Superficial Gray Layer Research Articles

Localization of dopamine D1A and D1B receptor mRNAs in the forebrain and midbrain of the domestic chick

The distribution and cellular localization of dopamine D1A and D1B receptor mRNAs in the forebrain and midbrain of the domestic chick were examined using in situ hybridization histochemistry with 35[S]-dATP labeled oligonucleotide probes, visualized with film and emulsion autoradiography. Labeling for D1A receptor mRNA was intense in the medial and lateral striatum, and moderately abundant in the pallial regions termed the archistriatum and the neostriatum, in the hypothalamic paraventricular nucleus region, and in the superficial gray layer of optic tectum of the midbrain. D1B receptor mRNA was abundant in the medial and lateral striatum, and in the pallial region termed the hyperstriatum ventrale, and moderately abundant in the intralaminar dorsal and posterior thalamus and in the superficial gray of the optic tectum. At the cellular level, about 75% of neurons in the medial striatum and 59% of neurons in the lateral striatum were labeled for D1A receptor mRNA, whereas about 39% of the neurons in the medial striatum and 21% in the lateral striatum were labeled for D1B receptor mRNA. Large striatal neurons were not labeled for D1A or D1B receptor mRNA. The data suggest that while both D1A and D1B receptors mediate dopaminergic responses in many neurons of the avian striatum, primarily D1A receptors mediate dopaminergic responses in the archistriatum and the neostriatum, while primarily D1B receptors mediate dopaminergic responses in the hyperstriatum ventrale and the thalamus.

Contribution of Superficial Layer Neurons to Premotor Bursts in the Superior Colliculus

In vitro whole-cell patch-clamp methods were used to examine the contribution of one component of intracollicular circuitry, the superficial gray layer, to the generation of bursts of action potentials that occur in the intermediate layer and that command head and eye movements in vivo. Applying a single brief (0.5 ms) pulse of current to the superficial layer of rat collicular slices evoked prolonged bursts of excitatory postsynaptic currents (EPSCs) in the cells of the intermediate layer. The EPSCs were sufficient to elicit bursts of action potentials that lasted as long as 300 ms and resembled presaccadic command bursts. To examine the contribution of neurons within the superficial layer to the production of these bursts, we determined how superficial neurons respond to the same current pulses that evoke bursts in the intermediate layer. Recordings from 61 superficial layer cells revealed 19 neurons that produced multiple action potentials following stimulation. Nine of these 19 neurons were wide- and narrow-field vertical cells, which are known to project to the intermediate layer and could contribute to producing the EPSC bursts. The remaining cells (n = 42) did not generate trains of action potentials and 21 of these showed only subthreshold potential changes in response to the stimulus. Our results indicate that most superficial cells do not directly contribute to production of the EPSC bursts, but a small number do have the properties necessary to provide a prolonged excitatory drive to the premotor neurons.

The functional influence of nicotinic cholinergic receptors on the visual responses of neurones in the superficial superior colliculus.

In the rat, the superficial gray layer (SGS) of the superior colliculus receives glutamatergic projections from the contralateral retina and from the visual cortex. A few fibers from the ipsilateral retina also directly innervate the SGS, but most of the ipsilateral visual input is provided by cholinergic afferents from the opposing parabigeminal nucleus (PBG). Thus, visual input carried by cholinergic afferents may have a functional influence on the responses of SGS neurones. When single neuronal extracellular recording and iontophoretic drug application were employed to examine this possibility, cholinergic agonists were found to depress responses to visual stimulation. Lobeline and 1-acetyl-4-methylpiperazine both depressed visually evoked activity and had a tendency to reduce the background firing rate of the neurones. Carbachol depressed the visual responses without any significant effect on the ongoing activity, while the muscarinic receptor selective agonist methacholine increased the background activity of the neurones and reduced their visual responses. Lobeline was chosen for further studies on the role of nicotinic receptors in SGS. Given that nicotinic receptors are associated with retinal terminals in SGS, and that the activation of presynaptic nicotinic receptors normally facilitates transmitter release (in this case glutamate release), the depressant effects of nicotinic agonists are intriguing. However, many retinal afferents contact inhibitory neurones in SGS; thus it is possible that the increase in glutamate release in turn facilitates the liberation of GABA which goes on to inhibit the visual responses. We therefore attempted to reverse the effects of lobeline with GABA receptor antagonists. The depressant effects of lobeline on the visual response could not be reversed by the GABA(A) antagonist bicuculline, but the GABA(B) antagonist CGP 35348 reduced the effects of lobeline. We hypothesize that cholinergic drive from the parabigeminal nucleus may activate presynaptic nicotinic receptors on retinal terminals, thereby facilitating the release of glutamate onto inhibitory neurones. Consequently GABA is released, activating GABA(B) receptors, and thus the ultimate effect of nicotinic receptor activation is to depress visual responses.

