GluR1 and GluR2/3 subunits of the AMPA‐type glutamate receptor are associated with particular types of neurone in laminae I–III of the spinal dorsal horn of the rat

GluR1 and GluR2 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor are expressed at high levels by neurones in laminae I–III of rat spinal dorsal horn, an area which contains numerous, densely packed small neurones. In order to determine whether these subunits are expressed by inhibitory or excitatory neurones, we combined pre-embedding immunocytochemistry with antibodies that recognize either GluR1, or an epitope common to GluR2 and 3, with postembedding detection of γ-aminobutyric acid (GABA) and glycine. Most (78%) of the neurones with GluR1-immunoreactivity were GABA-immunoreactive, and some of these were also glycine-immunoreactive, whereas nearly all (97%) of the GluR2/3-immunoreactive neurones were not GABA- or glycine-immunoreactive. We carried out double-immunofluorescence and confocal microscopy to provide further information on the neurochemistry of cells that express these subunits. As expected, all neurotensin- and virtually all somatostatin-immunoreactive cells (which are thought to be excitatory interneurones) were GluR2/3- but not GluR1-immunoreactive, whereas parvalbumin-containing cells (most of which are GABAergic) possessed GluR1-, but usually not GluR2/3-immunoreactivity. Neurones that contained nitric oxide synthase (most of which are GABAergic) were more variable, with 57% GluR1-immunoreactive and 41% GluR2/3-immunoreactive. Cholinergic neurones in lamina III (which are also GABAergic) invariably showed each type of GluR-immunoreactivity. These results suggest that neuronal populations in laminae I–III have characteristic patterns of GluR expression: GluR1 is particularly associated with inhibitory neurones, and GluR2 with excitatory neurones. This makes it likely that some of the AMPA receptors present on the inhibitory interneurones lack the GluR2 subunit, and may therefore have significant Ca²⁺-permeability.     

Immunology: Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizingantibodies in the sera of infertile women and monoclonal antispermantibodies against humans and mice on fertilization were investigated.The hemizona assay (HZA) and sperm penetration assay (SPA) wereused to study the inhibitory effects of sera from 22 infertilepatients positive for sperm immobilizing antibodies. Use ofthese tests allowed us to differentiate whether the antibodyblocked sperm—zona pellucida tight binding and/or spermpenetration into the ooplasm. The zona pellucida penetrationassay (ZPA) was also used to study the effects of four monoclonalantibodies (mAbs) on human sperm penetration into the zona pellucida.Seven mAbs against murine spermatozoa were tested for theirinhibitory effects on in-vitro fertilization (IVF) and HZA inmice. Of 22 patient sera with sperm immobilizing antibodies,21 (95.5%) inhibited HZA attachment and penetration, whereasthis did not occur in any of 13 patient sera without these antibodies.However, 19 of 22 (86.4%) patient sera with sperm immobilizingantibodies and eight of 13 (61.5%) patient sera without theseantibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs againsthuman spermatozoa showed strong inhibitory effects in all theassays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZAbut blocked ZPA and SPA. Another mAb (H6-3C4) seemed to haveno inhibitory effects on fertilization. Two (Vx 5 and Vx 8)of seven mAbs against murine spermatozoa inhibited IVF in micebut did not block mouse HZA. These findings suggest that antispermantibodies block fertilization at specific stages. Some of themmay inhibit sperm capacitation and thus prevent all processesof fertilization that follow. Some other antibodies may notaffect capacitation and sperm binding to zona pellucida butinhibit the acrosome reaction, followed by the blocking of spermpenetration through zona pellucida and ooplasm.     

Role of the ventromedial nucleus of the thalamus in motor behaviour—I. Effects of focal injections of drugs

An assortment of drugs was injected into one or both ventromedial nuclei of the thalamus, to see how these influenced stereotypy, locomotion and posture in spontaneously behaving and actively rotating rats. Unilateral intrathalamic muscimol promoted weak ipsiversive circling, while bilateral treatment gave catalepsy. Similar injections of 4-amino-hex-5-enoic acid, which inhibits γ-aminobutyrate metabolism, raised γ-aminobutyrate levels in the ventromedial nuclei more than three-fold yet had none of these behavioural effects. The indirectly acting γ-aminobutyrate agonists flurazepam and cis-1,3-aminocyclohexane car☐ylic acid had little effect on posture and locomotion and, like muscimol and 4-amino-hex-5-enoic acid, elicited only very weak stereotypies. Procaine behaved like the γ-aminobutyrate antagonist bicuculline, provoking vigorous locomotor hyperactivity and teeth chattering if given uni- or bilaterally. Pretreatment of one ventromedial nucleus with muscimol or 4-amino-hex-5-enoic acid, and to a lesser extent flurazepam or cis-1,3-aminocyclohexane car☐ylic acid, gave rise to pronounced ipsilateral asymmetries when combined with a large systemic dose of apomorphine. Contraversive rotations were initiated by unilateral stereotaxic injection of muscimol into the substantia nigra pars reticulata, or with apomorphine from the supersensitive striatum in unilaterally 6-hydroxydopamine lesioned rats. Drug treatments in the ipsilateral ventromedial nucleus showed a similar rank order of potency at inhibiting these circling behaviours, seemingly by reducing apomorphine-induced posture and muscimol-induced hypermotility. The suppression of circling by muscimol in these tests was highlighted by introducing the compound into the ventromedial nucleus at the height of circling activity. Both types of circling stimulus lost the capacity to increase locomotion, but still caused head turning and stereotypy in rats made cataleptic with bilateral ventromedial muscimol. Treating one ventromedial thalamus with muscimol greatly intensified any pre-existing posture directed towards that side, and vice versa.

