Characterisation of interneurons in lamina II of the rat spinal cord

Lamina II of the dorsal horn contains numerous small neurons with varying morphologies, most of which have axons that remain within the spinal cord. It can be distinguished from the other laminae by its lack of myelinated fibres and its constituent interneurons that are densely packed. This region is the major termination site for unmyelinated (C) primary afferent fibres, which convey mostly nociceptive information. It also receives inputs from thinly myelinated (Aδ) fibres, some of which are nociceptive. In spite of its importance and several past attempts, little is known of its neuronal circuitry. This is mainly due to the great functional and morphological diversity of lamina II interneurons, which has made characterisation difficult. A comprehensive classification scheme is essential to identify discrete functional populations of lamina II interneurons, and to enable understanding of their roles in the local neuronal circuitry. The present study aims to investigate the physiological, pharmacological and morphological properties of lamina II interneurons recorded in an in vitro slice preparation from adult rat spinal cord. These properties were correlated with the neurotransmitter content of each cell, which was identified by detection of vesicular transporters in axonal boutons, in order to distinguish discrete functional subpopulations of cells in this region. Both inhibitory and excitatory interneurons were identified in lamina II, based on their expression of vesicular GABA transporter (VGAT) or vesicular glutamate transporter (VGLUT2), respectively. None of the cells that had VGAT-immunoreactive axons displayed staining for VGLUT2, and vice-versa. Injection of depolarising current evoked tonic-, transient-, delayed-, gap-, reluctant- and single spike-firing among these cells. Discharge pattern was strongly related to neurotransmitter phenotype, since most excitatory cells, but very few inhibitory cells had firing patterns that could be attributed to A-type potassium (IA) currents (i.e. delayed, gap or reluctant-firing). This suggests that excitatory lamina II interneurons with IA –type firing patterns are involved in plasticity that contributes to pain states. The majority of inhibitory cells displayed tonic-firing pattern in response to depolarisation. There was also an obvious difference in the response of lamina II neurons to hyperpolarisation, since the majority of inhibitory cells showed inward currents while most excitatory cells displayed transient outward currents. Noradrenaline and serotonin hyperpolarised both inhibitory and excitatory neurons, while only inhibitory neurons responded to somatostatin. This is consistent with the findings of a previous study that had shown that the somatostatin 2 receptor (sst2a) is only expressed by inhibitory neurons in lamina II, and suggests that the pro-nociceptive effects of somatostatin are mediated by ‘disinhibition’. The somatodendritic morphology of 61 lamina II interneurons was reconstructed from projected confocal images of Neurobiotin labelling and assessed according to the morphological scheme developed by Grudt and Perl (2002). Although cells in the islet, central, vertical and radial class were identified, a substantial number of cells (19/61) had morphology that was atypical or intermediate between two classes and therefore could not be classified. Certain morphological types were consistently found in the inhibitory or excitatory population: all islet cells were GABAergic, while all radial cells and most vertical cells were glutamatergic. However, the correlation between these properties may be complex, since there was a considerable diversity in the remaining cells. Some glutamatergic interneurons had axons that contained somatostatin and many of these also contained enkephalin. Somatostatin-expressing glutamatergic cells included various morphological types, while enkephalin was detected in the axons of vertical and radial cells. All cells with axons that were somatostatin- and enkephalin-immunoreactive had delayed-firing patterns. Taken together with the pharmacological data from the present study, this suggests that somatostatin released from these glutamatergic neurons would hyperpolarise subsets of inhibitory neurons and causes disinhibition. This could lead to alterations of pain thresholds. The results from this study demonstrate that distinctive populations of inhibitory and excitatory interneurons can be recognised in lamina II, and these cells are most likely to correspond to discrete functional groups. Electrophysiological, neurochemical, morphological and pharmacological properties of neurons can be correlated but this is likely to be very complex. Future investigations that combine various approaches should allow further understanding of the specific roles of lamina II interneurons in nociceptive processing within the spinal cord

Kcnab1 Is Expressed in Subplate Neurons With Unilateral Long-Range Inter-Areal Projections