Postnatal development of nitric oxide synthase expression in the mouse superior colliculus

Since nitric oxide has a role in the refinement of the retinal projection to the superior colliculus (SC), we studied the onset of neuronal nitric oxide synthase (nNOS) expression in the mouse SC in order to compare its development with that of the refinement process. Sections from animals at ages P1, P5, P8, P11, P15, and P21 and adults were examined with nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry or immunocytochemistry using an antibody directed against nNOS. At all ages there was a wedge of labeled neurons in the dorsolateral periaqueductal gray extending into the deep layers of the SC. At P1 there was also a single superficial band of labeled neurons within the region that will become the intermediate gray layer (IGL). By P5, labeled neurons were also seen in what will become the superficial gray layer. There was a ventral to dorsal progression in nNOS expression with substantial changes in the numbers of labeled neurons in the different laminae between P5 and adulthood. The number of labeled neurons in the IGL peaked at P15, whereas in the superficial layers the numbers continued to increase through P21 and then declined in adults. At all ages these neurons represented a variety of morphological cell types. The onset of nNOS expression in the different laminae is earlier than has been reported in studies using NADPHd as a marker for nNOS. The temporal and spatial patterns of nNOS expression reported here match more closely the time course of pathway refinement in the SC, providing additional evidence for the involvement of nitric oxide in this process.

Normal development of the ipsilateral retinocollicular pathway and its disruption in double endothelial and neuronal nitric oxide synthase gene knockout mice.

The development of the ipsilateral retinocollicular pathway involves activity-dependent refinement in which misdirected axons retract to form a precise retinotopic map in adults. This refinement is altered by disruption of genes for the endothelial and neuronal isoforms of nitric oxide synthase (e,nNOS), but the extent of disruption during early development is not known. Therefore, we studied the refinement of this pathway in normal C57/BL6 and e,nNOS double knockouts from P4 to P21 and in adults. Anterograde tracers were injected into one eye to localize the ipsilateral retinal projection (IRP) within the superior colliculus (SC). At P4, the IRP in normal mice was distributed throughout the dorsoventral extent of the superficial gray layer (SGL) across most of the rostrocaudal axis of SC. Between P4 and P9, the pathway retracted to the rostromedial SC, and retracted further between P15 and P21, such that multiple patches of label were seen only in the rostral 200-300 microm. Refinement also began to occur between P4 and P9 in e,nNOS double knockout mice, but labeling was more extensive in P9, P15, and P21 knockout animals. This delay in refinement was confirmed quantitatively at P15 where differences in the area occupied by the pathway were statistically significant. The refinement process is therefore in progress in both normal and e,nNOS knockout mice before eye opening but is significantly delayed in the double knockouts. The IRP in normal mice is also more exuberant at early ages, and the process of refinement more protracted than has been previously reported, suggesting that there is a prolonged critical period of synaptic plasticity.

The NMDAR1 subunit of the N-methyl-D-aspartate receptor is localized at postsynaptic sites opposite both retinal and cortical terminals in the cat superior colliculus.

