Development of the visual pathway is disrupted in mice with a targeted disruption of the calcium channel beta(3)-subunit gene.

Refinement of the retinal pathways to the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) is mediated by nitric oxide (NO). Long-term depression (LTD) can also be induced in SC and LGN during the time at which these pathways are refined, and this LTD is partially dependent on NO and L-type Ca(2+) channel function. In an effort to determine whether NO-mediated pathway refinement is also mediated by Ca(2+) channel function, we have examined the refinement of the retinocollicular and retinogeniculate pathways in mice which lack the gene for the Ca(2+) channel beta(3) subunit (CCKO) and which have significantly reduced L-type Ca(2+) currents. Injections of the anterograde tracer cholera toxin subunit B/HRP were made into one eye of these knockout animals and in wild-type mice ages postnatal day (P) 13, P19, and P26. After 48 hours, mice were perfused and sections processed by using tetramethylbenzidine histochemistry. Labeling distribution in some animals was analyzed quantitatively. Obvious differences in the distribution of the ipsilateral retinocollicular pathway were observed at P15, with the pathway being more exuberant in CCKO mice. This difference was statistically significant. More subtle differences were seen at P21 and P28. Obvious differences were also seen in the contralateral retinogeniculate pathway which in CCKO mice filled most of the domain normally occupied by ipsilateral eye fibers. This difference was also statistically significant. We conclude that reduction in L-type Ca(2+) currents has an effect on axonal refinement similar to that which occurs in NO knockout mice, which supports the possibility that L-type Ca(2+) channel-dependent LTD mediates NO-dependent axonal refinement.

Prenatal and postnatal expression of nitric oxide in the developing kitten superior colliculus revealed with NADPH diaphorase histochemistry.

Nitric oxide (NO) is a neuronal messenger molecule that mediates pathway refinement in some brain regions. We used nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry to examine the development of NO expression in the superior colliculus (SC) of kittens aged E28-E58 and P2-P57 and adults in order to determine if NO expression is correlated with pathway refinement. At E28, labeled cells were seen only within the subventricular zone (SVZ). At E36-E41, labeled cells were also found within the deep gray layer (DGL) of SC. At E51 and E58, a few labeled neurons were also present in the intermediate gray layer (IGL). These neurons already had extensive dendritic fields and well-developed morphologies at the time that they first expressed nitric oxide synthase (NOS). The number of neurons labeled in the DGL and IGL increased postnatally, reaching a peak density between P14 and P35. Neurons within the optic (OL) and superficial gray layers (SGL) were first visible at P7 and increased slightly in number until adulthood. However, SGL-labeled neurons were relatively limited in number and lightly labeled at all ages examined. We conclude that (1) NADPHd expression occurs in SC beginning in the second trimester in kittens and progresses in a ventral to dorsal pattern between E36-P35; (2) few neurons in kitten SGL are labeled by NADPHd and these appear relatively late in postnatal development; and (3) there is no correlation between NOS expression and retinocollicular pathway refinement in kittens, a result different from that seen in rodents.

Nitric oxide, impulse activity, and neurotrophins in visual system development(1).

Topographic refinement of synaptic connections within the developing visual system involves a variety of molecules which interact with impulse activity in order to produce the precise retinotopic maps found in the adult brain. Nitric oxide (NO) has been implicated in this process, as have various growth factors. Within the subcortical visual system, we have recently shown that nitric oxide contributes to pathway refinement in the superior colliculus (SC). Long-term potentiation (LTP) and long-term depression (LTD) are also expressed in SC during the time that this pathway undergoes refinement. The role of NO has been demonstrated by showing that refinement of ipsilateral fibers in the retinocollicular pathway is significantly delayed in gene knockout mice in which both the endothelial and neuronal isoforms of nitric oxide synthase (NOS) have been disrupted. The effect also depends upon Ca(2+) channels because refinement of both the ipsilateral retinocollicular and retinogeniculate pathways is disrupted in genetic mutants in which the beta3 subunit of the Ca(2+) channel has been deleted. LTD may also be involved in this process, because the time course of its expression correlates with that of pathway refinement and LTD magnitude is depressed by nitrendipine, an L-type Ca(2+) channel blocker. LTP is also expressed during early postnatal development in the LGN and SC and may contribute to synaptic stabilization. The role of neurotrophins in pathway refinement in the visual system is also reviewed.

