Substance P receptor expression by inhibitory interneurons of the rat hippocampus: enhanced detection using improved immunocytochemical methods for the preservation and colocalization of GABA and other neuronal markers

Two unresolved issues regarding the identification and characterization of hippocampal interneurons were addressed in this study. One issue was the longstanding inability to detect gamma-aminobutyric acid (GABA) in the somata of several hippocampal interneuron subpopulations, which has prevented the unequivocal identification of all hippocampal interneurons as GABA neurons. The second issue was related to the identification of the hippocampal interneurons that constitutively express substance P (neurokinin-1) receptors (SPRs). The recent development of neurotoxins that specifically target SPR-expressing cells suggests that it may be possible to destroy hippocampal inhibitory interneurons selectively for experimental purposes. Although SPRs are apparently expressed in the hippocampus only by interneurons, colocalization studies have found that most interneurons of several subtypes and hippocampal subregions appear SPR-negative. Thus, the identities and locations of the inhibitory interneurons that are potential targets of an SPR-directed neurotoxin remain in doubt. Using newly developed methods designed to copreserve and colocalize GABA and polypeptide immunoreactivities with increased sensitivity, the authors report that virtually all hippocampal interneuron somata that are immunoreactive for parvalbumin (PV), calbindin, calretinin, somatostatin (SS), neuropeptide Y, cholecystokinin, and vasoactive intestinal peptide exhibited clearly detectable, somal, GABA-like immunoreactivity (LI). Hippocampal SPR-LI was detected only on the somata and dendrites of GABA-immunopositive interneurons. All glutamate receptor subunit 2-immunoreactive principal cells, including dentate granule cells, hilar mossy cells, and hippocampal pyramidal cells, were devoid of detectable SPR-LI, even after prolonged electrical stimulation of the perforant pathway that induced the expression of other neuronal proteins in principal cells. Thus, hippocampal interneurons of all subtypes and subregions were found to be SPR-immunoreactive, including the PV-positive interneurons of the dentate hilus and hippocampus, and the SS-positive cells of area CA1, both of which were previously reported to lack SPR-LI. Only minor proportions of hippocampal interneurons appeared clearly devoid of detectable SPR-LI. These results demonstrate for the first time that all identified interneuron subpopulations of the rat hippocampus are GABA-immunoreactive, and that many inhibitory interneurons of all subtypes in all subregions of the rat hippocampus express SPRs constitutively.     

Transient expression of A-type K channel alpha subunits Kv4.2 and Kv4.3 in rat spinal neurons during development

A-type K(+) currents (I(A)s) have been detected from the ventral horn neurons in rat spinal cord during embryonic day (E) 14 to postnatal day (P) 8 but not in adulthood. It is not known which types of neurons and which A-type K(+) channel alpha subunits express the I(A)s and what the possible function might be. Here, we examined the expression of two A-type K(+) channel alpha subunits, Kv4.2 and Kv4.3, in rat spinal cord at various developmental stages by immunohistochemistry. We found a transient expression of Kv4.2 in somatic motoneurons during E13.5-P8 with a peak around E17.5, which coincides temporally with the natural selection of motoneurons. Transient expression of Kv4.2 and Kv4.3 was also observed in the intermediate gray (IG) interneurons. During E19.5-P14, some IG interneurons express Kv4.2, some express Kv4.3 and a subset co-express Kv4.2 and Kv4.3. Peak expression of Kv4.2 and Kv4.3 in the IG interneurons was detected around P1, which coincides temporally with the developmental selection of IG interneurons. In contrast to the I(A)-expressing subunits Kv4.2 and Kv4.3, a delayed-rectifier K(+) channel alpha subunit Kv1.6 is persistently expressed in somatic motoneurons and IG interneurons. Together, these data support the hypothesis that expression of I(A)s may protect I(A)-expressing somatic motoneurons, and possibly also IG interneurons, from naturally occurring cell death during developmental selection.     

Subcellular localization of the K channel subunit Kv3.1b in selected rat CNS neurons

Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K⁺ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts of the axon. The distribution in the cells analyzed supports a role in action potential transmission by regulating action potential duration.     

Subunit-dependent axonal trafficking of distinct alpha heteromeric potassium channel complexes

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.     

