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Background: Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood., Methodology/principal Findings: We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45-60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. CONCLUSIONS/KEY FINDINGS: We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin's entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.

We studied the development of optic tract evoked field potentials (FP) in the rodent superior colliculus (SC) and the effect of GABA antagonists upon their development and upon induction of long-term depression (LTD). Brain slices were cut from Lister Hooded rats. The optic tract was stimulated while recording from the superficial grey layer. GABAergic inhibition was assessed by adding 100 microm picrotoxin and 3 microm CGP55845 antagonists to block GABA A,B,C receptors. LTD was induced with a 50 Hz, 20 s tetanus. At age P2, the FP consisted only of a presynaptic spike. The GABA antagonists had no effect. By P4, the FP consisted of a presynaptic spike, a longer latency population spike, and a field excitatory postsynaptic potential (fEPSP). The fEPSP was slightly prolonged by the GABA antagonists at this age. By P7-P14, a prominent FP with trailing fEPSP was recorded. The GABA antagonists usually had a large effect, with the fEPSP increasing in both amplitude and duration. A mature FP was usually recorded in P15-P23 slices where the GABA antagonist effect remained substantial. LTD could be induced in 17 of 30 control slices from rats aged P4-P26. The average fEPSP amplitude after tetanus was 77.9% of control. Pre-treatment with GABA antagonists produced a short-term potentiation (average 114.0%), rather than LTD, in 14 of 19 cases. This STP was followed by a more prolonged potentiation in 12 of the 14 cases. We conclude that GABAergic inhibitory circuits mature before eye opening and that GABA contributes to induction of LTD in the developing SC.

Developmental refinement of the retinogeniculate and retinocollicular pathways is partially dependent upon Ca(2+) channel function [J. Comp. Neurol. 440 (2001) 177-191]. We have examined the development of the L-type voltage gated Ca(2+) channel to determine if the onset of expression matches this period of refinement. Labeling by an antibody directed against the alpha 1C subunit of this channel was examined in the superior colliculus (SC), lateral geniculate nucleus (LGN), visual cortex (CTX), hippocampus (HC) and cerebellum (CB) in mice aged P3-4, P8-9, P15, P21, P28, and adults. At P3-4, labeled cells within the SC were concentrated within a dense band in the retinorecipient zone of the superficial gray layer. More lightly labeled neurons were seen in other layers. This dense band was still seen at P15, while more labeled neurons were seen in other layers. By P21-P28, labeled neurons were fairly uniformly distributed throughout all layers of SC. Neuronal cell types appeared to be labeled at all ages examined within the LGN. Within CTX, putative layer V-VI pyramidal neurons were well labeled at P4 and later ages, and labeled layer II-III pyramids could be distinguished by P9 and later ages. The dendrites and cell bodies of pyramidal neurons within CA1-CA3 of HC, granule neurons in the dentate gyrus, and Purkinje neurons in CB were labeled at all ages examined. We conclude that the L-type Ca(2+) channel is expressed in many neurons within retinorecipient targets as well as in other brain regions during the developmental period in which pathway refinement and synaptic plasticity occurs.

The developing retinocollicular pathway undergoes synaptic refinement in order to form the precise retinotopic pattern seen in adults. To study the mechanisms which underlie refinement, we investigated long-term changes in retinocollicular transmission in rats aged P0-P25. Field potentials (FPs) in the superior colliculus (SC) were evoked by stimulation of optic tract fibers in an in vitro isolated brainstem preparation. High intensity stimulation induced long-term depression (LTD) in the SC after both low (1000 stimuli at 1 Hz) and higher (1000 stimuli at 50 Hz) frequency stimulation. The induction of LTD was independent of activation of NMDA and GABA(A) receptors, because D-APV (100 microM) and bicuculline (10 microM) did not block LTD. Induction of LTD was dependent upon activation of L-type Ca(2+) channels as 10 microM nitrendipine, an L-type Ca(2+) channel blocker, significantly decreased the magnitude of LTD. LTD was down-regulated during development. LTD magnitude was greatest in rats aged P0-P9 and significantly less in rats aged P10-P25. Long-term potentiation (LTP) was induced by low intensity stimulation and only after high frequency tetanus (1000 stimuli at 50 Hz). LTP was NMDA receptor dependent because d-APV (100 microM) completely abolished it. LTP induction was also blocked by the L-type Ca2+ channel blocker nitrendipine. The magnitude of LTP first increased with age, being significantly greater at P7-P13 than at P0-3 and then decreased at P23-25. In summary, both LTD and LTP are present during retinocollicular pathway refinement, but have different transmitter and ionic mechanisms and time courses of expression.

