Phenotypic diversity and expression of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67-green fluorescent protein mice.

The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67-green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67-GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67-GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages >67% of GFP(+) neurons were also GABA(+). With increasing age; (i) GFP(+) and GABA(+) cell numbers declined, (ii) ventral horn GFP(+) and GABA(+) neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in approximately 63% and approximately 35% of GFP(+) cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca(2+) binding proteins examined. Notably, most laminae I-II parvalbumin(+) neurons were also GFP(+). Neurogranin, a protein kinase C substrate, was found in approximately 1/2 of GFP(+) cells. Lastly, while only 7% of GFP(+) cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS(+) cells. We conclude that GAD67-GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67-GFP(+) neurons comprise a phenotypically diverse population.

Reversal of the circadian expression of tyrosine-hydroxylase but not nitric oxide synthase levels in the spinal cord of dopamine D3 receptor knockout mice.

Circadian rhythms have been described for numerous transmitter synthesizing enzymes in the brain but rarely in spinal cord. We measured spinal tyrosine-hydroxylase (TH) and nitric oxide synthase (NOS) levels in the thoracic intermediolateral nucleus, the location of sympathetic preganglionic neurons, in male wild type (WT) and dopamine D(3) receptor knockout mice (D(3)KO). TH and NOS levels both displayed circadian patterns in WT and D(3)KO animals with overall reduced TH and increased NOS expression in the D(3)KO mice. The circadian pattern of NOS expression was similar in WT and D(3)KO mice. In contrast, TH expression was inverted in D(3)KO mice, with TH levels consistently lower than in WT throughout the day, but strongly increased temporarily 1 h prior to daylight. TH is the rate-limiting enzyme for the production of dopamine. Spinal dopamine dysfunction is implicated in a sleep disorder called restless legs syndrome (RLS). RLS follows a circadian rhythm and is relieved clinically by dopamine D(3) receptor agonists. Our observations of an altered circadian pattern in spinal dopamine synthesis in D(3)KO animals may provide insight into putative dopaminergic mechanisms contributing to RLS.

Development of L-type calcium channels and a nifedipine-sensitive motor activity in the postnatal mouse spinal cord.

Intrinsic membrane properties are important in the regulation of motoneuronal output during such behaviours as locomotion. A conductance through L-type calcium channels has been implicated as an essential component in the transduction of motoneuronal input to output during locomotion. Given the developmental changes in calcium currents occurring postnatally in some neurons, and the increasing interest in the study of spinal locomotor output in neonatal preparations, experiments were conducted to investigate the postnatal development of L-type calcium channels in mouse motoneurons. This was assessed both physiologically, using a chemically induced rhythmic motor output, and anatomically, using immunohistochemical methods. The electrophysiological data were obtained during rhythmic bursting produced by application of N-methyl-D-aspartate (NMDA) and strychnine to the isolated spinal cord at various postnatal ages. The L-type calcium channel blocker nifedipine has no effect on this ventral root bursting in postnatal day (P) P2-P5 animals, but reversibly reduced the amplitude and/or burst duration of this activity in animals greater than P7. The immunohistochemical evidence demonstrates a dramatic change in the cellular profile of both the alpha1C and alpha1D subunits of L-type calcium channels during postnatal development; the labelling of both subunits increases with age, approximating the adult pattern by P18. These results demonstrate that in the spinal cord, the L-type calcium channel profile develops both physiologically and anatomically in the early postnatal period. This development parallels the development of the mature functional behaviours of weight bearing and walking, and may be necessary for the production of complex motor behaviour in the mature mammal.

Astrocytic gap junction removal, connexin43 redistribution, and epitope masking at excitatory amino acid lesion sites in rat brain.

We previously reported that kainic acid (KA) lesion sites in rat brain exhibit an absence of astrocytic gap junctions at 1 week post-lesion. Loss of immunocytochemical reactivity with a sequence-specific antibody against the astrocytic gap junctional protein connexin43 (Cx43) suggested epitope masking since persistence of Cx43 was observed on Western blots. Here, we determined the fate of Cx43 at various times after thalamic KA and striatal NMDA lesions. In normal tissue and at 6 hr post-KA lesion, Cx43 immunoreactivity predominated at typical astrocytic gap junctions. Immunolabelled junctions were still seen at 3 days, with epitope masking already present, and were virtually absent by 6 days post-lesion. Gap junction remodeling was indicated by the appearance of intracellular immunostained annular profiles and uncharacteristically extensive gap junctions between symmetrically immunolabelled membranes and between labelled astrocytic and unlabelled oligodendrocytic membranes. Labelled multivesicular clusters emerged at 2 days, were numerous at 3 days and constituted the sole Cx43 sequestration site by post-lesion day 6. Ultrastructural disruption and gap junction disassembly progressed more slowly in NMDA-injected tissue where immunoreactivity persisted, albeit at markedly decreasing levels until the final survival time examined (16 days). Intense Cx43 immunolabelling was seen in filopodia of putative reactive astrocytes at the lesion periphery at 6-8 days and was associated at 16 days with an increased number of gap junctions primarily between fine astrocytic processes. These results demonstrate that massive neuronal loss alone or in conjunction with direct actions of excitotoxins on astrocytes precipitates an astrocytic reaction accompanied initially by removal of their gap junctions followed by redistribution of Cx43, and suggest that the astrocytic syncytium may undergo reorganization in a manner leading to isolation of the lesion site.