Increased Kv1 channel expression may contribute to decreased sIPSC frequency following chronic inhibition of NR2B-containing NMDAR

Numerous studies have documented the effects of chronic N-methyl-D-aspartate receptor (NMDAR) blockade on excitatory circuits, but the effects on inhibitory circuitry are not well studied. NR2A- and NR2B-containing NMDARs play differential roles in physiological processes, but the consequences of chronic NR2A- or NR2B-containing NMDAR inhibition on glutamatergic and GABAergic neurotransmission are unknown. We investigated altered GABAergic neurotransmission in dentate granule cells and interneurons following chronic treatment with the NR2B-selective antagonist, Ro25,6981, the NR2A-prefering antagonist, NVP-AAM077, or the non-subunit-selective NMDAR antagonist, D-APV, in organotypic hippocampal slice cultures. Electrophysiological recordings revealed large reductions in spontaneous inhibitory postsynaptic current (sIPSC) frequency in both granule cells and interneurons following chronic Ro25,6981 treatment, which was associated with minimally altered sIPSC amplitude, miniature inhibitory postsynaptic current (mIPSC) frequency, and mIPSC amplitude, suggesting diminished action potential-dependent GABA release. Chronic NVP-AAM077 or D-APV treatment had little effect on these measures. Reduced sIPSC frequency did not arise from downregulated GABA(A)R, altered excitatory or inhibitory drive to interneurons, altered interneuron membrane properties, increased failure rate, decreased action potential-dependent release probability, or mGluR/GABA(B) receptor modulation of GABA release. However, chronic Ro25,6981-mediated reductions in sIPSC frequency were occluded by the K+ channel blockers, dendrotoxin, margatoxin, and agitoxin, but not dendrotoxin-K or XE991. Immunohistochemistry also showed increased Kv1.2, Kv1.3, and Kv1.6 in the dentate molecular layer following chronic Ro25,6981 treatment. Our findings suggest that increased Kv1 channel expression/function contributed to diminished action potential-dependent GABA release following chronic NR2B-containing NMDAR inhibition and that these Kv1 channels may be heteromeric complexes containing Kv1.2, Kv1.3, and Kv1.6.     

Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus

PURPOSE: Mice lacking the Kv1.1 potassium channel alpha subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1(-/-) (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. METHODS: Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1(-/-) animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1(-/-) brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E. coli lacZ reporter gene. Vector-injected mice were examined to determine the extent of neuronal infection. RESULTS: Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1(-/-) mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed "ectopic" Kv1.1 alpha channel subunit expression. CONCLUSIONS: Kcna1 deletion in mice results in a seizure disorder that resembles--electrographically and neuropathologically--the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection.     

Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic I_SAs. It has been proposed that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits, K⁺ channel‐interacting proteins (KChIPs), and dipeptidyl peptidase‐like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin‐ and somatostatin‐positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of I_SA‐expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. J. Comp. Neurol. 523:608–628, 2015. © 2014 Wiley Periodicals, Inc.     

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains.     

Axonal sorting of Kir3.3 defines a GABA-containing neuron in the CA3 region of rodent hippocampus

Hippocampal interneurons comprise a heterogeneous group of locally acting GABAergic neurons. In addition to their variability in cotransmitter content and receptor profile, they express a variety of potassium channels that specify their individual properties. Here we describe a new type of large GABA-containing neuron in rodent hippocampus that is characterized by an axonal sorting of the potassium channel Kir3.3. The parent cell bodies of the Kir3.3-positive axons are located in CA3, as assessed by primary cultures derived from hippocampal subareas. At postnatal day 14 these neurons appear at the border between stratum oriens and stratum pyramidale of CA3, from where their axons pass through stratum pyramidale to join the mossy fiber tract. In adult hippocampus, high levels of Kir3.3 channel protein exist in axons that run with the mossy fiber tract. Kir3.3 and the vesicular GABA transporter could be identified in subpopulations of large synaptic terminals that probably derive from Kir3.3 neurons. Axonal sorting of Kir3.3 appears to be typical of a group of large inhibitory neurons, including Purkinje cells and a novel type of interneuron in CA3. Kir3.3 neurons might modulate the activity of CA3 circuitries and consequently memory processing in the hippocampus.     

Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development

Dopaminergic neurons of the substantia nigra pars compacta (SNc) are involved in the control of movement, sleep, reward, learning, and nervous system disorders and disease. To date, a thorough characterization of the ion channel phenotype of this important neuronal population is lacking. Using immunohistochemistry, we analyzed the somatodendritic expression of voltage‐gated ion channel subunits that are involved in pacemaking activity in SNc dopaminergic neurons in 6‐, 21‐, and 40‐day‐old rats. Our results demonstrate that the same complement of somatodendritic ion channels is present in SNc dopaminergic neurons from P6 to P40. The major developmental changes were an increase in the dendritic range of the immunolabeling for the HCN, T‐type calcium, Kv4.3, delayed rectifier, and SK channels. Our study sheds light on the ion channel subunits that contribute to the somatodendritic delayed rectifier (Kv1.3, Kv2.1, Kv3.2, Kv3.3), A‐type (Kv4.3) and calcium‐activated SK (SK1, SK2, SK3) potassium currents, I_H (mainly HCN2, HCN4), and the L‐ (Cav1.2, Cav1.3) and T‐type (mainly Cav3.1, Cav3.3) calcium currents in SNc dopaminergic neurons. Finally, no robust differences in voltage‐gated ion channel immunolabeling were observed across the population of SNc dopaminergic neurons for each age examined, suggesting that differing levels of individual ion channels are unlikely to distinguish between specific subpopulations of SNc dopaminergic neurons. This is significant in light of previous studies suggesting that age‐ or region‐associated variations in the expression profile of voltage‐gated ion channels in SNc dopaminergic neurons may underlie their vulnerability to dysfunction and disease. © 2014 Wiley Periodicals, Inc.     

Calcium-permeable AMPA receptors provide a common mechanism for LTP in glutamatergic synapses of distinct hippocampal interneuron types

Glutamatergic synapses on some hippocampal GABAergic interneurons exhibit activity-induced long-term potentiation (LTP). Interneuron types within the CA1 area expressing mutually exclusive molecular markers differ in LTP responses. Potentiation that depends on calcium-permeable (CP) AMPA receptors has been characterized in oriens-lacunosum moleculare (O-LM) interneurons, which express parvalbumin and somatostatin (SM). However, it is unknown how widely CP-AMPAR-dependent plasticity is expressed among different GABAergic interneuron types. Here we examine synaptic plasticity in rat hippocampal O-LM cells and two other interneuron types expressing either nitric oxide synthase (NOS) or cholecystokinin (CCK), which are known to be physiologically and developmentally distinct. We report similar CP-AMPAR-dependent LTP in NOS-immunopositive ivy cells and SM-expressing O-LM cells to afferent fiber theta burst stimulation. The potentiation in both cell types is induced at postsynaptic membrane potentials below firing threshold, and induction is blocked by intense spiking simultaneously with afferent stimulation. The strong inward rectification and calcium permeability of AMPARs is explained by a low level of GluA2 subunit mRNA expression. LTP is not elicited in CCK-expressing Schaffer collateral-associated cells, which lack CP-AMPARs and express high levels of the GluA2 subunit. The results show that CP-AMPAR-mediated synaptic potentiation is common in hippocampal interneuron types and occurs in interneurons of both feedforward and feedback inhibitory pathways.     

Contribution of Kv channel subunits to glutamate‐induced apoptosis in cultured rat hippocampal neurons

Potassium channel dysfunction has been implicated in apoptosis in many pathological conditions. However, which Kv channel subunit is involved in glutamate‐induced apoptosis remains unknown. In this study, the contributions of nine Kvα and three Kvβ subunits to glutamate‐induced hippocampal neuronal apoptosis were investigated. Results showed that neuronal apoptosis was not obvious with 12 hr incubation of glutamate but increased markedly after 18 hr, which was attenuated by the Kv channel blocker TEA. Among all the Kv subunits investigated, gene and protein expression of Kv2.1 increased significantly before the appearance of neuronal apoptosis, whereas the Kv1.1 mRNA level decreased quickly, and protein expression was reduced gradually after the insult. Seven other Kvα subunits and three Kvβ subunits were not obviously affected over time. In addition, Kv1.1 overexpression could reduce glutamate‐induced hippocampal neuronal apoptosis. Therefore, the alterations of Kv1.1 and Kv2.1 might contribute to glutamate‐induced toxicity in hippocampal neurons. These findings suggest that these two Kv channel subunits may represent potential therapeutic targets for neuropathological conditions in which glutamate‐induced toxicity is thought to contribute to neuronal dysfunction. © 2009 Wiley‐Liss, Inc.     

Modulation of Kv1.5 currents by Src tyrosine phosphorylation: potential role in the differentiation of astrocytes.