These data suggest that the ventromedial nucleus is not involved with the expression of stereotyped behaviours, but can profoundly influence posture and locomotion, especially in the presence of some other motor stimulus. The recovery of circus movements in rats with impaired ventromedial nucleus function implies this nucleus is not essential for the execution of circling in these models.     

Correlation between dopaminergic neurons, acetylcholinesterase and nicotinic acetylcholine receptors containing the alpha3- or alpha5-subunit in the rat substantia nigra

The aim of this study was to investigate the relationship between the cells possessing the alpha3 or alpha5 nicotinic acetylcholine receptor subunits and the enzyme acetylcholinesterase, with respect to tyrosine hydroxylase immunoreactive dopaminergic neurons in the rat substantia nigra. Most, but certainly not all, acetylcholinesterase immunoreactive cells were located in the pars compacta. In the substantia nigra pars compacta there were in turn two populations of acetylcholinesterase containing neurons: those that were tyrosine hydroxylase reactive and those that were not. Double label studies, that included an antibody immunoreactive against a common immunogen on alpha1 of muscle and alpha3 and alpha5 neuronal nicotinic acetylcholine receptor subunits, revealed that nearly all nicotinic receptor positive cells were also tyrosine hydroxylase neurons. However, a minority non-tyrosine hydroxylase population was alpha3- and/or alpha5-nAChR positive and these were always AChE-immunoreactive. In summary, there appears to be a close correlation between nicotinic receptors and acetylcholinesterase in the substantia nigra, irrespective of the transmitter phenotype in different neuronal subpopulations.     

The control exerted by the substantia nigra on the quiet biting attack elicited by hypothalamic stimulation in the cat

The effects of substantia nigra (SN) (pars compacta) stimulation on the quiet biting attack evoked by hypothalamic activation in the cat were studied. The measure of the aggressive behavior was the latency of the biting which did not greatly change from one animal to another when hypothalamic stimulation was performed with the same parameters. Concurrent activation of the SN determined an inhibitory effect on the studied behavior in the form of an increase in the biting latency or loss of the attack pattern. The inhibitory role of the SN on predatory attack behavior is discussed.     

Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

ABSTRACT: In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium-3,5-diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona-IgG was raised by immunization of female rabbits with 500 heat-solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (M_r = 42,000), ZP II/1 (M_r = 67,000), ZP II/2 (M_r = 32,000), ZP III/1 (M_r = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high-pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O-glycosidic-glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.     

Bicuculline-induced circling from the rat superior colliculus is blocked by GABA microinjection into the deep cerebellar nuclei

In a recent electrophysiological experiment, we showed the deep cerebellar nuclei to be a major source of excitatory input to the superior colliculus. Furthermore, target neurons in the colliculus were found, in every case, to receive convergent tonic inhibitory input from the substantia nigra pars reticulata. In the present study, we investigated these effects in the awake rat. We asked whether circling behaviour, induced by unilateral injection of a GABA antagonist into the lateral colliculus, could be suppressed by concurrent cerebellar inactivation. Rats were chronically implanted with bilateral guide cannulae located above the superior colliculus and deep cerebellar nuclei. Bicuculline methiodide (25 pmol) was microinjected unilaterally into intermediate layers of the colliculus at increasing depths until an optimal contralateral circling response was elicited. This behaviour was taken as the baseline response and was the first of three treatments. The second was an identical manipulation of the colliculus with a concurrent 200-nl microinjection of 1 M GABA into the contralateral deep cerebellar nuclei. The third was a repeat of BIC alone into the colliculus or, if rotation had been suppressed by more than 50% on test 2, the treatment was collicular BIC plus deep cerebellar saline. This latter treatment was used as a control for possible non-pharmacological injection effects. The effect of cerebellar GABA at 26 sites (17 within cerebellar nuclei and 9 outside) on BIC-induced rotation at 15 collicular sites was studied in ten animals. Only GABA injections at sites that fell within the cerebellar nuclei significantly reduced turning (P<0.0001). A full behavioural analysis showed that this was a specific suppression of turning, not the result of general motor impairment. These results provide clear behavioural evidence that opposing, convergent influences from the basal ganglia and cerebellum interact in the lateral superior colliculus to control head and body movements. They furthermore suggest that the tonic deep cerebellar excitation of the superior colliculus could be the driving force in the expression of rotation induced by manipulations of the basal ganglia.     

Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons

Summary Extracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-a-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-Dglutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihy-droxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.     

IL-1β诱导体外培养的大鼠中脑黑质神经元c-jun表达

应用细胞原位杂交技术，观察经重组小鼠白细胞介素-19（IL-1β）处理后的体外培养的新生1d大鼠中脑黑质神经元c－jun基因的表达．结果显示，培养的黑质细胞多为酪氨酸羟化酶阳性神经元，IL-1β可诱导体外培养的黑质神经元c－junmRNA表达，高水平的表达出现在IL-1β处理后2～4h。说明IL－1β有兴奋黑质神经元的作用，并提示黑质神经元上可能存在IL－1β受体．