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that is important in neurotransmission as well as in processes of synaptic plasticity in the mammalian superior colliculus (SC). Despite the importance of this receptor in synaptic transmission, there is as yet no evidence that demonstrates directly the synaptic localization of the NMDAR receptor in SC. We have used electron-microscope (EM) immunocytochemistry to localize the NMDAR1 subunit of this receptor protein and its association with sensory afferents in the cat SC. Retinal synaptic terminals were identified by normal morphology and cortical synaptic terminals by degeneration after lesions of areas 17-18 of the visual cortex. At the light-microscope level, label was densest within the superficial gray and upper optic layers, but also present in all other layers. Label was contained within cell bodies, dendrites, and a few putative axons. At the EM level, antibody labeling was found along postsynaptic densifications and internalized within the cytoplasm of a variety of dendrites and some cell bodies. Postsynaptic profiles labeled by NMDAR1 included conventional dendrites and presynaptic dendrites which contained pleomorphic synaptic vesicles and are known to be GABAergic. Many of the labeled postsynaptic densifications of both of these profile types received synaptic inputs from retinal or cortical terminals. Virtually no NMDAR1 immunoreactivity was found on thin dendritic thorns or putative spines, even when these were postsynaptic to retinal or cortical terminals. In summary, these results show that the NMDAR1 subunit is postsynaptic to both retinal and cortical afferents, which are known to be glutamatergic, and are consistent with physiological evidence showing that stimulation of either pathway can activate the NMDA receptor.

Calbindin 28kD and parvalbumin immunoreactive neurons receive different patterns of synaptic input in the cat superior colliculus

Recent evidence suggests that neurons containing the calcium binding proteins calbindin 28kD (CB) and parvalbumin (PV) have differing distributions which match respectively the distribution of W and Y retinal ganglion cell inputs to the cat superior colliculus (SC). In this study we have used electron microscope immunocytochemistry to study directly the synaptic inputs to neurons containing CB and PV. Aspiration lesions of areas 17–18 of visual cortex were made 4 days prior to sacrifice in order to identify degenerating cortical terminals (CT). Retinal terminals (RTs) were identified by their characteristic morphology including large round synaptic vesicles and pale mitochondria. We photographed RTs and CTs that were in contact with immunoreactive profiles sampled in both the superficial gray and optic layers (ol) of SC. CB immunoreactive (ir) dendrites were usually of small to medium caliber and were found to receive synaptic input from RTs. These RTs were all small profiles forming a single synaptic contact with asymmetric densifications. CBir profiles also received other synaptic input, including from terminals with dark mitochondria that contained flattened synaptic vesicles (F profiles). No CBir dendrites were found to receive CT input even though degenerating CTs were found in the vicinity of CBir profiles. By contrast, both RT and CT were found to contact PVir dendrites. RT terminals contacting PVir dendrites were both small and larger profiles with round synaptic vesicles and asymmetric synaptic densifications. CT were undergoing electron dense degeneration but still sometimes formed asymmetric synaptic densifications with PV neurons. PV cells also received F profile synaptic input. We conclude that CB neurons receive small RT synapses that are probably of W origin, while PV neurons receive both RT and CT synapses which are likely related to the Y pathway.

Excitatory effects of adenosine are not mediated by inhibition of GABAergic system in slices of superior colliculus and hippocampus from guinea pig

Neurotransmission in the superior colliculus (SC) and the hippocampus is enhanced by application of adenosine. To investigate the possibie involvement of disinhibition by adenosine on γ-aminobutyric acid (GABA) -ergic action, the effects of adenosine and picrotoxin, a GABA antagonist, were studied using slices of SC and hippocampus of the guinea pig. Application of adenosine on SC slices (concentration at 100 μM) and hippocampus (concentration at 0.1 μM) enhanced the amplitude of population spikes (PS) in the superficial gray layer (SGL) of SC and in the granule cell layer of dentate gyrus of hippocampus. The application of picrotoxin at 100 μM increased the amplitude of both slices even after previous treatment with adenosine which once increased the amplitude. This was also true for adenosine with the previous treatment of picrotoxin. In SC slices, the increased amplitude by adenosine was reduced to the original amplitude after application of 3-[1-[3-(Dimetylamino) propyl]-1H-indol-3-yl-]4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (GF109203X) a protein kinase C inhibitor, the previous treatment by the drug counteracted the excitatory action of adenosine. However GF109203X failed to counteract the excitatory effects of picrotoxin. These results indicate that the excitatory mechanism of adenosine is different from that of picrotoxin and is not mediated by disinhibition of the GABAergic system through inhibitory action of adenosine on GABAergic interneurons.