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We examined the pattern of NADPH-diaphorase (NADPH-d) staining in the lateral geniculate nucleus (LGN) of dorsal thalamus in fetal and newborn kittens, and adult cats. This staining visualizes the synthesizing enzyme of nitric oxide (NO), a neuromodulator associated with central nervous system (CNS) development and synaptic plasticity. In the adult, very few LGN cells stained for NADPH-d, and these were restricted to interlaminar zones and ventral C layers. NADPH-d labeled a dense network of fibers and axon terminals throughout the LGN and adjacent thalamic nuclei. The source of such labelling has been reported to be cholinergic neurons from the parabrachial region of the brain stem (Bickford et al., 1993). A very different pattern of staining was observed in prenatal and early postnatal kittens. Between embryonic (E) day 46-57, lightly stained cells appeared throughout the LGN. From this age, through about the first month of life, the number of stained cells in the LGN rose rapidly. The density (cells/mm2) of labeled cells peaked at postnatal day (P) 28 (P28), and was about 150 times greater than the level measured in the adult LGN. After P28, cell staining declined rapidly, and fell to adult levels at P41. The reduction in cell staining that occurred between P35-41 was accompanied by the appearance of fine-caliber fiber staining, similar to that observed in the adult LGN. NADPH-d staining, which reveals the presence of nitric oxide synthase (NOS), and thus NO activity, may reflect two processes. In the adult LGN, the labeling of cholinergic axons arising from the brain-stem parabrachial region coupled with a paucity of the LGN cellular staining suggests that NO operates in an orthograde manner, being co-released with ACh to influence the gain and efficacy of retinogeniculate transmission. By contrast, in developing kitten, NADPH-d staining of LGN cells suggests that NO acts in a retrograde fashion, perhaps playing a role in maintaining associative processes underlying activity-dependent refinement of retinogeniculate connections.

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 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.

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.

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.

Projection neurons from the superior colliculus (SC) to the cuneiform region (CFR) of the midbrain form distinct cell clusters in the intermediate gray layer which precisely overlap the cholinergic fiber patches in this region of the cat SC (Jeon and Mize, J. Comp. Neurol., 337 (1993) 127-150). The acetylcholine (ACh)-containing fiber patches do not express ACh until after birth. It is not known when the cell clusters first appear. In this study, we examined the development of the clusters by retrogradely labeling the cells with a tracer injected into the CFR. Kittens aged from embryonic day E34 to E57 and postnatal day P1 to P14 were perfused with aldehyde fixatives and crystals of the carbocyanine dye DiI were placed into the CFR. After 5- to 8-week incubations, the SC was sectioned and examined using epi-fluorescent microscopy. At E34, very few labeled cells and fibers were present in SC. Between E41 and E46, both labeled cells and fibers were found within the intermediate gray (IGL) and deep gray (DGL) layers of SC. In one E46 case, a few labeled cells were grouped together within the IGL, suggesting the onset of clusters seen at later ages. By E51, DiI labeling produced an obvious pattern of clustered cells. Up to 4 clusters were seen in a single section, each containing 4-12 labeled neurons. By E57, labeled cell clusters were conspicuous in the IGL in caudal sections and the number of cells per cluster was increased. This pattern was also present at birth (P1). Scattered labeled neurons were also found outside the clusters and throughout the deep layers as were fibers innervating this region of SC. In summary, the axonal projections of the IGL clustered cells appear to reach the cuneiform region as early as E46 and are well-developed by E57. The clustering of efferent neurons precedes the formation of cholinergic fiber patches in the cat SC which is first identifiable using ChAT antibodies around the second postnatal week of life.

Although the excitatory neurotransmitter glutamate is known to be present in the cat superior colliculus (SC), the types of synapses that contain glutamate have not been examined. We, therefore, studied the ultrastructure of synaptic profiles labeled by a glutamate antibody by using electron microscopic postembedding immunocytochemistry. In addition, unilateral aspiration lesions of areas 17-18 were made at 5-28 days before death in order to determine whether degenerating terminals from visual cortex were glutamate immunoreactive (Glu-ir). Three types of axon terminal were glu-ir: 1) those containing large, round synaptic vesicles and pale mitochondria, characteristic of retinal terminals (RT profiles); 2) those containing small, round synaptic vesicles and dark mitochondria (RSD profiles); and 3) those containing large, round synaptic vesicles and dark mitochondria (RLD profiles). Measures of mean gold particle density revealed that RT, RSD, and RLD profiles had similar average grain densities (11.3-12.7 particles/unit area). Other labeled profile types included cell bodies, large-calibre dendrites, and myelinated axons. Axon terminals containing flattened synaptic vesicles and vesicle-containing presynaptic dendrites, both of which contain gamma-aminobutyric acid (GABA), had many fewer gold particles (3.6 and 4.8 mean particles/unit area, respectively). Following unilateral removal of visual cortex, normal RSD terminals were observed infrequently in the SC ipsilateral to the lesion. Synaptic terminals in the initial stages of degeneration were heavily labeled by the glutamate antibody, as were axon terminals and myelinated axons undergoing hypertrophied or neurofilamentous degeneration. These results show that both major sensory afferents to the superficial layers of cat SC contain glutamate--RT terminals from the retina and RSD terminals from visual cortex. The origin of RLD terminals is unknown.