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.

Refinement of the ipsilateral retinocollicular projection is disrupted in double endothelial and neuronal nitric oxide synthase gene knockout mice.

Development of retinal connections to the superior colliculus (SC) requires an activity dependent refinement process in which axons gradually become restricted to appropriate retinotopic locations. Nitric oxide has been implicated in this process. We tested this possibility by studying the refinement of the ipsilateral retinocollicular projections (IRP) in normal C57-BL/6 mice and in double knockout mice in which the genes for the edothelial and neuronal isoforms of nitric oxide synthase (e, nNOS) were disrupted. Mice aged between P19 and adulthood were perfused 44-48 h after anterograde injections of WGA-HRP into one eye in order to measure the distribution of the labeled IRP. In normal mice, segregation of the IRP was complete at P21, with the ipsilateral projection restricted to the rostro-medial SC. By contrast, the ipsilateral projection was spread over much more of the SC in double e, nNOS knockouts at P21 with patches of label distributed across the entire medio-lateral axis of the rostral 700 microm. Although the distribution of the ipsilateral projection became more restricted in knockout animals at later ages, it was still more extensive than that of normal mice of the same age at P28 and P42. In the adult, the distribution of axons was similar in both normal and double knockout animals. These results show that refinement of the IRP is delayed when expression of eNOS and nNOS is disrupted, presumably to axons with uncorrelated activity because nitric oxide serves as a repellant molecule during normal development.

Synaptic regulation of L-type Ca(2+) channel activity and long-term depression during refinement of the retinocollicular pathway in developing rodent superior colliculus.

The retinocollicular pathway undergoes activity-dependent refinement during postnatal development, which results in the precise retinotopic order seen in adults. This process is NMDA- and nitric oxide-dependent. Recent studies have shown that L-type Ca2+ channels may also play a role in synaptic plasticity, but such channel activity has not previously been reported in the developing superior colliculus (SC). Here we report the presence of a postsynaptic plateau potential mediated by L-type Ca2+ channels using whole-cell current clamp of the SC in an isolated brainstem preparation of rats. Seventy percent of SC neurons showed these potentials as early as postnatal day 0 (P0)-P2. The potential was blocked by nitrendipine and/or APV and facilitated by bicuculline, showing that the channel is activated by NMDA receptor-mediated EPSPs and deactivated by GABAA receptor-mediated IPSPs. Blockade of L-type Ca2+ channels also diminished long-term depression, which we could induce in the retinocollicular pathway in neonatal animals. The incidence of plateau potentials decreased to 39% of neurons by P10-P14, suggesting that L-type calcium channels may contribute to retinocollicular pathway refinement in the developing SC.

Failure to disrupt development of cholinergic fiber patches in the superior colliculus in nitric oxide synthase deficient mice.

Nitric oxide (NO) has been shown to mediate refinement of glutamatergic axonal pathways during development. In this study, we investigated whether the development of a cholinergic pathway in the intermediate gray layer (IGL) of the mouse superior colliculus (SC) is also mediated by NO. The pathway was labeled using an antibody directed against choline acetyltransferase (ChAT) and its distribution examined in normal C57/BL6 mice and in knockout mice in which the genes for the neuronal isoform of nitric oxide synthase (NOS) or both the endothelial and neuronal isoforms of NOS had been disrupted. We also examined the development of expression of NOS using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) staining. NADPHd labeled cells were found within the IGL by P8 and formed loose clusters of cells by P12-P15. ChAT and NADPHd labeled fibers were first observed at P12 and gradually established their characteristic two-tiered patchy pattern between P14 and P21. Comparison of the ChAT labeled fiber distribution in normal, single nNOS and double e,nNOS knockout mice revealed no differences between these three groups. We therefore conclude that nitric oxide does not mediate refinement of this cholinergic pathway.

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.

Retinal input induces three firing patterns in neurons of the superficial superior colliculus of neonatal rats.