In situ transblot and immunocytochemical comparisons of astrocytic connexin-43 responses to NMDA and kainic acid in rat brain.

Intracerebral injection of kainic acid (KA) in rat brain was previously found to cause altered immunohistochemical recognition of connexin-43 (Cx43) epitopes (epitope masking) with different sequence-specific antibodies against this gap junction protein. We demonstrate here that similar alterations occur when nitrocellulose membranes containing protein transferred from fresh cryostat sections of KA-injected brain are probed with these antibodies (in situ transblotting), indicating that epitope masking is not a result of epitope alteration due to fixation conditions used in earlier studies. Alterations in immuno-recognition of astrocytic Cx43 subsequent to injections of NMDA were also observed and were similar to those seen with KA in some, but not all respects. The results provide further indications of Cx43 molecular modification in excitotoxin-lesioned tissue and suggest that the sequelae of reactions by astrocytes and their gap junctions in these tissues is dependent on cell-type susceptibility to excitotoxin action.

Kainic acid induced alterations in antibody recognition of connexin43 and loss of astrocytic gap junctions in rat brain.

Intracerebral administration of kainic acid (KA) in rats was previously shown to abolish immunohistochemical labelling for the astrocytic gap junction protein connexin43 (Cx43) at sites depleted of neurons (Vukelic et al: Neurosci Lett 130:120-124, 1991). This response of Cx43 has now been further investigated with a number of different sequence-specific anti-Cx43 antibodies. At lesion sites in the thalamus, striatum, and hippocampus examined immunohistochemically with an antibody against amino acids (aa's) 346-363 in the Cx43 sequence, the antibody used in the earlier study, Cx43-immunoreactivity was increased 5 h after KA injections, absent by 24 h and for up to 2 weeks post-injection, and began to return to less than normal levels by 2 to 3 weeks post-injection. Analyses of KA lesion sites with antibodies against other sequences of Cx43 (amino acids 283-298, 253-270, 241-260, 113-123, and 49-61) revealed not only the presence but in some cases an increased density of Cx43 immunoreactivity after a survival time of 1 week. Immunolabelling patterns at these sites consisted of relatively large, coarse profiles rather the fine punctate labelling typically seen in sections of normal brain. In homogenates of KA-injected striatum analyzed by Western blots, Cx43 was detected at near normal or slightly increased levels at various survival times examined. The 43 kDa phosphorylated form of Cx43 and its faster migrating 41 kDa dephosphorylated form which is generated post-mortem by a brain phosphatase were both present after standard methods of tissue preparation for Western blot analysis, while only the 43 kDa form was present in normal and KA-injected striatum after inactivation of brain metabolism by focused cranial microwave irradiation. Ultrastructural investigations of lesions sites within the thalamus revealed a virtual absence of astrocytic gap junctions. These results demonstrate that Cx43 levels initially increase after intracerebral KA treatment, that its molecular organization in resident astrocytes is altered such that epitopes that are normally accessible to antibody are hidden while those that may be hidden or relatively inaccessible are exposed, and that this molecular alteration in Cx43 is associated with loss of astrocytic gap junctions.

Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain.

We have shown by immunohistochemical methods that the gap junction protein connexin43 is heterogeneously distributed in rat brain (Yamamoto et al: J Comp Neurol 302:853, 1990). Here we have compared quantitatively the relative amount of connexin43 detected on Western blots of seven central nervous system (CNS) regions with the density of connexin43-immunoperoxidase reactivity in these regions. As has been observed on Western blots of several cell types, homogenates of these CNS regions contained two forms of connexin43, its dephospho form with an apparent mobility of approximately 41 kDa and its approximately 43 kDa phosphorylated form. While the relative quantities of connexin43 varied considerably among the brain regions, the ratio of the 43/41 kDa forms, 0.71, was relatively uniform (correlation coefficient, r = 0.92). Sections of brain processed for connexin43-immunolocalization by the peroxidase-antiperoxidase (PAP) method showed that chromogen deposition was linear with incubation time in reaction medium. Optical density of tissue connexin43-immunoreactivity in each of the seven areas plotted against the density of connexin43 bands on Western blots gave a correlation coefficient of r = 0.90. Connexin43-immunoreactivity had a similar appearance in sections processed by PAP or immunofluorescence procedures and consisted of isolated or aggregates of puncta.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic analysis of [3H]Ryanodine binding sites in rat brain: regional distribution and the effects of lesions on sites in the hippocampus.

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12-18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5-9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.