Counter-regulation of the AP-1 monomers pATF2 and Fos: Molecular readjustment of brainstem neurons in hearing and deaf adult rats after electrical intracochlear stimulation.

Expression of the immediate-early gene fos (also known as c-fos) and phosphorylation of the product of the early response gene atf2 (pATF2) in the adult auditory brainstem can be modulated by electrical intracochlear stimulation. The Fos and pATF2 proteins are competitive monomers of the heterodimeric activator protein-1 (AP-1) transcription factor that triggers the expression of genes related to neural plasticity. Our previous findings showed that the stimulation-induced spatio-temporal pattern of Fos expression in the adult auditory system depends on hearing experience. In this study, we aimed to identify a possible correlation of pATF2 and Fos expression. Adult normal hearing and neonatally deafened rats were unilaterally stimulated with a cochlear implant (CI) for 45 min, 73 min, or 2h. The numbers of Fos- and pATF2-positive neurons in the anteroventral cochlear nucleus (AVCN), the lateral superior olive (LSO), and the central inferior colliculus (CIC) were evaluated. Following stimulation, an increased Fos expression was demonstrated in all these regions in hearing and deaf rats. However, in neonatally deafened rats, significantly more Fos-positive neurons emerged that did not obey a tonotopic order. Independent of hearing experience, Fos expression correlated with a locally matching decrease of pATF2 expression in AVCN and LSO, but not in CIC. We suggest that these changes in gene expression result in a shift of AP-1 dimer composition from ATF2:Jun to Fos:Jun. This change in AP-1 constellation is expected to invoke different transcriptional cascades leading to distinct modes of tissue reorganization and plasticity responses in the mature central auditory system under stimulation.

[The utilization of brain plasticity by cochlear implants : Molecular and cellular changes due to electrical intracochlear stimulation]. / Nutzung der Plastizität des Gehirns durch Cochleaimplantate : Molekulare und zelluläre Veränderungen durch elektrische intracochleäre Stimulation.

BACKGROUND AND OBJECTIVES: During pre- and postnatal development, a high level of growth-associated protein 43 (Gap43) is expressed in the brain. This neuron-specific protein is expressed in somata, axons, and growth cones and plays a key role in neurite outgrowth and synaptogenesis. With maturation of the brain, Gap43 is down-regulated by most neurons, except in brain areas such as the hippocampal CA3 region or the binaural auditory regions lateral superior olive (LSO) and central inferior colliculus (CIC). This study investigated how changes in sensory activity levels and patterns can modulate the adult plasticity response. METHODS: To study the effect of sensory activity on adult Gap43 expression, mRNA and protein levels were determined in LSO and CIC of hearing-experienced rats, unilaterally and bilaterally deafened rats, or rats unilaterally stimulated by a cochlear implant (CI). RESULTS: Unilateral hearing loss of an adult auditory system causes asymmetrical expression of Gap43 mRNA between ipsi- and contralateral LSOs or CICs of the brain stem. While the mRNA level rose on the contralateral side of the LSO, CIC neurons increased their gap43 transcription ipsilaterally compared to the control level (p<0.001). Compensation of the lost sensory input by way of CI stimulation resulted in a bilaterally symmetric but increased gap43 transcription. CONCLUSIONS: Our data indicate that Gap43 is not only a marker for neuronal growth and synaptogenesis, but also reflects modified patterns of synaptic activities on auditory neurons. Thus, unilateral deafness directly results in an asymmetrical adaptation of the gap43 transcription between both sides of the auditory brain stem. This can be prevented by simple-patterned stimulation of the auditory nerve via a CI.

Cell death or survival: molecular and connectional conditions for olivocochlear neurons after axotomy.