By using an in vitro isolated brain stem preparation, we recorded extracellular responses to electrical stimulation of the optic tract (OT) from 71 neurons in the superficial superior colliculus (SC) of neonatal rats (P1-13). At postnatal day 1 (P1), all tested neurons (n = 10) already received excitatory input from the retina. Sixty-nine (97%) superficial SC neurons of neonatal rats showed three response patterns to OT stimulation, which depended on stimulus intensity. A weak stimulus evoked only one spike that was caused by activation of non-N-methyl-D-aspartate (NMDA) glutamate receptors. A moderate stimulus elicited a short train (<250 ms) of spikes, which was induced by activation of both NMDA and non-NMDA receptors. A strong stimulus gave rise to a long train (>300 ms) of spikes, which was associated with additional activation of L-type high-threshold calcium channels. The long train firing pattern could also be induced either by temporal summation of retinal inputs or by blocking gamma-aminobutyric acid-A receptors. Because retinal ganglion cells show synchronous bursting activity before eye opening at P14, the retinotectal inputs appear to be sufficient to activate L-type calcium channels in the absence of pattern vision. Therefore activation of L-type calcium channels is likely to be an important source for calcium influx into SC neurons in neonatal rats.

Physiological properties of neurons in the optic layer of the rat's superior colliculus.

We made intracellular recordings from 74 neurons in the optic layer of the rat superior colliculus (SC). Resting membrane potentials were -62.3 +/- 6.2 (SD) mV, and input resistances were 37.9 +/- 10.1 MOmega. Optic layer neurons had large sodium spikes (74.2 +/- 12.3 mV) with an overshoot of 12 mV and a half-amplitude duration of 0.75 +/- 0.2 ms. Each sodium spike was followed by two afterhyperpolarizations (AHPs), one of short duration and one of longer duration, which were mediated by tetraethylammonium (TEA)-sensitive (IC) or apamin-sensitive (IAHP) calcium-activated potassium currents, respectively. Sodium spikes were also followed by an afterdepolarization (ADP), which was only revealed when the AHPs were blocked by TEA or apamin. In response to hyperpolarizing current pulses, optic layer neurons showed an inward rectification mediated by H channels. At the break of the current pulse, there was a rebound low-threshold spike (LTS) with a short duration of <25 ms. The LTS usually induced two sodium spikes (doublet). Most optic layer neurons (84%) behaved as intrinsically bursting cells. They responded to suprathreshold depolarization with an initial burst (or doublet) followed by a train of regular single spikes. The remaining 16% of cells acted as chattering cells with high-frequency gamma (20-80 Hz) rhythmic burst firing within a narrow range of depolarized potentials. The interburst frequency was voltage dependent and also time dependent, i.e., showed frequency adaptation. Unmasking the ADP with either TEA or apamin converted all of the tested intrinsically bursting cells into chattering cells, indicating that the ADP played a crucial role in the generation of rhythmic burst firing. Optic layer neurons receive direct retinal excitation mediated by both N-methyl--aspartate (NMDA) and non-NMDA receptors. Optic tract (OT) stimulation also led to gamma-aminobutyric acid-A (GABAA) receptor-mediated inhibition, the main effect of which was to curtail the excitatory response to retinal inputs by shunting the excitatory postsynaptic current. Intracellular staining with biocytin showed that the optic layer neurons that we recorded from were mostly either wide-field vertical neurons or other cells with predominately superficially projecting dendrites. These cells were similar to calbindin immunoreactive cells seen in the optic layer. The characteristics of these optic layer neurons, such as prominent AHPs, strong shunting effect of inhibition, and short-lasting LTS, suggest that they respond transiently to retinal inputs. This is consistent with a function for these cells as the first relay station in the extrageniculate visual pathway.

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.

A web-accessible digital atlas of the distribution of nitric oxide synthase in the mouse brain.

