Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Localization of nectin‐2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage‐gated A‐type K+ channel Kv4.2 in the medial habenula

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin‐like cell adhesion molecules (CAMs) nectin‐2α and nectin‐2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin‐2α, but not nectin‐2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage‐gated A‐type K⁺ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin‐2α at this boundary was not colocalized with the nectin‐2α‐binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin‐2α forms a cell adhesion apparatus different from the Kv4.2‐associated MSs. Genetic ablation of nectin‐2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin‐2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.     

Comprehensive analysis of area‐specific and time‐dependent changes in gene expression in the motor cortex of macaque monkeys during recovery from spinal cord injury

The present study aimed to assess the molecular bases of cortical compensatory mechanisms following spinal cord injury in primates. To accomplish this, comprehensive changes in gene expression were investigated in the bilateral primary motor cortex (M1), dorsal premotor cortex (PMd), and ventral premotor cortex (PMv) after a unilateral lesion of the lateral corticospinal tract (l‐CST). At 2 weeks after the lesion, a large number of genes exhibited altered expression levels in the contralesional M1, which is directly linked to the lesioned l‐CST. Gene ontology and network analyses indicated that these changes in gene expression are involved in the atrophy and plasticity changes observed in neurons. Orchestrated gene expression changes were present when behavioral recovery was attained 3 months after the lesion, particularly among the bilateral premotor areas, and a large number of these genes are involved in plasticity. Moreover, several genes abundantly expressed in M1 of intact monkeys were upregulated in both the PMd and PMv after the l‐CST lesion. These area‐specific and time‐dependent changes in gene expression may underlie the molecular mechanisms of functional recovery following a lesion of the l‐CST.     

Projections between visual cortex and pulvinar in the rat

The extrageniculate visual pathway, which carries visual information from the retina through the superficial layers of the superior colliculus and the pulvinar, is poorly understood. The pulvinar is thought to modulate information flow between cortical areas, and has been implicated in cognitive tasks like directing visually guided actions. In order to better understand the underlying circuitry, we performed retrograde injections of modified rabies virus in the visual cortex and pulvinar of the Long‐Evans rat. We found a relatively small population of cells projecting to primary visual cortex (V1), compared to a much larger population projecting to higher visual cortex. Reciprocal corticothalamic projections showed a similar result, implying that pulvinar does not play as big a role in directly modulating rodent V1 activity as previously thought.     

In vitro characterization of intrinsic properties and local synaptic inputs to pyramidal neurons in macaque primary motor cortex

Primates (including humans) have a highly developed corticospinal tract, and specialized motor cortical areas which differ in key ways from rodents. Much work on motor cortex has therefore used macaque monkeys as a good animal model for human motor control. However, there is a paucity of data describing the fundamental functional architecture of primate primary motor cortex, which is best addressed with in vitro approaches. In this study we examined the cellular properties and the micro‐circuitry of the adult macaque primary motor cortex by carrying out in‐vitro intracellular recordings. We aimed to characterize the basic properties of the cortical circuitry by studying the intrinsic properties of its pyramidal neurons and their physiological interconnectivity. We studied the passive and active electrophysiological properties of pyramidal neurons in both superficial and deep cortical layers. Both superficial and deep pyramidal neurons exhibited bursting behaviour that could act as powerful excitation for downstream targets. Synaptic connections were lamina specific. Neurons in the deep layers had convergent excitatory inputs from all cortical layers whereas superficial neurons had only significant inputs from superficial layers. This sheds light on the functional architecture of the primate primary motor cortex and how its output is shaped. We also took the unique opportunity in our recording technique to characterize the relationship between intracellular and extracellular spike waveforms, with implications for cell‐type identification in studies in awake behaving monkey. Our results will aid the interpretation of primate studies into motor control involving extracellular spike recordings and electrical stimulation in primary motor cortex.     