The cat superior colliculus (SC) plays an important role in visual and oculomotor functions, including the initiation of saccadic eye movements. We have studied the organization of neurochemical specific circuits in SC that underly these functions. In this chapter we have reviewed three microcircuits that can be identified by cell type, chemical content, and synaptic input from specific afferents. The first is located within the upper sgl and is related to the W retinal pathway to this region of SC. This circuit includes relay and interneurons that contain the calcium binding protein calbindin (CB), GABA containing presynaptic dendrites, and retinal terminals that have a distribution and size typical of W retinal terminals in the cat SC. This circuit is a typical synaptic triad that mediates feedforward inhibition, possibly to regulate outflow of the W pathway to the lateral geniculate nucleus. CB neurons in SC and other structures may be uniquely related to low threshold calcium currents in these neurons. The second microcircuit consists of neurons that contain parvalbumin (PV), another calcium binding protein. These neurons are located in a dense tier with the deep sgl and upper ol and they receive input from retinal terminals that are likely from 'Y' retinal ganglion cells. Some of these neurons also project to the lateral posterior nucleus and some colocalize glutamate. We speculate that these neurons also receive cortical 'Y' input although we have yet to prove this experimentally. The role of PV in these cells is unknown, but PV has been shown to be contained in fast spiking, non-accomodating neurons in visual cortex which have very rapid spike discharges that are also characteristic of SC neurons innervated by 'Y' input. The third microcircuit consists of a group of clustered neurons within the igl of the cat SC that overlaps the patch-like innervation of afferents to this region that come from the pedunculopontine tegmental and lateral dorsal tegmental nuclie, the substantia nigra, and the cortical frontal eye fields. These clustered neurons project through the tectopontobulbar pathway and terminate within the cuneiform region (CFR) of the midbrain tegmentum. They transiently express NOS during development. Ongoing studies in our laboratory suggest that these cells receive synaptic inputs directly from the PPTN and SN and may represent functional modules involved in the initiation of saccadic eye movements.

Neurons within the superior colliculus (SC) contain a variety of neurochemicals, including the amino acid neurotransmitters GABA and glutamate, the calcium binding proteins calbindin and parvalbumin, and the neuromodulator nitric oxide. We have examined the development of expression of these substances using antibody immunocytochemistry. These results are summarized in Fig. 10. GABA and calbindin are expressed very early in development, at a time when cells are still dividing and migrating from the subventricular zone. The expression of both GABA and CB is maximal at around E40-46, the age at which these cells have just established their adult lamination and extrinsic afferents have begun to grow into the tectum. GABA and CB likely play diverse roles during this stage of development, including the regulation of intracellular calcium during cell migration and neurite outgrowth. Glutamate is expressed somewhat later in development while parvalbumin immunoreactivity does not appear until shortly after birth. These two substances continue to increase in density throughout the period of postnatal growth, at a time when synapse formation and evoked electrical activity are beginning to develop. Both PV and glutamate may be involved in one or both of these activity-dependent processes. Nitric oxide synthase (NOS) is expressed at different times in different cell groups. NOS appears very early in prenatal development in cells within the SVZ and in the deep gray layer of SC. On the other hands, cells within the intermediate gray layer of SC do not express NOS until shortly before birth. The igl cells that express NOS at this age are clustered neurons similar to those that project to the CFR in the adult. NOS expression occurs in these cells at precisely the time when axons begin to form patches that innervate these clusters. Based upon this temporal correlation, we hypothesize that nitric oxide may regulate synapse formation in this cell group.

Both the nucleus of the optic tract (NOT) and the superior colliculus (SC) are thought to play important roles in the regulation of eye movements. The superior colliculus contributes to visual orientation and saccades, and the nucleus of the optic tract contributes to the detection of slow movements of the visual surround. Recently, a GABAergic projection has been described between these two nuclei in the cat, a species with frontal vision. The present study aimed at determining whether a similar GABAergic pathway exists in the rabbit, a species with lateral vision. To study this pathway we used the retrograde tracer cholera-toxin (CTB) to identify NOT neurons projecting to the SC and GABA-antibody immunostaining to identify GABA-containing neurons and processes. CTB injections into the superficial laminae of the SC showed that GABAergic and non-GABAergic neurons in the NOT project to the SC. Both types of neurons have structural characteristics similar to other projection neurons in the NOT. In contrast to the NOT neurons projecting to the inferior olive (IO) which are mainly located in the rostral NOT, the GABAergic and non-GABAergic NOT-SC neurons are situated throughout the nucleus. The somata and principal dendrites of both neuron types receive numerous synaptic contacts from GABAergic terminals and only a few from retinals. The NOT projection neurons to the SC thus establish prominent excitatory and inhibitory links between the two structures, suggesting the existence of separate circuits that could interact through a GABAergic and non-GABAergic NOT-SC projection. It is further suggested that these circuits may be involved in the regulation of saccades in the SC during optokinetic nystagmus.