We have produced a digital atlas of the distribution of nitric oxide synthase (NOS) in the mouse brain as a reference source for our studies on the roles of nitric oxide in brain development and plasticity. NOS was labeled using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In addition, choline acetyltransferase (ChAT) immunocytochemistry was used to identify cholinergic cells because many of the NADPHd positive cells were thought to colocalize acetylcholine. Some sections were also labeled with antibodies to either the neuronal (nNOS) or endothelial (eNOS) isoforms of NOS. Series of sections from 11 C57/BL6 mice were collected and labeled for NADPHd and/or ChAT. We collected two types of data from this material: color digital photographs illustrating the density of cell and fiber labeling, and computer/microscope plots of the locations of all the labeled cells in selected sections. The data can be viewed as either a series of single-section maps produced by combining the plots with the digital images, or as 3-D views derived from the cell plots. The atlas of labeled cell maps, together with selected color photographs and 3-D views, is available for viewing via the World Wide Web (http:@nadph.anatomy.lsumc.edu). Examination of the atlas data has revealed several points about the distribution of NOS throughout the mouse brain. Firstly, different populations of NADPHd-positive neurons can be distinguished by different patterns of staining. In some brain areas neurons are intensely stained by the NADPHd technique where label fills the cell bodies and much of the dendritic trees. In other brain regions labeling is much lighter, is principally confined to the cytoplasm of the cell soma, and extends only a short distance within proximal dendrites. Intense labeling is typical of neurons in the caudate/putamen and mesopontine tegmental nuclei. Most of the labeled neurons in the cortex also stain this way. Lighter, "granular" label is found in many other nuclei, including the medial septum, hippocampus, and cerebellum. In addition to staining pattern, we have also noted that different subpopulations of NOS-neurons can be distinguished on the basis of colocalization with ChAT. Substantial overlap of the distributions of these two substances was observed although very little colocalization was found in most cholinergic cell groups except the mesopontine tegmental nuclei. Other points of interest arising from this project include the apparent lack of NADPHd labeling in the CA1 pyramidal cells of the hippocampus or the Purkinje neurons in the cerebellum. This observation is especially relevant given that synaptic plasticity in these regions is reported to be nitric-oxide dependent.

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.

The distribution of the GABA(A) beta2,beta3 subunit receptor in the cat superior colliculus using antibody immunocytochemistry.

GABA-containing synaptic terminals in the cat superior colliculus include two varieties of presynaptic dendrite and at least one type of axon terminal with flattened vesicles. These anatomically distinct synaptic profiles probably also mediate different types of inhibition. Whether they are associated with different types of GABA receptor is unknown and one objective of the present paper. We used the antibody mAb 62-361 directed against the beta2,beta3 subunits of the GABA(A) receptor complex to determine whether the distribution of this receptor subunit is specific to one or more types of GABA-containing synapse. At the light microscope level, beta2,beta3 immunoreactivity was densely distributed within the neuropil of the zonal and superficial gray layers, and more lightly within the optic, intermediate, and deep gray layers. No cell bodies were labelled by the antibody in the zonal and superficial gray layers, but numerous cells contained internalized cytoplasmic immunoreactivity in the optic, intermediate gray, and deeper layers. At the ultrastructural level, synaptic sites opposite axon terminals that contained flattened synaptic vesicles (F profiles) were often beta2,beta3 immunoreactive, while postsynaptic sites opposite presynaptic dendrites (PSD profiles) were never immunoreactive. The label at F profiles usually filled the synaptic cleft and coated the postsynaptic plasma membrane. Some membrane-associated label was also found at non-synaptic sites. We conclude that this receptor subunit is selectively associated with flattened vesicle axon terminals and not with presynaptic dendrites, a result which supports evidence that those terminal types mediate different types of inhibition.

Glutamate containing neurons in the cat superior colliculus revealed by immunocytochemistry.

Glutamate is the probable neurotransmitter of both retinal and cortical afferents to the cat superior colliculus (SC). The present study shows that glutamate is also contained in many postsynaptic neurons in SC. The distribution, morphology, and ultrastructure of neurons in SC were examined using glutamate antibody immunocytochemistry. Labeled cells were widely distributed throughout, but a specific laminar pattern was evident. Relatively few cells were found in the zonal and upper superficial gray layers (SGL). A dense band of intensely labeled neurons was found within the deep superficial gray and upper optic layers. Many cells were also labeled in the deeper layers. Labeled cells had varied sizes and morphologies. Soma diameters ranged from 9-67 microns, with a mean of 22 microns. Cells with stellate, vertical fusiform, and multipolar morphologies were labeled. Cells in the deep subdivision all had morphologies and sizes typical of projection neurons. To determine if labeled cells in the dense band were also projection neurons, WGA-HRP was injected into the lateral posterior nucleus and these sections were double-labeled with the glutamate antibody. Over one-half of cells in the dense band that were labeled by HRP were also obviously labeled by antibody. At the electron-microscope level, both medium- and large-sized neurons were also labeled by glutamate antibodies. These cells had different but characteristic morphologies.