Dorsal and ventral parts of thalamic nucleus submedius project to different areas of rat orbitofrontal cortex: A single neuron‐tracing study using virus vectors

The rodent orbitofrontal cortex is involved in a variety of cognitive and behavioral functions that require thalamic input to be successfully expressed. Although the thalamic nucleus submedius (Sm) is a major source of afferents to the orbitofrontal cortex, thalamocortical projection from the Sm has not been fully elucidated. In the present study, we first divided the rat Sm into dorsal and ventral parts according to the distribution of vesicular glutamate transporter 2‐immunoreactive varicosities, which were somatosensory afferents from the brain stem. Subsequently we investigated dendritic and axonal arborizations of individual dorsal and ventral Sm neurons by visualizing the processes with Sindbis virus vectors expressing membrane‐targeted fluorescent proteins. The number of dendritic processes of ventral Sm neurons was greater than that of dorsal Sm neurons. In the cerebral cortex, all the reconstructed Sm neurons sent axons primarily to layers 2–5. Interestingly, dorsal Sm neurons formed a single axon arbor exclusively within the ventrolateral orbital area, whereas ventral Sm neurons made two axon arbors in the lateral orbital and ventral orbital areas simultaneously. The spread of each axon arbor was 500–1000 µm in diameter in the direction tangential to the cortical surface. These results indicate that the dorsal and ventral Sm comprise two distinct thalamocortical pathways. The dorsal Sm pathway relay somatosensory information to the ventrolateral orbital area and may be involved in emotional and aversive aspects of nociceptive information processing, whereas the ventral Sm pathway seems to co‐activate distant orbitofrontal cortical areas, and may link their functions under certain circumstances.     

Parvalbumin-like immunoreactivity of layer V pyramidal cells in the motor and somatosensory cortex of adult primates

Most previous immunocytochemical studies have indicated that the calcium-binding protein parvalbumin is present only in non-pyramidal neurons of the adult cerebral cortex. Using nickel and cobalt to enhance the diaminobenzidine reaction product, we observed large layer V pyramidal cells with parvalbumin-like immunoreactivity in the primary motor cortex (area 4) and somatosensory cortex of adult macaque monkeys and galagos, including giant Betz cells in area 4.     

Immunohistochemical localisation of the voltage gated potassium ion channel subunit Kv3.3 in the rat medulla oblongata and thoracic spinal cord

Voltage gated K+ channels (Kv) are a diverse group of channels important in determining neuronal excitability. The Kv superfamily is divided into 12 subfamilies (Kv1-12) and members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique expression pattern. Since the localisation of Kv subunits is important in defining the roles they play in neuronal function, we have used immunohistochemistry to determine the distribution of the Kv3.3 subunit in the medulla oblongata and spinal cord of rats. Kv3.3 subunit immunoreactivity (Kv3.3-IR) was widespread but present only in specific cell populations where it could be detected in somata, dendrites and synaptic terminals. Labelled neurones were observed in the spinal cord in laminae IV and V, in the region of the central canal and in the ventral horn. In the medulla oblongata, labelled cell bodies were numerous in the spinal trigeminal, cuneate and gracilis nuclei whilst rarer in the lateral reticular nucleus, hypoglossal nucleus and raphe nucleus. Regions containing autonomic efferent neurones were predominantly devoid of labelling with only occasional labelled neurones being observed. Dual immunohistochemistry revealed that some Kv3.3-IR neurones in the ventral medullary reticular nucleus, spinal trigeminal nucleus, dorsal horn, ventral horn and central canal region were also immunoreactive for the Kv3.1b subunit. The presence of Kv3.3 subunits in terminals was confirmed by co-localisation of Kv3.3-IR with the synaptic vesicle protein SV2, the vesicular glutamate transporter VGluT2 and the glycine transporter GlyT2. Co-localisation of Kv3.3-IR was not observed with VGluT1, tyrosine hydroxylase, serotonin or choline acetyl transferase. Electron microscopy confirmed the presence of Kv3.3-IR in terminals and somatic membranes in ventral horn neurones, but not motoneurones. This study provides evidence supporting a role for Kv3.3 subunits in regulating neuronal excitability and in the modulation of excitatory and inhibitory synaptic transmission in the medulla oblongata and spinal cord.     