Image analysis hardware, software, and procedures are described for analysis of tissue reacted for antibody immunocytochemistry and in situ hybridization. A Magiscan image analyzer is used to process images viewed with a light microscope. LUT functions, spatial filters (parabola) and gray level convolutions (sharpen, laplacian, mexican hat) are applied in order to extract immunoreaction product or autoradiographic grains. These objects are then thresholded and binary operators (erosion, dilation, separation) are applied to separate closely apposed objects. Measurements routines are used to estimate the optical density and size of labeled profiles or to count grains and compute grain density per profile. A JEOL 1210 electron microscope is used to view tissue treated for post-embedding immunochemistry. Digital images are captured with a Kodak 1K CCD camera, archived, transported across a local area network, stored on optical disks and analyzed on a MacIntosh IIci. NIH Image is used to process these images. Results show that the optical density of GABA antibody labeling is reduced by monocular deprivation, that substance P mRNA hybridization labeling is increased by scopolamine, and that retinal terminals are densely labeled by antibodies to glutamate. These techniques are thus useful for measuring the amount of change in labeling after experimental manipulations and for distinguishing labeled from unlabeled profiles.

The advent of microcomputers has brought about a revolution in the computing power available to the average user. Image analysis is a very resource-intensive process, making great demands on computing power, memory, and display capabilities of most computers. Thus, in the past, dedicated, single-use hardware and software had to be custom made for environments requiring image analysis. We present here an easy-to-use image analysis protocol available to most users with a Macintosh II series computer and access to IMAGE (a public domain image analysis program). The protocol allows for semi-quantitation of silver grains over cells used in the interpretation of in-situ hybridization results. We show that the method provides a quick and reliable means of counting grains over mRNA-positive cells in an automated fashion. We also provide evidence that the method can be used to detect differences between experimental treatments.

We have developed a computer image analysis procedure for counting autoradiographic grains in in situ hybridization experiments. The procedure automatically estimates the number of autoradiographic grains over cells and measures cell number and size so that grain density per unit cell area can be calculated. Advantages include the clear separation of grains and cells, using chromatic and spatial filters to enhance the image; the use of gray level operators to extract cells from grains; and the use of binary operators for separating apposed or partially overlapping cells and grains. Comparison of manual and automated grain counts revealed a significant correlation between human and computer estimations of grain number. However, the automatic grain counting technique consistently underestimated the number of grains when grain density was high. Measures of the fractional area occupied by grains normalized by the average area of a single grain were a better estimate at high grain densities. The procedure can be modified easily to operate on most image analyzers.

We have studied the organization of gamma-aminobutyric acid (GABA)ergic profiles in the superior colliculus of the rabbit to determine whether the synaptic types found in cat and monkey also exist in a mammalian species whose visual system has a different organization. Ultrastructure of GABAergic profiles was examined by use of a polyclonal antibody to GABA and quantitative postembedding immunocytochemistry. Three distinct types of vesicle-containing profiles were labeled by the GABA antibody in the rabbit superior colliculus. One type was a putative presynaptic dendrite (PSD profile) that received synaptic input from other profiles and contained pleomorphic synaptic vesicles scattered throughout the profile. These PSD profiles frequently received retinal input and formed dendrodendritic synapses. A second type of profile was a large caliber dendrite, often horizontal in orientation (H profile), that had one or more discrete clusters of pleomorphic synaptic vesicles at sites of synaptic contact with conventional dendrites. These H profiles received few synaptic contacts. A third profile type was a putative axon terminal (F profile) with smaller, more flattened synaptic vesicles that densely and uniformly filled the profile. Quantitative analysis of gold particle density revealed that F profiles had a significantly higher gold particle density (14.3/microns 2) than did PSD or H profiles (10.4 and 10.2/microns 2), suggesting that GABAergic profile types contain different concentrations of GABA. The vesicle density of these profile types also differed, but no obvious relationship between vesicle and particle distributions was observed. We conclude that the profiles labeled by GABA in rabbit superior colliculus are similar to those in cat and monkey and must represent a phylogenetically conserved organization common to many mammals, and that particle density analysis of postembedding immunocytochemistry can distinguish different GABAergic profile types.

Fibers containing acetylcholine (ACh) form distinct patches in the dorsal intermediate gray layer (IGL) of the cat superior colliculus (SC). Although these patches are known to overlap several afferent projections to SC, it is not known whether they are associated with specific postsynaptic cell groups. We have examined the relationship of these ACh fiber patches to specific efferent cell groups by combining retrograde transport of horseradish peroxidase (HRP) with choline acetyltransferase (ChAT) immunocytochemistry. Successful HRP injections were made into the predorsal bundle (PB), the tecto-pontine-bulbar pathway (TPB) and the cuneiform region (CFR), the inferior olive (IO), the dorsolateral pontine gray nucleus (PGD), and the pedunculopontine tegmental nucleus (PPTN). The distribution of HRP-labeled neurons which project to these targets was mapped by a computer-based microscope plotter. Distinct clusters of HRP-labeled neurons in the IGL were seen after three injections into the mesencephalic reticular formation that involved the caudal TPB and cuneiform region (CFR), and after one injection into the medial accessory nucleus of IO. As many as seven clusters of labeled neurons were found in some sections through the caudal one-half of SC after the TPB/CFR injections. Each cluster consisted of 3-20 cells, all of which were small to medium in size. In sections also tested for ChAT, the cell clusters in the TPB/CFR cases were found to overlap precisely the ACh patches in the IGL. In addition, SC neurons projecting to the IO formed clusters above the ChAT patches and in the intermediate white layer (IWL) of SC. None of the other HRP injections produced any obvious cell clusters in the deep layers of SC. These results are the first to show that specific cell groups, distinguished by size and projection site, form clusters that match the patch-like innervation of cholinergic afferents to SC. This modular organization may correspond to saccade-related cells that have also been reported to be organized into clusters in the cat SC.