Inhibition of nitric oxide synthase fails to disrupt the development of cholinergic fiber patches in the rat superior colliculus.

Nitric oxide may serve as a retrograde messenger to refine or stabilize synapses in the developing nervous system. Whether this action is dependent upon glutamate and the N-methyl-D-aspartate receptor is not yet established. We have used the patch-cluster system in the intermediate gray layer (IGL) of the rat superior colliculus (SC), a system receiving both glutamatergic and cholinergic input, to study this question. The normal distribution and development of nitric oxide synthase (NOS) in SC was examined using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry in Sprague-Dawley rats aged P4 to adulthood. Fibers containing acetylcholine (ACh) were identified using choline acetyltransferase (ChAT) immunocytochemistry. In addition, N omega-nitro-L-arginine, an inhibitor of NOS, was injected intraperitoneally from birth until P10, P14, P18, or P21-22 to determine if NOS inhibition would disrupt the formation of the ACh patches. Control animals were studied from the same age groups. Our results show NADPH-d-labeled cells within the periaqueductal gray and the deep gray layer of SC by P4, the earliest age examined. By P8-P9, cells in the IGL were well labeled by NADPH-d, while few in the superficial layers (SL) were labeled. SL cells were visible by P10 and were intensely labeled by P14. IGL cells transiently expressed NADPH-d in that the number of labeled cells increased from P8 to P35, then decreased in the adult. ChAT-labeled fibers first appeared in the IGL at P10, formed a characteristic two-tier pattern by P14, and established obvious patches by P21. Inhibition of NOS from birth produced no qualitative differences in the distribution or density of either ChAT-labeled fibers or NADPH-d-labeled cells and fibers at any of the ages examined. We therefore conclude that NO does not contribute to the refinement of cholinergic fiber patches in the rat SC, probably because the fiber system is not glutamatergic.

Glutamate-like immunoreactivity in the cat superior colliculus and visual cortex: further evidence that glutamate is the neurotransmitter of the corticocollicular pathway.

Biochemical studies provide evidence that the pathway from visual cortex to the superior colliculus (SC) utilizes glutamate as a neurotransmitter. In the present study, we have used immunocytochemistry, visual cortex lesions, and retrograde tracing to show directly by anatomical methods that glutamate or a closely related analog is contained in corticocollicular neurons and terminals. A monoclonal antibody directed against gamma-L-glutamyl-L-glutamate (gamma glu glu) was used to localize glutamate-like immunoreactivity in both the superior colliculus (SC) and visual cortex (VC). Unilateral lesions of areas 17-18 were made in four cats to determine if gamma glu glu labeling was reduced in SC by this lesion. WGA-HRP was injected into the SC of 10 additional cats in order to determine if corticocollicular neurons were also labeled by the gamma glu glu antibody. A distinctive dense band of gamma glu glu immunoreactivity was found within the deep superficial gray and upper optic layers of SC where many corticotectal axons are known to terminate. Both fibers and cells were labeled within the band. Immunoreactivity was also found in cells and fibers throughout the deep layers of SC. Measures of total immunoreactivity (i.e. optical density) in the dense band were made in sections from the SC both ipsilateral to and contralateral to the lesions of areas 17-18. A consistent reduction in optical density was found in both the neuropil and in cells within the dense band of the SC ipsilateral to the lesion. A large percentage of all corticocollicular neurons that were retrogradely labeled by WGA-HRP also contained gamma glu glu. These results provide further evidence that the corticocollicular pathway in mammals is glutamatergic. The results also suggest that visual cortex ablation alters synthesis or storage of glutamate within postsynaptic SC neurons, presumably as a result of partial deafferentation.