Magnitude of 5-HT 1B and 5-HT 1A receptor activation in guinea-pig and rat brain: evidence from sumatriptan dimer-mediated [ [formula omitted]]GTPγS binding responses

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT 1A and 5-HT 1B receptors in guinea-pig and rat brain using agonist-stimulated [ 35 S ]GTPγS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT 1B (p K i: 9.22 vs. 7.79 for sumatriptan), h 5-HT 1D (9.07 vs. 8.08) and also h 5-HT 1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht 1E (6.67 vs. 6.19) and h 5-ht 1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 μM) stimulated [ 35 S ]GTPγS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT 1B/1D receptor antagonist [ 3 H ] GR 125743. The observed [ 35 S ]GTPγS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT 1B receptor subtype, since they were antagonized by the 5-HT 1B inverse agonist SB 224289 (10 μM), and not by the 5-HT 2A/1D antagonist ketanserin (10 μM). Quantitative assessment of the [ 35 S ]GTPγS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51±10% and 35±13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT 1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT 1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT 1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT 1B receptors besides 5-HT 1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT 1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

Metabotropic glutamate receptor mGluR2/3 immunoreactivity in the mouse superior colliculus: co-localization with calbindin D28K.

We have studied the distribution of mGluR2/3 in the mouse superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on this distribution. We also compared this labeling to that for calbindin D28K. Anti-mGluR2/3-immunoreactive (IR) cells formed distinctive laminar patterns within the lower optic and upper intermediate gray layers. By contrast, anti-calbindin D28K-IR cells formed obvious laminar patterns in three layers: one within the zonal and upper superficial gray layers, a second within the optic and intermediate gray layers, and the third within the deep gray layer. The distribution of mGluR2/3-IR cells thus matches the second layer of calbindin D28K cells. Two-color immunofluorescence revealed that more than half (52.5%) of mGluR2/3-IR cells were also labeled with antibody to calbindin D28K. The majority of mGluR2/3-IR cells were small to medium-sized round/oval or stellate cells. Immunoreactivity for mGluR2/3 was clearly reduced in the contralateral SC following unilateral enucleation. The present results show that mGluR2/3 has a unique cellular sublaminar organization in SC that includes some calbindin D28K-IR cells. The effects of enucleation suggest that the retinal projection may control the expression of mGluR2/3 in some cells in the mouse SC.

Cellular and subcellular localization of 5-hydroxytryptamine 1B receptors in the rat central nervous system: immunocytochemical, autoradiographic and lesion studies