The cat superior colliculus (SC) receives a dense cholinergic input from three brainstem nuclei, the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus, and the parabigeminal nucleus (PBG). The tegmental inputs project densely to the intermediate gray layer (IGL) and sparsely to the superficial layers. The PBG input probably projects only to the superficial layers. In the present study, the morphology of choline acetyltransferase (ChAT)-immunoreactive axons and synaptic endings in the superficial and deep layers of the SC was examined by light and electron microscopy to determine whether these cholinergic afferents form different types of synapses in the superficial and deep layers. Two types of fibers were found within the zonal (ZL) and upper superficial gray layers (SGL): small diameter fibers with few varicosities and larger diameter fibers with numerous varicosities. Quantitative analysis demonstrated a bimodal distribution of axon diameters, with one peak at approximately 0.3-0.5 micron and the other at 0.9-1.0 micron. On the other hand, ChAT-immunoreactive fibers in the IGL were almost all small and formed discrete patches within the IGL. Two types of ChAT-immunoreactive synaptic profiles were observed within the ZL and upper SGL using the electron microscope. The first type consisted of small terminals containing predominantly round synaptic vesicles and forming asymmetric synaptic contacts, mostly on dendrites. The second type was comprised of varicose profiles that also contained round synaptic vesicles. Their synaptic contacts were always symmetric in profile. ChAT-immunoreactive terminals in the IGL patches contained round or pleomorphic synaptic vesicles, and the postsynaptic densities varied from symmetric to asymmetric, including intermediate forms. However, no large varicose profiles were observed. This study suggests that cholinergic fibers include at least two different synaptic morphologies: small terminals with asymmetric thickenings and large varicose profiles with symmetric terminals. The large varicose profile in the superficial layers is absent in the IGL. This result suggests that the cholinergic inputs that innervate the superficial layers and the patches in the IGL of the cat SC differ in their synaptic organization and possibly also in their physiological actions.

The calcium-binding proteins calbindin (CaBP) and parvalbumin (PV) are important in regulating intracellular calcium in brain cells. PV immunoreactivity is reduced by enucleation in the lateral geniculate nucleus (LGN) and by enucleation and visual deprivation in the striate cortex of adult monkeys. The effects of enucleation and visual deprivation on CaBP immunoreactivity in the LGN are not known. We therefore have studied cells and neuropil in the LGN that are labeled by antibodies to CaBP in normal and visually deprived Rhesus monkeys to determine if there is an effect on this calcium-binding protein. One group of monkeys had one eye removed 2 weeks to 4.3 years before sacrifice. A second group had one eye occluded with opaque lenses from infancy without enucleation. A final group had one eye occluded long-term followed by short-term enucleation 2 weeks before sacrifice. In normal monkeys, CaBP-immunoreactive neurons were found throughout the LGN. They were sparsely distributed within the six main laminae, and more densely distributed within layer S and the interlaminar zones (ILZ). The labeled ILZ neurons had a distinct morphology, with fusiform somata and elaborate dendritic trees that were confined primarily to the ILZ. Most CaBP-labeled neurons in the main layers had dendrites that radiated in all directions from the soma. ILZ and main layer cells labeled by CaBP thus probably represent two different cell types. Monocular enucleation with or without occlusion produced a significant reduction in antibody labeling in the deafferented laminae. Field measures revealed an average 11.5% reduction in optical density in each deafferented lamina compared to its adjacent, nondeprived layer. The differences in field optical density between deprived and nondeprived layers were statistically significant. CaBP neurons were still visible, but the optical density of antibody labeling in these cells also was reduced. Occlusion without enucleation had no effect. Thus, deafferentation, but not light deprivation, reduces concentrations of CaBP in monkey LGN. This effect is different than that seen in striate cortex of adult monkeys, where visual deprivation as well as enucleation alters CaBP immunoreactivity.