Immunocytochemical heterogeneity of somatostatin‐expressing GABAergic interneurons in layers II and III of the mouse cingulate cortex: A combined immunofluorescence/design‐based stereologic study

Many neurological diseases including major depression and schizophrenia manifest as dysfunction of the GABAergic system within the cingulate cortex. However, relatively little is known about the properties of GABAergic interneurons in the cingulate cortex. Therefore, we investigated the neurochemical properties of GABAergic interneurons in the cingulate cortex of FVB‐Tg(GadGFP)45704Swn/J mice expressing green fluorescent protein (GFP) in a subset of GABAergic interneurons (GFP‐expressing inhibitory interneurons [GINs]) by means of immunocytochemical and design‐based stereologic techniques. We found that GINs represent around 12% of all GABAergic interneurons in the cingulate cortex. In contrast to other neocortical areas, GINs were only found in cortical layers II and III. More than 98% of GINs coexpressed the neuropeptide somatostatin (SOM), but only 50% of all SOM + neurons were GINs. By analyzing the expression of calretinin (CR), calbindin (CB), parvalbumin, and various neuropeptides, we identified several distinct GIN subgroups. In particular, we observed coexpression of SOM with CR and CB. In addition, we found neuropeptide Y expression almost exclusively in those GINs that coexpressed SOM and CR. Thus, with respect to the expression of calcium‐binding proteins and neuropeptides, GINs are surprisingly heterogeneous in the mouse cingulate cortex, and the minority of GINs express only one marker protein or peptide. Furthermore, our observation of overlap between the SOM + and CR + interneuron population was in contrast to earlier findings of non‐overlapping SOM + and CR + interneuron populations in the human cortex. This might indicate that findings in mouse models of neuropsychiatric diseases may not be directly transferred to human patients. J. Comp. Neurol. 524:2281–2299, 2016. © 2015 Wiley Periodicals, Inc.     

Continuous measurement of net potassium movements in rat brain cortex suspensions: Effects of glutamate, veratridine, creatine and other substances

Net K fluxes in in vitro suspensions of sliced rat brain cortex were studied by means of a K-sensitive electrode. When incubation was in 3 mM K, a net K efflux occurred. It could be resolved into two first-order rate constants: k1 = 0.486 min-1, and k2 = 0.0102 min-1, that originated from compartments that contained 18% and 82% of tissue K, respectively. k1 Was suppressed by tetrodotoxin (TTX), and k2 was increased 38-fold by veratridine. The latter effect was blocked by TTX, methylphenidate (1 mM), creatine (25 mM), apamin (50 nM), quinine (100 microM), verapamil (22 microM) or D-600 (38 microM). Net K loss was greatly increased by 1 mM ouabain, and enhanced by sodium azide plus iodoacetamide, but not by 0.1 M ethanol. Glutamate (5 mM) induced a considerable and rapid net uptake of K, while aspartate or N-methylaspartate increased K efflux.     

Origins of callosal projections to the supplementary motor area (SMA): a direct comparison between pre-SMA and SMA-proper in macaque monkeys.

The two subdivisions of the supplementary motor area (SMA), the pre-SMA (rostrally) and SMA-proper (caudally), exhibit distinct functional properties and clear differences with respect to their connectivity with the spinal cord, the thalamus, and other homolateral motor cortical areas. The goal of the present study was to establish in monkeys whether these subdivisions also differ with regard to their callosal connectivity. Two fluorescent retrograde tracers (Fast Blue and Diamidino Yellow) were injected in each animal, one in the pre-SMA and the second in the SMA-proper. Tracer injections in the pre-SMA or in SMA-proper resulted in significant numbers of labeled neurons in the opposite SMA, premotor cortex (PM), cingulate motor areas (CMA), and cingulate gyrus. Labeled neurons in M1 were rare, being observed only after injection in the SMA-proper. The two subdivisions of the SMA differed in the proportion of labeled neurons found across areas providing their callosal inputs. The SMA-proper receives about half of its callosal inputs from its counterpart in the other hemisphere (42-65% across monkeys). A comparable proportion of neurons was found in the pre-SMA after injection in the opposite pre-SMA (32-47%). The pre-SMA receives more callosal inputs from the rostral halves of the dorsal PM, the ventral PM, and the CMA than from their caudal halves. In addition, the pre-SMA, but not the SMA-proper, receives callosal inputs from the prefrontal cortex. The SMA-proper receives more callosal inputs from the caudal halves of the dorsal PM and ventral PM than from their rostral halves. The two subdivisions of the SMA receive callosal inputs from the same cortical areas (except the prefrontal cortex and M1), but they differ with respect to the quantitative contribution of each area of origin. In conclusion, quantitative data now support the notion that pre-SMA receives more transcallosal inputs than the SMA-proper.     