The localization of 5-hydroxytryptamine 1B receptors in the rat central nervous system was investigated using anti-peptide antibodies that recognize a selective portion of the third intracytoplasmic loop of the receptor protein. At the light microscope level the densest 5-hydroxytryptamine 1B receptor-like immunoreactivity was observed in ventral pallidum, globus pallidus, substantia nigra and dorsal subiculum. In addition, moderate immunoreactivity was found in the entopeduncular nucleus, the superficial gray layer of the superior colliculus, the caudate–putamen and the deep nuclei of the cerebellum. This distribution matched perfectly that previously described from radioligand binding studies. At the ultrastructural level, 5-hydroxytryptamine 1B receptor-like immunoreactivity was associated with axons and axon terminals in the three areas examined: substantia nigra, globus pallidus and superficial gray layer of the superior colliculus. In all cases, immunostaining was located on the plasma membrane of unmyelinated axon terminals and in the cytoplasm close to the plasmalemma. Synaptic differentiations were never labelled, but, in some cases, 5-hydroxytryptamine 1B receptor-like immunoreactivity was found in their close vicinity. Injection of kainic acid into the neostriatum resulted in a marked decrease in receptor-like immunoreactivity in the globus pallidus and the substantia nigra, consistent with the location of 5-hydroxytryptamine 1B receptors on terminals of striatopallidal and striatonigral fibres, respectively. A reduction in 5-hydroxytryptamine 1B receptor-like immunoreactivity was also noted in the superficial gray layer of the superior colliculus after contralateral enucleation, as expected of the location of 5-hydroxytryptamine 1B receptors on the terminals of retinocollicular fibres. In both lesion experiments, immunolabelled degenerating terminals were observed in the projection areas. Anterograde labelling experiments coupled with immunocytochemical detection further showed that 5-hydroxytryptamine 1B receptors in the substantia nigra are located on axons of striatal neurons. These data provide anatomical supports to the idea that 5-hydroxytryptamine 1B receptors act as terminal receptors involved in presynaptic regulation of the release of various neurotransmitters, including 5-hydroxytryptamine itself.

Calretinin and calbindin D28K immunoreactivity in the superficial layers of the rabbit superior colliculus.

Calretinin and calbindin D28K were localized in the superficial layers of rabbit superior colliculus (SC). Calretinin and calbindin D28K-immunoreactive (-IR) neurons were concentrated in the upper superficial gray layer. Calretinin-IR fibers were found in the optic layer. The majority of calretinin-IR cells were small- to medium-sized vertical fusiform neurons and neurons with round or stellate-shaped somas with small varicose dendrites. The morphology of calbindin D28 K-IR neurons was different from that of calretinin neurons. Anti-calbindin D28K-IR neurons usually had fusiform cell bodies and a thick primary dendrite with small branches forming a dendritic bouquet. Two-color immunofluorescence revealed that no cells expressed both proteins. Following unilateral enucleation a marked reduction of calretinin-IR fibers in the contralateral side to the enucleation was found. Enucleation appeared to have no effect on the cell bodies labeled with either protein. The results suggest the anti-calretinin immunoreactivity in the superficial layer of rabbit SC contrasts starkly with that of other animals.

The Visuo-Motor Pathway in the Local Circuit of the Rat Superior Colliculus

Intrinsic circuit of the superior colliculus (SC), in particular the pathway from the optic tract (OT) to neurons in the intermediate layer (SGI), was investigated by whole-cell patch-clamp recording in slice preparations obtained from 17- to 24-d-old rats. Stimulation of the OT induced monosynaptic EPSPs in neurons in the superficial gray layer (SGS) and the optic layer (SO), and disynaptic or polysynaptic EPSPs in a majority of SGI neurons. Stimulation of the SGS induced monosynaptic or oligosynaptic EPSPs in the SGI neurons. Both the monosynaptic EPSPs induced in the SGS/SO neurons by stimulation of the OT and those induced in the SGI neurons by stimulation of the SGS were mediated by AMPA- and NMDA-type glutamate receptors. Thus, we have clarified the existence of the glutamatergic excitatory pathway from the OT to the SGI neurons via SGS and SO neurons. The EPSPs in the SGI neurons induced by stimulation of the OT or SGS were remarkably enhanced by bicuculline, suggesting that the signal transmission in this pathway is under strong suppression by the GABAergic system.