Using biophysical techniques, we previously have implicated outwardly rectifying potassium currents in the proliferation of cultured spinal cord astrocytes and have demonstrated that delayed rectifier potassium currents (I(Kd)), in particular, are upregulated on entry into the cell cycle and downregulated with cell cycle exit and differentiation. In the present study, using specific antibodies and antisense oligodeoxynucleotides, we show that this proliferation-dependent potassium current is mediated by the Shaker potassium channel Kv1.5. Downregulation of Kv1.5 protein by antisense oligodeoxynucleotides reduces astrocyte proliferation by approximately 50%, although no observed changes occur in Kv1.5 protein expression during spontaneous differentiation in culture. Tyrosine phosphorylation of Kv1.5, however, is downregulated markedly in differentiated cells but unaltered on cell cycle arrest. Using immunoprecipitation, we show that Kv1.5 is associated with Src family protein tyrosine kinases and that this association does not change with cell differentiation. Inhibition of kinase activity with the Src-specific inhibitor PP2 decreases Kv1.5 phosphorylation, reduces I(Kd), and inhibits astrocyte proliferation, specifically in the G(0)/G(1) phase of cell cycle. Conversely, I(Kd) are potentiated when active Src is present in the pipette. Transfection of quiescent astrocytes with constitutively active Src (Src Y529F) causes marked upregulation of astrocyte proliferation. These data suggest that Kv1. 5 is phosphorylated constitutively by Src kinases during growth and that downregulation of Src activity may underlie both astrocyte differentiation and the accompanying changes in delayed rectifier potassium channel activity.     

The loss of interneuron functional diversity in the piriform cortex after induction of experimental epilepsy

Interneuronal functional diversity is thought to be an important factor in the control of neural network oscillations in many brain regions. Specifically, interneuron action potential firing patterns are thought to modulate brain rhythms. In neurological disorders such as epilepsy where brain rhythms are significantly disturbed interneuron function is largely unexplored. Thus the purpose of this study was to examine the functional diversity of piriform cortex interneurons (PC; an area of the brain that easily supports seizures) before and after kindling-induced epilepsy. Using cluster analysis, we found five control firing behaviors. These groups were termed: non-adapting very high frequency (NAvHF), adapting high frequency (AHF), adapting low frequency (ALF), strongly adapting low frequency (sALF), and weakly adapting low frequency (wALF). A morphological analysis showed these spiking patterns were not associated with any specific interneuronal morphology although we found that most of the cells displaying NAvHF firing pattern were multipolar. After kindling about 40% of interneuronal firing pattern changed, and neither the NAvHF nor the wALF phenotypes were found. We also found that in multipolar interneurons a long-lasting potassium current was increased. A qPCR analysis indicated Kv1.6 subtype was up-regulated after kindling. An immunocytochemical analysis showed that Kv1.6 protein expression on parvalbumin (multipolar) interneurons increased by greater than 400%. We also examined whether these changes could be due to the selective death of a subset of interneurons but found that there was no change in cell number. These data show an important loss of the functional diversity of interneurons in the PC. Our data suggest that under pathophysiological condition interneurons are plastic resulting in the attenuation of high frequency network oscillations in favor of low frequency network activity. This may be an important new mechanism by which network synchrony is disturbed in epileptic seizures.     

Alpha7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons.

C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the alpha7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in alpha7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [(125)I]alpha-bungarotoxin. However, the distribution of the [(125)I]alpha-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [(125)I]alpha-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the alpha7 receptor gene. The distribution of [(125)I]alpha-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the alpha7 gene. These data suggest that the alpha7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.     

Preferential localization of the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are involved in the control of neuronal excitability and plasticity. In this study, we used immunoblotting and immunohistochemical techniques to reveal the developmental expression and subcellular distribution of the HCN1 subunit in the cerebellar cortex. During postnatal development, the spatio-temporal expression of HCN1 correlated well with the morphological events occurring during the ontogenesis of cerebellar interneurons. Using immunoblotting techniques, HCN1 was weakly detected during the first postnatal week and continued to increase throughout postnatal development, peaking at postnatal day (P)15. At the light-microscopic level, HCN1 immunoreactivity was very weak until P7 whereas from P10-12 to adulthood it was strongly detected in the lower third of the molecular layer and in the Purkinje cell layer. HCN1 was present in axons running through the molecular layer and in the pericellular basket around Purkinje cells at P12, but in the periaxonal plexus (the pinceau) surrounding their initial segment only after P15. Using immunofluorescence, HCN1 colocalized with GAD65 and synaptophysin, demonstrating that the subunit was present in inhibitory axons and axon terminals. At the electron-microscopic level, in adulthood, HCN1 immunoparticles were detected at postsynaptic sites in basket and Purkinje cells but most immunoparticles were found at presynaptic sites in basket cell axons and in terminals. In the axon terminals, the distribution of HCN1 was relatively uniform along the extrasynaptic plasma membrane; this was confirmed using quantitative techniques. The present findings suggest that HCN1 channels may provide a significant route for modulating co-ordinated cerebellar synaptic transmission through basket cells.     