Subplate (SP) neurons are among the earliest-born neurons in the cerebral cortex and heterogeneous in terms of gene expression. SP neurons consist mainly of projection neurons, which begin to extend their axons to specific target areas very early during development. However, the relationships between axon projection and gene expression patterns of the SP neurons, and their remnant layer 6b (L6b) neurons, are largely unknown. In this study, we analyzed the corticocortical projections of L6b/SP neurons in the mouse cortex and searched for a marker gene expressed in L6b/SP neurons that have ipsilateral inter-areal projections. Retrograde tracing experiments demonstrated that L6b/SP neurons in the primary somatosensory cortex (S1) projected to the primary motor cortex (M1) within the same cortical hemisphere at postnatal day (PD) 2 but did not show any callosal projection. This unilateral projection pattern persisted into adulthood. Our microarray analysis identified the gene encoding a β subunit of voltage-gated potassium channel (Kcnab1) as being expressed in L6b/SP. Double labeling with retrograde tracing and in situ hybridization demonstrated that Kcnab1 was expressed in the unilaterally-projecting neurons in L6b/SP. Embryonic expression was specifically detected in the SP as early as embryonic day (E) 14.5, shortly after the emergence of SP. Double immunostaining experiments revealed different degrees of co-expression of the protein product Kvβ1 with L6b/SP markers Ctgf (88%), Cplx3 (79%), and Nurr1 (58%), suggesting molecular subdivision of unilaterally-projecting L6b/SP neurons. In addition to expression in L6b/SP, scattered expression of Kcnab1 was observed during postnatal stages without layer specificity. Among splicing variants with three alternative first exons, the variant 1.1 explained all the cortical expression mentioned in this study. Together, our data suggest that L6b/SP neurons have corticocortical projections and Kcnab1 expression defines a subpopulation of L6b/SP neurons with a unilateral inter-areal projection

Morphometric Study of Hippocampal CA1 Pyramidal Neurons after Tualang Honey Administration

Tualang honey can be collected from the hives of Apis dorsata bee species on Tualang trees. Its various nutritional and curative properties could probably be due to its antioxidant effects. Subsequent to previous studies demonstrating its positive effects on spatial memory performance and hippocampal neuronal count, the current study investigated whether it has morphometric effects on the hippocampal cornu ammonis 1 (CA1) pyramidal neurons. It is important to evaluate the characteristics of hippocampal constituent neurons since this brain structure, which is primarily involved in memory processing, is most vulnerable towards oxidative stress. Male Sprague Dawley rats were force-fed five days a week for 12 consecutive weeks with 1.0ml/100g body weight of 70% Tualang honey (HON) or with 0.9% saline (SAL) as control. Nissl’s stained dorsal transverse hippocampal sections (8µm thick) of both groups were visualized under Olympus BX51 light microscope. Images were captured using Analyzer Life Science software and morphometric analysis was conducted using Image-Pro Premier 9.1 64-bit software. Only neuronal somas with clear nucleus and nucleolus were included in the morphometric analysis. Significant differences were observed between the groups for all five parameters selected (somatic area [SA], somatic perimeter [SP], somatic aspect ratio [SAR], somatic circularity index [SCI], and somatic roundness [SRo]). Values of SA and SP of HON group indicated significantly bigger sized CA1 neurons. Values of SAR, SCI and SRo, which indicated the shape of the neuronal somas, are biased towards less rounded shape. These values demonstrated HON has effects at the neuronal morphometric level

Kcnab1 Is Expressed in Subplate Neurons With Unilateral Long-Range Inter-Areal Projections

Subplate (SP) neurons are among the earliest-born neurons in the cerebral cortex and heterogeneous in terms of gene expression. SP neurons consist mainly of projection neurons, which begin to extend their axons to specific target areas very early during development. However, the relationships between axon projection and gene expression patterns of the SP neurons, and their remnant layer 6b (L6b) neurons, are largely unknown. In this study, we analyzed the corticocortical projections of L6b/SP neurons in the mouse cortex and searched for a marker gene expressed in L6b/SP neurons that have ipsilateral inter-areal projections. Retrograde tracing experiments demonstrated that L6b/SP neurons in the primary somatosensory cortex (S1) projected to the primary motor cortex (M1) within the same cortical hemisphere at postnatal day (PD) 2 but did not show any callosal projection. This unilateral projection pattern persisted into adulthood. Our microarray analysis identified the gene encoding a β subunit of voltage-gated potassium channel (Kcnab1) as being expressed in L6b/SP. Double labeling with retrograde tracing and in situ hybridization demonstrated that Kcnab1 was expressed in the unilaterally-projecting neurons in L6b/SP. Embryonic expression was specifically detected in the SP as early as embryonic day (E) 14.5, shortly after the emergence of SP. Double immunostaining experiments revealed different degrees of co-expression of the protein product Kvβ1 with L6b/SP markers Ctgf (88%), Cplx3 (79%), and Nurr1 (58%), suggesting molecular subdivision of unilaterally-projecting L6b/SP neurons. In addition to expression in L6b/SP, scattered expression of Kcnab1 was observed during postnatal stages without layer specificity. Among splicing variants with three alternative first exons, the variant 1.1 explained all the cortical expression mentioned in this study. Together, our data suggest that L6b/SP neurons have corticocortical projections and Kcnab1 expression defines a subpopulation of L6b/SP neurons with a unilateral inter-areal projection. © 2019 Tiong, Oka, Sasaki, Taniguchi, Doi, Akiyama and Sato