Antibody labeling of the calcium-binding protein calbindin 28kD (CaBP) and gamma-aminobutyric acid (GABA) is altered by short-term monocular deprivation in the lateral geniculate nucleus and visual cortex of adult primates. It is not known whether these alterations occur in other subcortical visual structures. We therefore have examined antibody labeling to CaBP and GABA in the superior colliculus (SC) of visually deprived Rhesus monkeys. One group was monocularly enucleated as adults. The other monkeys experienced different types of monocular and binocular deprivation from birth, including occlusion of one eye, and/or surgically induced aphakia, optically corrected with extended-wear contact lenses, or an intraocular lens implant. Some of these monkeys also had one eye enucleated prior to perfusion. In the SC of normal monkeys, CaBP-immunoreactive neurons formed three laminar tiers within SC, one within the zonal layer (ZL) and upper superficial gray layer (SGL), another bridging the optic and intermediate gray layers, and a third within the deep gray layer. CaBP neurons within the upper tier had small pyriform or stellate morphologies while those in the deeper tiers were slightly larger neurons, most with a stellate morphology. GABA-immunoreactive neurons were densely distributed within the SGL and more sparsely distributed within the deeper layers. These cells were mostly small neurons with horizontal, pyriform, or stellate morphologies. Neither monocular enucleation nor occlusion nor aphakia combined with continuous occlusion of the fellow eye produced any visible reduction in antibody labeling in cells or neuropil within the SC. Full-field measures of labeling intensity (optical density) within the ZL and upper SGL revealed no consistent differences between the SC contralateral or ipsilateral to the affected eye in either CaBP- or GABA-labeled sections. Measures of the optical density, number, and size of labeled neurons also showed no consistent effects of enucleation and/or occlusion. We therefore conclude that the retino-geniculostriate and retino-collicular systems differ in their response to deprivation which is likely due to the significant overlap of retinal axons from the two eyes that occurs in the SC of the Rhesus monkey.

Parvalbumin (PV) and calbindin-D 28K (CaBP) are calcium binding proteins involved in calcium regulation in the brain. In some regions they coexist in the same neuron, while in other regions they are found in different cell types. We have studied the distribution and morphology of PV labeled neurons in the cat superior colliculus (SC) with antibody immunocytochemistry and compared this labeling to that of CaBP. PV neurons were concentrated in a dense tier within the deep superficial gray and upper optic layers. Scattered PV neurons also were found within the deep layers of SC. By contrast, CaBP neurons were concentrated in three tiers: one within the zonal and upper superficial gray layers, a second within the deep optic and upper intermediate gray layers (IGL), and a third within the deep gray layer. The distribution of PV neurons is thus complementary to that of CaBP neurons, with the CaBP cell tiers bordering the dense tier of PV neurons. PV neurons varied in size and morphology. The average diameter of labeled cells was 20 microns, almost twice the size of CaBP neurons. The cells were predominantly round, vertical fusiform, or stellate, and included the very large neurons found scattered in the IGL. Horseradish peroxidase injections into the lateral geniculate nucleus, the lateral posterior nucleus, the opposite superior colliculus, the dorsal lateral pontine gray nucleus, and two descending pathways--the crossed predorsal bundle and the tecto-ponto-bulbar tracts--each labeled SC neurons that were also labeled by PV. A large percentage (84%) of projection neurons contained PV. This result also differs from CaBP neurons in SC, most of which are interneurons. Two antigen double-label experiments did not produce any cells that contained both PV and CaBP. The two calcium binding proteins thus reveal a unique sublaminar organization in SC that consists of alternating small cell interneuron groups and large cell projection neuron groups.

GABA is an important inhibitory neurotransmitter in the mammalian superior colliculus. As in the lateral geniculate nucleus, GABA immunoreactive neurons in SC are almost all small and are distributed throughout the structure in all mammalian species studied to date. Unlike the LGN, GABA-labeled neurons in SC have a variety of morphologies. These cells have been best characterized in cat, where horizontal and two granule cell morphologies have been identified. Horizontal cells give rise to one class of presynaptic dendrite while granule C cells give rise to another class of spine-like presynaptic dendrite. Granule A cells may be the origin of some GABAergic axon terminals. GABA containing synaptic profiles form serial synapses, providing a possible substrate for disinhibition. The distribution of GABAA and GABAB receptor subtypes appears similar to that of GABA neurons, with the densest distribution found within the superficial gray layer. However, antibody immunocytochemistry of the beta 2 and beta 3 subunits of the GABAA receptor reveals that it is located at both synaptic and non-synaptic sites, and may be associated with membrane adjacent to terminals with either flattened or round vesicles. A few GABA containing neurons in SC colocalize the pentapeptide leucine enkephalin or the calcium binding protein calbindin. However, none appear to co-localize parvalbumin, a situation different from GABA containing interneurons in the LGN and visual cortex. The diversity of GABA neurons in SC rivals that found in visual cortex, although unlike visual cortex, the pattern of co-occurrence does not distinguish GABA cell types in SC. The superior colliculus also differs from both LGN and visual cortex in that GABA and calbindin immunoreactivity is not altered by either long-term occlusion and/or short-term enucleation in adult Rhesus monkeys. No consistent differences have been found in the optical density of GABA labeling in either cells or neuropil. To conclude, GABA neurons in the superior colliculus share some properties like those in LGN and others like those in visual cortex. In other properties, they differ from GABA neurons in both the LGN and visual cortex. The GABA systems in the superior colliculus are similar in all mammalian species studied, suggesting that they are phylogenetically conserved systems which are not amenable to plastic alterations, a situation different to that in the geniculostriate system.