Expression pattern of membrane-associated guanylate kinases in interneurons of the visual cortex

GABAergic interneurons are key elements regulating the activity of local circuits, and abnormal inhibitory circuits are implicated in certain psychiatric and neurodevelopmental diseases. The glutamatergic input that interneurons receive is a key determinant of their activity, yet its molecular structure and development, which are often distinct from those of glutamatergic input to pyramidal cells, are poorly defined. The membrane-associated guanylate kinase (MAGUK) homologs PSD-95/SAP90, PSD-93/chapsyn110, SAP97, and SAP102 are central organizers of the postsynaptic density at excitatory synapses on pyramidal neurons. We therefore studied the cell-type-specific and developmental expression of MAGUKs in the nonoverlapping parvalbumin (PV)- and somatostatin (SOM)-positive interneurons in the visual cortex. These interneuron subtypes account for the vast majority of interneurons in the cortex and have different functional properties and postsynaptic structures, being either axodendritic (PV(+)) or axospinous (SOM(+)). To study cell-type-specific MAGUK expression, we used DIG-labeled riboprobes against each MAGUK along with antibodies against either PV or SOM and examined tissue from juvenile (P15) and adult mice. Both PV(+) and SOM(+) interneurons express mRNA for PSD-95, PSD-93, and SAP102 in P15 and adult tissue. In contrast, these interneuron subtypes express SAP97 at P15, but for adult visual cortex we found that most PV(+) and SOM(+) interneurons show low or no expression of SAP97. Given the importance of SAP97 in regulating AMPA receptor GluA1 subunit and NMDA receptor subunits at glutamatergic synapses, these results suggest a developmental shift in glutamate receptor subunit composition and regulation of glutamatergic synapses on PV(+) and SOM(+) interneurons.     

束缚应激上调小鼠脾脏淋巴细胞钾离子通道(Kv1.3)的表达

目的观察束缚应激条件下小鼠脾脏细胞Kv1.3表达的变化。方法采用RT-PCR、Western blot等分子生物学及免疫组化实验方法,分别从基因和蛋白质水平揭示束缚应激对小鼠脾脏细胞Kv1.3表达的调节。结果束缚应激16 h后小鼠脾脏细胞Kv1.3 mRNA和蛋白质表达水平明显上调(P<0.05)。但是,相同束缚应激条件下小鼠脑组织Kv1.3表达无明显变化(P>0.05)。结论束缚应激状态下小鼠脾脏细胞Kv1.3 mR-NA和蛋白质表达水平呈明显组织特异性上调,提示束缚应激条件下免疫功能的调节可能与钾通道有关。这为进一步研究神经内分泌系统调节淋巴细胞功能的机制提供了新的思路。     

束缚应激上调小鼠脾脏淋巴细胞钾离子通道（Kvl．3）的表达

目的观察束缚应激条件下小鼠脾脏细胞Kvl．3表达的变化。方法采用RT—PCR、Western blot等分子生物学及免疫组化实验方法，分别从基因和蛋白质水平揭示束缚应激对小鼠脾脏细胞Kvl．3表达的调节。结暴束缚应激16h后小鼠脾脏细胞Kvl．3 mRNA和蛋白质表达水平明显上调（P〈0．05）。但是。相同束缚应激条件下小鼠脑组织Kvl．3表达无明显变化（P〉0．05）。结论束缚应激状态下小鼠脾脏细胞Kvl．3 mRNA和蛋白质表达水平呈明显组织特异性上调．提示束缚应激条件下免疫功能的调节可能与钾通道有关。这为进一步研究神经内分泌系统调节淋巴细胞功能的机制提供了新的思路。