Hypergravity exposure decreases γ-aminobutyric acid immunoreactivity in axon terminals contacting pyramidal cells in the rat somatosensory cortex: A quantitative immunocytochemical image analysis

Quantitative evaluation of γ-aminobutyric acid immunoreactivity (GABA-IR) in the hindlimb representation of the rat somatosensory cortex after 14 days of exposure to hypergravity (hyper-G) was conducted by using computer-assisted image processing. The area of GABA-IR axosomatic terminals apposed to pyramidal cells of cortical layer V was reduced in rats exposed to hyper-G compared with control rats, which were exposed either to rotation alone or to vivarium conditions. Based on previous immunocytochemical and behavioral studies, we suggest that this reduction is due to changes in sensory feedback information from muscle receptors. Consequently, priorities for muscle recruitment are altered at the cortical level, and a new pattern of muscle activity is thus generated. It is proposed that the reduction observed in GABA-IR of the terminal area around pyramidal neurons is the immunocytochemical expression of changes in the activity of GABAergic cells that participate in reprogramming motor outputs to achieve effective movement control in response to alterations in the afferent information. J. Neurosci. Res. 53:135–142, 1998. © 1998 Wiley-Liss, Inc.     

Dopamine-β-hydroxylase-like immunoreactivity in the fetal cerebral cortex of the rat: Noradrenergic ascending pathways and terminal fields

A topographical analysis of the noradrenergic innervation in the fetal rat cerebral cortex was carried out from embryonic day 15 (E15) until birth using antibodies raised against dopamine-β-hydroxylase (DBH). During late gestation DBH-like immunoreactive axons were coursing through the basal forebrain along three pathways:

1. (1) a medial component reached the medial cortex and then ran caudally along the anlage of the cingulum bundle;

2. (2) a lateral component reached the frontal pole and curved ventro-dorsally in the primordium of the external capsule;

3. (3) a few fibers were observed along the ventral amygdaloid bundle toward the amygdaloid complex and the surrounding cortex. No DBH positive fibers were observed in the main body of the internal capsule.

The first noradrenergic axons were seen at E17 in the frontal pole, the lateral frontal cortex, and in the medial frontal cortex which also receives a dopaminergic input. The innervation then extended caudally, but the dorsal part of the cortex was reached after a 2-day delay when compared to the medial and lateral parts. The arrival of noradrenergic axons did not parallel the gradient of cortical neurogenesis; however, all cortical areas were innervated at birth. DBH positive fibers reached a given cortical region simultaneously through the marginal and intermediate zones and then invaded the cortical plate.     

A comparison of GAD- and GABA-immunoreactive neurons in the first somatosensory area (SI) of the rat cortex

Neurons immunoreactive for anti-glutamic acid decarboxylase (anti-GAD) and anti-gamma-aminobutyric acid (anti-GABA) were compared in adjacent sections from the rat somatosensory cortex (SI). GAD- and GABA-positive neurons in animals either treated or not treated with colchicine were found to occur at a ratio of 1:2. Measurement of areas of GAD- and GABA-immunoreactive neurons confirmed the presence of an 'exuberant' GABA-positive neuronal population not visualized by the GAD antiserum.     