Experience-dependent changes in the importance of N-methyl- d-aspartate (NMDA) receptors for visual transmission in superior colliculus

The excitatory amino acid transmitter glutamate mediates visual activity in the superficial grey layer (SGS) of superior colliculus. At eye opening N-methyl- d-aspartate receptors (NMDA-rs) convey little of the visual response, but with age their role in visual transmission increases to a peak at P21, then falls to the lower adult level. Visual deprivation which begins before eye opening causes NMDA-rs to assume a greater importance for visual transmission in SGS. Here we explore the possibility that these experience-dependent changes in the role of NMDA-rs in the SGS are limited by age. We find that the effects of visual deprivation on NMDA-r mediated visual activity are recoverable even after extensive dark rearing. Also, a short episode of visual experience is sufficient to allow the normal situation to be established and subsequent dark rearing is ineffective. Four-day periods of visual experience beginning at P14 or P25 have the same effect. Given that NMDA-rs take little part in visual transmission prior to P18, these data prompt a reconsideration of the role of NMDA-r mediated sensory transmission in the mechanisms by which early environmental experience influences the development of the visual system.

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Developmental changes in NMDA receptor-mediated visual activity in the rat superior colliculus, and the effect of dark rearing.

N-methyl-D-aspartate (NMDA) receptor-mediated activity is considered important for experience-dependent plasticity in the developing visual system. We investigated the influence of age and experience on the role of NMDA receptors in the visual transmission in the superficial grey layer of the superior colliculus (SGS) of the superior colliculus, where, in the adult, NMDA receptors mediate a substantial part of the visual response. In normally reared (postnatal day 14, P14, to adult) rats, visual responses were challenged with NMDA receptor-selective iontophoretic applications of the antagonist D-2-amino-5-phosphonovalerate (AP5). After eye opening (at P14), there was a significant increase in the number of neurones whose visual responses were reduced during AP5 ejection, which peaked at P22 (85%; n = 21), and then declined to adult levels (66%; n = 47) at P25. The mean reduction of the response (from control levels) by AP5 was similar at all ages (approximately 40%). Dark rearing had striking effects on the role of NMDA receptors in visual transmission, especially when comparisons were made between age-matched subjects greater than P25. In these subjects, AP5 ejection reduced the visual responses of all neurones studied. In addition, AP5 ejection caused a significantly larger reduction of visual responses in dark-reared rats (mean reduction 62 +/- 4; n = 29) compared with age-matched controls (mean reduction 44 +/- 8; n = 23). The D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the visual responses of every neurone studied and there were no age- or experience-dependent effects. We conclude that NMDA receptors, but not AMPA receptors, assume greater importance for visual transmission in the SGS of dark-reared rats.

CalbindinD28k- and parvalbumin-immunoreactive neurons form complementary sublaminae in the rat superior colliculus

By using light microscopic immunocytochemistry and computer analysis, we have mapped the distributions of two calcium-binding proteins (CaBPs), calbindinD28k (CB) and parvalbumin (PV), in the rat superior colliculus (SC). The patterns of CaBP expression were complementary. A band of heavily labeled, medium-sized CB-immunoreactive cells (CB-cells) was centered in the optic layer (OL), whereas PV-immunoreactive cells (PV-cells) were found predominantly in the intermediate gray layer (IGL), where they were clustered within patches of PV-labeled fibers. The superficial gray layer (SGL) could be divided into two sublaminae. CB-cells were found mostly in the dorsal half of the SGL, whereas PV-cells were scattered throughout the ventral SGL and the dorsal OL. Most of the CaBP-immunoreactive cells in the SGL were small bipolar cells with vertically oriented dendrites; however, there were also some PV-cells with horizontally oriented dendrites. Quantitative analysis of the CaBP distributions reinforced our observations that these cells are distributed in complementary tiers that are not restricted to the traditional laminae. The size and shape of some of these tiers were determined from a three-dimensional reconstruction of serial sections. The complementarity of the CaBP-immunoreactive tiers was also confirmed by fluorescence microscopy of double-labeled sections, in which few if any double-labeled neurons were observed. Complementary tiers of CB-cells and PV-cells have been observed previously in the SC of the cat. The present results demonstrate them in another species and further suggest that there are functional sublaminae in the SC that can be distinguished by CaBP content.

Depressor responses to endothelin-1 into the superior colliculus of rats: predominant role of endothelin ET(B) receptors.

