cortical and Subcortical Projections from Granular Insular Cortex Receiving Orofacial Proprioception.

We have recently revealed that the proprioceptive signal from jaw-closing muscle spindles (JCMSs) is conveyed to the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of secondary somatosensory cortex (dGIrvs2) via the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM) in rats. However, it remains unclear to which cortical or subcortical structures the JCMS proprioceptive information is subsequently conveyed from the dGIrvs2. To test this issue, we injected an anterograde tracer, biotinylated dextranamine, into the electophysiologically identified dGIrvs2, and analyzed the resultant distribution profiles of labeled axon terminals in rats. Labeled terminals were distributed with an ipsilateral predominance. In the cerebral cortex, they were seen in the primary and secondary somatosensory cortices, lateral and medial agranular cortices and dorsolateral orbital cortex. In the basal ganglia, they were found in the caudate putamen, core part of accumbens nucleus, lateral globus pallidus, subthalamic nucleus, and substantia nigra pars compacta and pars reticulata. They were also observed in the central amygdaloid nucleus and extended amygdala (the interstitial nucleus of posterior limb of anterior commissure and the juxtacapsular part of lateral division of bed nucleus of stria terminalis). In the thalamus, they were seen in the reticular nucleus, ventromedial nucleus, core VPM, parvicellular part of ventral posterior nucleus, oval paracentral nucleus, medial and triangular parts of posterior nucleus, and zona incerta as well as the VPMcvm. These data suggest that the JCMS proprioceptive information through the dGIrvs2 is transmitted to the emotional 'limbic' regions as well as sensorimotor regions.

Orofacial proprioceptive thalamus of the rat.

The ascending pathway mediating proprioception from the orofacial region is still not fully known. The present study elucidated the relay of jaw-closing muscle spindle (JCMS) inputs from brainstem to thalamus in rats. We injected an anterograde tracer into the electrophysiologically identified supratrigeminal nucleus (Su5), known to receive JCMS input. Many thalamic axon terminals were labeled and were found mainly contralaterally in a small, unpredicted area of the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM). Electrical stimulation of the masseter nerve and passive jaw movements induced large responses in the VPMcvm. The VPMcvm is far from the rostrodorsal part of ventral posterolateral thalamic nucleus (VPL) where proprioceptive inputs from the body are represented. After injection of a retrograde tracer into the electrophysiologically identified VPMcvm, many neurons were labeled almost exclusively in the contralateral Su5, whereas no labeled neurons were found in the principal sensory trigeminal nucleus (Pr5) and spinal trigeminal nucleus (Sp5). In contrast, after injection of a retrograde tracer into the core of VPM, many neurons were labeled contralaterally in the Pr5 and Sp5, but none in the Su5. We conclude that JCMS input excites trigeminothalamic projection neurons in the Su5 which project primarily to the VPMcvm in marked contrast to other proprioceptors and sensory receptors in the orofacial region which project to the core VPM. These findings suggest that lesions or deep brain stimulation in the human equivalent of VPMcvm may be useful for treatment of movement disorders (e.g., orofacial tremor) without affecting other sensations.

Direct projection from the lateral habenula to the trigeminal mesencephalic nucleus in rats.

Trigeminal mesencephalic nucleus (Vmes) neurons are primary afferents conveying deep sensation from the masticatory muscle spindles or the periodontal mechanoreceptors, and are crucial for controlling jaw movements. Their cell bodies exist in the brain and receive descending commands from a variety of cortical and subcortical structures involved in limbic (emotional) systems. However, it remains unclear how the lateral habenula (LHb), a center of negative emotions (e.g., pain, stress and anxiety), can influence the control of jaw movements. To address this issue, we examined whether and how the LHb directly projects to the Vmes by means of neuronal tract tracing techniques in rats. After injections of a retrograde tracer Fluorogold in the rostral and caudal Vmes, a number of neurons were labeled in the lateral division of LHb (LHbl) bilaterally, whereas a few neurons were labeled in the medial division of LHb (LHbm) bilaterally. After injections of an anterograde tracer, biotinylated dextranamine (BDA) in the LHbl, a small number of labeled axons were distributed bilaterally in the rostral and caudal levels of Vmes, where some labeled axonal boutons contacted the cell body of rostral and caudal levels of Vmes neurons bilaterally. After the BDA injection into the LHbm, however, no axons were labeled bilaterally in the rostral and caudal levels of Vmes. Therefore, the present study for the first time demonstrated the direct projection from the LHbl to the Vmes and the detailed projection patterns, suggesting that jaw movements are modulated by negative emotions that are signaled by LHbl neurons.

Jaw-opening and -closing premotoneurons in the nucleus of the solitary tract making contacts with laryngeal and pharyngeal afferent terminals in rats.

This study clarified the neural mechanisms underlying jaw movements in pharyngolaryngeal reflexes such as swallowing in rats. After retrograde tracer injections into the ventromedial division (Vmovm) of the trigeminal motor nucleus (Vmo) containing jaw-opening (JO) motoneurons or into the dorsolateral division (Vmodl) of Vmo containing jaw-closing (JC) motoneurons, JO and JC premotoneurons were labeled with an ipsilateral predominance in the medial and intermediate subnuclei of the rostrocaudal middle two-thirds of the nucleus of the solitary tract (Sol); JC premotoneurons were also in the lateral subnucleus of Sol. After anterograde tracer injections into the Sol, axons were labeled with an ipsilateral predominance in the Vmovm and Vmodl, prominently in the ipsilateral Vmovm. After transganglionic tracer applications to the superior laryngeal nerve (SLN) or the cervical trunk of the glossopharyngeal nerve (GpN-ct), labeled afferents were seen in the medial, intermediate, lateral and interstitial subnuclei of Sol at the rostral three-fourths of Sol, indicating considerable overlap with the JO and JC premotoneurons in the Sol. Double labeling experiments demonstrated contacts between the afferent terminals and the JO and JC premotoneurons. The present study has for the first time revealed the differential distribution of JO and JC premotoneurons in the Sol and features of their projections from the Sol, as well as their connections with SLN and GpN-ct afferent inputs. The JO and JC premotoneurons in the Sol may play an important role in generation and organization of jaw movements in pharyngolaryngeal reflexes evoked by SLN and GpN-ct inputs, such as swallowing.