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

Nigella sativa supplementation attenuates recognition memory and cellular morphometric impairments induced by toluene administration in mice

Toluene exposure has been associated with detrimental effects on the central nervous system. Discovering natural products that can offer neuroprotection following toluene exposure is an essential alternative. Nigella sativa (NS), a popular natural supplement, is a good candidate due to its antioxidant and neuroprotective properties. The study aimed to investigate the protective potentials of NS against toluene, on recognition memory performance in the Novel Object Recognition (NOR) test and cellular morphometric measurements of hippocampal CA1 pyramidal neurons. Adult male ICR mice (n30) were randomly divided into five groups; G1: corn oil (CO), G2: toluene (TOL), G3: toluene and NS seed suspension (TOL-NSS), G4: toluene and NS oil (TOL-NSO), G5: toluene and thymoquinone (TOL-TQ). NS supplementations were administered orally once daily for 14 consecutive days while 500 mg/kg b.w. toluene was administered intraperitoneally from day 8 until day 14. Behavioural NOR test was conducted. Subsequently, mice were intracardially perfused and the brains were dissected, then histologically processed. The somatic size and shape of hippocampal CA1 pyramidal neurons were quantified according to specific morphometric parameters. Toluene reduced recognition memory performance of mice and somatic size of hippocampal CA1 pyramidal neurons. Contradictorily, TQ, NSO, and NSS improved mice recognition memory and somatic size of hippocampal CA1 pyramidal neurons. Somatic shape of hippocampal CA1 pyramidal neurons was unaffected by the different treatments. Although nonsignificant differences were observed, the results indicated the tendency for toluene to cause impairment, while NS supplementations improved mouse recognition memory performance and hippocampal neuronal neuron structure

Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord

Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted similar to 7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in similar to 6% of GABAergic boutons in laminae I-IIo, and similar to 1% of those in laminae IIi-III. Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina I

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn

Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia

Interstitial Axon Collaterals of Callosal Neurons Form Association Projections from the Primary Somatosensory to Motor Cortex in Mice

Association projections from cortical pyramidal neurons connect disparate intrahemispheric cortical areas, which are implicated in higher cortical functions. The underlying developmental processes of these association projections, especially the initial phase before reaching the target areas, remain unknown. To visualize developing axons of individual neurons with association projections in the mouse neocortex, we devised a sparse labeling method that combined in utero electroporation and confocal imaging of flattened and optically cleared cortices. Using the promoter of an established callosal neuron marker gene that was expressed in over 80% of L2/3 neurons in the primary somatosensory cortex (S1) that project to the primary motor cortex (M1), we found that an association projection of a single neuron was the longest among the interstitial collaterals that branched out in L5 from the earlier-extended callosal projection. Collaterals to M1 elongated primarily within the cortical gray matter with little branching before reaching the target. Our results suggest that dual-projection neurons in S1 make a significant fraction of the association projections to M1, supporting the directed guidance mechanism in long-range corticocortical circuit formation over random projections followed by specific pruning.Yuichiro Oka, Miyuki Doi, Manabu Taniguchi, Sheena Y X Tiong, Hisanori Akiyama, Takuto Yamamoto, Tokuichi Iguchi, Makoto Sato, Interstitial Axon Collaterals of Callosal Neurons Form Association Projections from the Primary Somatosensory to Motor Cortex in Mice, Cerebral Cortex, 2021;, bhab153, https://doi.org/10.1093/cercor/bhab153

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