We used in vitro autoradiography to identify the endothelin-1 receptor subtype(s) in the superficial gray layer of the superior colliculus of rats. These studies showed dense binding of (3-[125I]iodotyrosyl13)-[Ala11,15]Ac-endothelin-1-(6-2 1) (BQ3020) (for endothelin ETB receptors), while tissues incubated with [125I]( N-(hexahydro-1-azepinyl)carbonyl)L-Leu(1-Me)D-Trp-D-Tyr (PD151242) (for endothelin ETA receptors) had low binding. In addition, we examined the effects of the endothelin receptor antagonists, (R)-2-[(R)-2-[(S)-2-[[1-hexahydro-1H-azepinyl)]carbonyl]amino-4-++ +methylpentanoyl]-amino-3-(2-pyridyl)propionic acid (FR139317) (endothelin ETA receptor-selective), (+)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenediox yphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid (SB209670) (endothelin ETA/ETB receptor non-selective) and N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-metLeu-D-1-m ethoxycarbonylTrp-D-Nle (BQ-788) (endothelin ETB receptor-selective antagonist) on the responses following administration of endothelin-1 into the superficial layer of the superior colliculus. Endothelin-1 microinjected into this nucleus causes decreases in blood pressure (control, 109 +/- 6 mmHg, n = 6; endothelin-1, 72 +/- 5 mmHg, n = 6). These effects were greatly reduced by pre-administration into the superior colliculus of BQ-788 (5 nmol/rat) or SB209670 (3 nmol/rat) (94 +/- 5 and 98 +/- 6%, respectively) but were not affected by FR139317 (5 nmol/rat) (6 +/- 3%). Therefore, together with autoradiography, our functional data showed that endothelin ETB receptors are the predominant mediators of depressor responses induced by endothelin-1 injected into the superior colliculus of the rat.

Expression of GABA receptor rho subunits in rat brain.

The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

Chapter 10 The role of nitric oxide in development of the patch—cluster system and retinocollicular pathways in the rodent superior colliculus

Nitric oxide (NO) has been implicated as a retrograde signal in the process of refining axonal pathways during brain development. To determine some of the factors involved in this process, we have used two model pathway systems in the rat and mouse superior colliculus (SC). The first, the patch-cluster system, consists of clusters of neurons in the intermediate gray layer (igl) which transiently express NO during development and which receive input from a cholinergic pathway from the parabrachial brainstem as well as from other pathways containing different transmitters. The second system, the retinocollicular pathway, consists of glutamatergic fibers that project to the superficial gray layer. We have used both nitric oxide synthase inhibition (nw-nitro-L-arginine, NoArg) and single (nNOS) and double (nNOS and eNOS) gene knockout mice to examine the effect that reduction in NOS has upon the development of these two systems. The onset of NOS expression in rat, as revealed by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeling, occurred in igl cells as early as postnatal day P5, with clusters being well-established by P14. Cholinergic fibers were first visible at P10 and formed obvious patches and tiers by P14. Intraperitoneal injections of NoArg from P1-P22 had no effect upon the development of these cholinergic patches. The pathway also developed normally in both single and double-knockout mice. In contrast, the ipsilateral retinocollicular pathway was altered in the double, but not in the single knockout mouse. This pathway is exuberant during the first week of life, being distributed across much of the mediolateral axis of the rostral SC. By P8-P15, this pathway has retracted to the most mediorostral SC. This refinement was delayed substantially in the double NOS gene knockout mouse. Ipsilateral fibers were found within 3-5 separate medio-lateral patches within the rostral 600 microns of SC at P15, and patches of abnormal size and extent were also seen at P18. We conclude from these results that NO plays a role in pathway development in the rodent SC, but only in glutamatergic pathways and only when both endothelial and neuronal forms of NOS have been deleted. The mechanism of this effect must involve pathway elimination in situations where there is non-correlated electrical activity. It is likely that NO promotes fiber retraction rather than fiber stabilization in these developing nerve fibers.