The calcium binding protein calbindin-D 28K (CaBP) has been localized in the cat superior colliculus (SC). Four important features of SC organization have been revealed by using CaBP immunocytochemistry. 1) CaBP neurons formed three laminar tiers in SC, one within the upper one half of the superficial gray layer (SGL), the second bridging the deep optic (OL) and intermediate gray layers (IGL), and the third within the deep gray layer (DGL). 2) CaBP labeled several classes of interneuron in SC. In the upper CaBP tier, the labeled neurons were all small, but they varied in morphology and included horizontal, pyriform, and stellate neurons. A unique class of interneuron was labeled by anti-CaBP in the OL-IGL tier. This cell was stellate-like with highly varicose dendrites and broad dendritic trees. Other labeled neurons in the intermediate and deep tiers included nonvaricose stellate neurons and rare large neurons in the DGL. 3) A few anti-CaBP neurons were projection neurons. Virtually no CaBP neurons were retrogradely labeled after injections of HRP into the predorsal bundle and dorsolateral midbrain tegmentum or into the lateral posterior nucleus. However, 2.4% of anti-CaBP neurons were retrogradely labeled after HRP injections into the dorsal and ventral lateral geniculate nuclei. These represented 14.7% of all neurons projecting to the LGN complex. 4) A small percentage of CaBP neurons co-localized GABA. A two-chromagen double-labeling technique showed that about 4.0% of labeled neurons were labeled by both antibodies. In summary, antibodies to CaBP densely labeled subpopulations of neurons in the cat SC, most of which were interneurons, some of which projected to the LGN, and a few of which co-localized GABA.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is found in the superior colliculus (SC) of many mammalian species. In cat, several distinct classes of putative GABAergic neuron have been identified using antibodies directed against the neurotransmitter. It is not known whether these classes are found in other species. To study this, we examined the distribution, morphology, ultrastructure, and synaptic organization of GABA immunoreactive neurons in the SC of the Rhesus monkey (Macaca mulatta). Antibody-labeled neurons were distributed throughout the monkey SC, but were most densely concentrated within the zonal and superficial gray layers (32.5% of the total). These neurons were all small cells ranging from 6.6-16.3 microns in average diameter, and had granule, pyriform, and horizontal morphologies. Four types of labeled profile were identified in single ultrathin sections with the electron microscope. Presynaptic dendrites (PSDs) contained pleomorphic vesicles, received synaptic input from unlabeled axon terminals, and sometimes formed symmetric synaptic contacts with postsynaptic profiles. Two subtypes were found. One type contained loose accumulations of synaptic vesicles throughout the profile and had a distinctive varicose shape. The other type contained small discrete clusters of synaptic vesicles near the site of synaptic apposition. The former were much more common. Profiles with typical axon terminal morphology were also found. These profiles usually contained numerous flattened vesicles and formed symmetric synapses with postsynaptic profiles, both dendrites and cell bodies. Some conventional dendrites and myelinated axons were also labeled. Serial ultrathin section reconstructions revealed that PSDs formed complex synaptic relationships with other elements. Retinal terminals, identified by their characteristic pale mitochondria, established synaptic contacts with both types of PSD. These PSDs also established contact with each other, providing a possible anatomical substrate for disinhibition. We conclude that the monkey SC has multiple GABAergic cell types, similar to those found in cat, and may represent an organization common to both mammals and some other vertebrate species. The circuitry established by these cell types may provide a mechanism for disinhibition as well as inhibition in the mammalian SC.

A microcomputer system has been developed for the quantitative analysis of neurons, synapses, and other biological profiles. The outlines of profiles are traced from micrographs using a digitizer. From these traces, the system calculates cross-sectional area, perimeter (length), average diameter, and form factor. Data values for the number of profile contacts, the number of elements within a profile, the number of elements associated with a profile, profile type, and profile depth are also entered. The program calculates contact and element densities from these values. The data are printed-out on a thermal printer and stored on magnetic tape or floppy disk. The software is written entirely in BASIC. The system uses commercially available hardware, including a Hewlett-Packard 9845T microcomputer and H-P 9874A digitizer. The programs can be adapted to a wide variety of other microcomputer systems.

We have designed a computer-based electron microscope plotting system which maps the locations of organelles in tissue specimens and analyzes their distribution. The system includes: (1) two optical incremental shaft encoders which translate stage drive rotation to electrical pulses; (2) a Display/Control unit used to convert encoder pulses to binary code for computer input; (3) two 16-bit parallel interfaces for transferring data to the computer; (4) a Hewlett-Packard 9845T microcomputer, used to control data input and to store, graph, and analyze the plots. The software for the plotter is written in enhanced BASIC. The plotter system is driven by 4 programs called Trace, Plot, Analyze, and Density. The Trace program "draws" an outline of the edges of the tissue. The Plot program maps the positions of profiles within the tissue. The Analyze program compares trace and plot data and calculates the depth and medial-lateral distance of each plotted profile from the surfaces of the tissue. The Density program sorts and counts profile types, measures surface areas, and calculates profile densities. Commercial statistical software is used to analyze the data. Our laboratory uses the system to map the spatial distribution of synapses and neurons in the central nervous system. The plotting system will also be of value in other areas of neurobiology research.