Modulation of the ability of clozapine to facilitate NMDA- and electrically evoked responses in pyramidal cells of the rat medial prefrontal cortex by dopamine: pharmacological evidence

Previous studies have shown that dopamine (DA) may play an important role in mediating or modulating the facilitating action of clozapine in glutamatergic transmission. This possibility was tested further in the present study by pharmacological manipulation of the DA system. When rats were pretreated with reserpine (which blocks storage of biogenic amines) and alpha-methyl para-tyrosine (AMPT, which inhibits tyrosine hydroxylase, the rate-limiting enzyme for the DA synthesis), the ability of clozapine to augment glutamatergic transmission in pyramidal cells of the medial prefrontal cortex (mPFC) was totally abolished. Furthermore, the application of l-dihydroxyphenylalanine (L-DOPA, the immediate precursor of DA which bypasses the synthesis step inhibited by AMPT) reversed the effect produced by reserpine plus AMPT and reinstated the facilitating action of clozapine, whereas administration of 5-hydroxytryptophan (5-HTP), the immediate precursor of 5-HT, was ineffective. In addition, DA D1 receptor antagonist SCH 23390 also completely prevented clozapine-induced facilitating action in the mPFC pyramidal cells. The present results demonstrate that newly synthesized DA and DA D1 receptors are required for clozapine to elicit its facilitating action on glutamatergic neurotransmission in the mPFC.     

Differential Na+ channel β1 subunit mRNA expression in stellate and flat astrocytes cultured from rat cortex and cerebellum: A combined in situ hybridization and immunocytochemistry study

Astrocytes have been shown to express voltage-sensitive Na⁺ channels, but the molecular structure of these channels is not yet known. Recent studies have demonstrated the expression of rat brain voltage-sensitive Na⁺ channel mRNAs in astrocytes. In this study, we used a combined non-radioactive in situ hybridization immunocytochemistry method to investigate the expression of voltage-sensitive Na⁺ channel ß1 subunit (Naß1) mRNA in definitively identified, GFAP-positive astrocytes cultured from two different regions of the rat brain, cerebrum and cerebellum. In general, two morphologically distinct types of GFAP-positive astrocytes were observed in culture: flat, fibroblast-like and stellate, process-bearing. We observed a differential expression ofNaß1 mRNA in GFAP-positive astrocytes: (1) stellate astrocytes expressed Naß1 mRNA, although the level of Naß1 mRNA expression was variable, and (2) flat astrocytes generally did not express Naß1 mRNA. Moreover, Bergmann-like cells from cerebellum did not express Naß mRNA, while the granule cells associated with Bergmann-like cell expressed Naß mRNA. These observations indicate that Naß mRNA is differentially expressed in rat astrocytes with various morphologies in vitro. © 1995 Wiley-Liss, Inc.     

Electrophysiological evidence that the mediodorsal nucleus of the thalamus is a relay between the ventral pallidum and the medial prefrontal cortex in the rat

The neural connections from the ventral pallidum (VP) through the mediodorsal nucleus of the thalamus (MD) to the medial prefrontal cortex (MPC) were investigated. Extracellular recordings were made from 219 neurons in the medial and lateral portions of the MD and the VP and the MPC were stimulated. The most frequent response to VP stimulation was inhibition and inhibition preceded by excitation. Also, the most frequent response of MD units to MPC stimulation was inhibition and inhibition preceded by excitation. Nineteen of 26 MD units, activated antidromically by MPC stimulation, responded orthodromically to VP stimulation. The most frequent orthodromic response of these MD output neurons was inhibition and inhibition preceded by excitation. GABA iontophorized onto MD neurons reduced their rate of discharge. GABA and picrotoxin iontophorized onto MD neurons did not influence the inhibitory or excitatory responses to VP stimulation. These electrophysiological results support previous anatomical findings of connections between the VP and the MPC by way of the MD. MD output neurons to the MPC receive mostly inhibitory inputs from VP afferents. A high proportion of MD neurons respond orthodromically to both VP and MPC stimulation, suggesting the convergence of synaptic inputs from these structures to the same MD units.