We have developed a computer system which enters and aligns serial sections and displays the completed reconstructions at different rotations in space. The system uses commercially available hardware, including a Hewlett-Packard 9845T microcomputer and an H-P 9874A digitizer. The software for the system is written in the BASIC language. The system consists of two programs, one for section digitization, the other for rotation and display of the reconstructions. Sections are digitized directly from micrographs or back-projected slides. The outlines of cells or other structures are traced from these media using a hand-held cursor on the digitizer. The positions of elements (inputs) which contact the structure and fiducials are also digitized. The sections are aligned by simultaneously displaying two consecutive sections on the graphics CRT screen. The sections are coarsely superimposed by centering around screen center using a centering algorithm. They are precisely aligned by rotating and translating the images with a reference cursor. Special functions for inserting and deleting sections and rapid section scanning are available for editing. The aligned sections are stored using a linked-list file structure on either floppy disks or tape cartridges. The rotation program replots the completed reconstructions on the graphics CRT or digital plotter. The program will reproduce the reconstructions at any scale and at any rotation in the x-, y- or z-planes. A hidden line algorithm removes hidden lines to give a 3-dimensional (3-D) perspective to the reconstructions. The positions of inputs and fiducials are represented by symbols. We use the system to reconstruct cells and neural processes. The 3-D reconstructions allow us to: (a) examine the spatial distribution and density of synaptic contacts on neurons; (b) study complex neuronal shapes; (c) examine the vectors of neural processes. The computer reconstruction system, which is moderately priced, should also prove useful for reconstructing many other types of biological profile.

The retinal terminals of the medial interlaminar nucleus (MIN) and ventral lateral geniculate nucleus ( VLG ) have been examined quantitatively to determine if there are morphological differences in their synaptic ultrastructure which reflect their distinctive physiologies . The cross-sectional area and density (number per unit area) of synaptic contact zones with conventional and presynaptic dendrites (F2 profiles) were measured for each retinal terminal. The densities of F2 presynaptic dendrites and F1 flattened vesicle axon terminals were also measured. Retinal terminals in MIN were often large (mean size = 2.7 micron2 area) and had a high density of synaptic contacts (0.14 per micron surface area) with conventional dendrites, presynaptic dendrites, and dendritic spines. A high density of F2 presynaptic dendrites (0.08 per micron2 area) was found in MIN. F1 axon terminals were also found frequently (0.04 per micron2). MIN retinal terminals were often organized in glomeruli like those of the dorsal lateral geniculate nucleus. The retinal terminals in VLG were almost always small (mean size = 0.94 micron2 area), although they also had a high density of synaptic contacts (0.17 per micron surface area). They frequently synapsed on small dendrites and dendritic spines and less frequently on large dendrites. Unlike MIN, retinal terminals in VLG rarely contacted F2 presynaptic dendrites which were much less frequent in VLG (0.01 per micron2 area). Like MIN, VLG contained numerous F1 axon terminals (0.06 per micron2 area). No typical retinal glomeruli were found in VLG . These results show that MIN, which contains many Y cells, has a population of large retinal terminals and many F2 presynaptic dendrites. VLG , which apparently has only W cells, contains only small retinal terminals and has fewer F2 presynaptic dendrites. Both have a high density of F1 flat vesicle axon terminals.

We have examined by autoradiography the labeling pattern in the cat superior colliculus following injection of tritiated gamma-aminobutyric acid (GABA). Silver grains were heavily distributed within the zonal layer and the upper 200 micrometer of the superficial gray. Fewer grains were observed deeper within the superficial gray, and still fewer were found within the optic and intermediate gray layers. The accumulation of label was restricted to certain classes of neuron and glia. Densely labeled neurons were small (8-12 micrometer in diameter) and located primarily within the upper 200 micrometer. Dark oligodendrocytes and astrocytes showed a moderate accumulation of label while pale oligodendrocytes and microglia were unlabeled. Label was also selectively accumulated over several other types of profile within the neuropil, including presynaptic dendrites, axons, and axon terminals.

We examined the pattern of [3H]muscimol labeling in cat superior colliculus to determine if it matches that of [3H] gamma-aminobutyric acid ([3H]-GABA) labeling or GABA antibody immunoreactivity. Injections in the superficial layers labeled cell bodies in only the superficial layers. Of 204 labeled cells, 68% were located within the upper 200 microns of the superior colliculus, 31% within the deep superficial gray layer, and only 1% below that layer, a pattern similar to that seen with [3H]GABA labeling. By contrast, an injection in the deep layers of the colliculus resulted in cell labeling in both the superficial and deep layers, including 68% in the superficial gray layer, 24% in the optic layer, and 8% in the intermediate and deep gray layers. This pattern approximates that seen with GABA immunocytochemistry. We conclude that the pattern of accumulation of [3H]muscimol depends critically upon the location of the injection and reasonably matches the pattern of GABA immunoreactivity if the injection involves the deep layers of the colliculus.