We aimed to determine whether rat olivocochlear neurons survive axotomy inflicted through cochlear ablation, or if they degenerate. To estimate their intrinsic potential for axonal regeneration, we investigated the expression of the transcription factor c-Jun and the growth-associated protein-43 (GAP43). Axonal tracing studies based on application of Fast Blue into the cochlea and calcitonin gene-related peptide immunostaining revealed that many, but not all, lateral olivocochlear neurons in the ipsilateral lateral superior olive degenerated upon cochleotomy. A decrease of their number was noticed 2 weeks after the lesion, and 2 months postoperative the population was reduced to approximately one quarter (27-29%) of its original size. No further reduction took place at longer survival times up to 1 year. Most or all shell neurons and medial olivocochlear neurons survived axotomy. Following cochleotomy, 56-60% of the lateral olivocochlear neurons in the ipsilateral lateral superior olive were found to co-express c-Jun and GAP43. Only a small number of shell and medial olivocochlear neurons up-regulated c-Jun expression, and only a small number of shell neurons expressed GAP43. Up-regulation of c-Jun and GAP43 in lateral olivocochlear neurons upon axotomy suggests that they have an intrinsic potential to regenerate after axotomy, but cell counts based on the markers Fast Blue and calcitonin gene-related peptide indicate that this potential cannot be exploited and degeneration is induced instead. The survival of one quarter of the axotomized lateral olivocochlear neurons and of all, or almost all, shell and medial olivocochlear neurons appeared to depend on connections of these cells to other regions than the cochlea by means of axon collaterals, which remained intact after cochleotomy.

[The cochlear implant. Molecular arguments favouring early implantation]. / Kochleaimplantat. Molekularbiologische Argumente für eine Frühversorgung.

BACKGROUND: An important factor in the clinical outcome of cochlear implantation is the age of the patient. Compared to older patients, children with congenital deafness have a better outcome when the implantation is made before the age of 2 years. The cause may lie in the molecular biology of the brain, which changes during postnatal maturation. METHODS: Protein probes were obtained from tissue of the rat inferior colliculus at different ages. The probes were analyzed using 2-dimensional SDS electrophoresis. RESULTS: The expression of GAP-43, a protein expressed by neurons during axonal outgrowth and synaptogenesis, and the total number of the protein species showed a significant reduction during ontogenesis. This shows that while neurons gradually assume their specific function, they downregulate GAP-43 and the molecular complexity decreases. CONCLUSIONS: Due to a lack of neuronal pluripotency at later developmental stages, the flexibility to adapt to the afferent activation provided by a cochlear implant is increasingly limited.

In vivo visualization of the cochlear nerve and nuclei with fluorescent axonal tracers.

In recent years multichannel neuroprostheses have been developed which directly stimulate the central auditory pathway. Substantially these have been used in cases of total hearing loss caused by neurofibromatosis type 2 where bilateral damage to the auditory nerve prevents more peripheral stimulation. The electrode carrier of the auditory brainstem implant (ABI) is designed to be placed on the cochlear nucleus complex residing at the lateral brainstem surface. Despite altered anatomy due to tumor growth or preceding surgery, correct electrode placement is essential to maximize the variety of pitch percept elicited during electrical stimulation with the ABI without producing side-effects. In order to assist intraoperative identification of the proximal auditory nerve and cochlear nuclei, the non-toxic fluorescent axonal tracers Fast Blue or Fluorogold were injected into the cochlea of rats and Java monkeys. Four to seven days after tracer application, labeling of the eighth cranial nerve, its entrance into the brainstem and the primary radiation of auditory fibers into the cochlear nucleus could be demonstrated as colored fluorescence on the living brain under appropriate ultraviolet illumination. Additional histological processing revealed groups of retrogradely labeled neuronal cell bodies in both species. Our results suggest that this method could also be used in humans in order to aid surgeons with the proper positioning of the electrode array.

Modulation of P-CREB and expression of c-fos in cochlear nucleus and superior olive following electrical intracochlear stimulation.

Investigating activity-dependent plasticity in the auditory brain stem of the adult rat, we observed that electrical intracochlear stimulation led to a tonotopically localized modulation of the phosphorylation of the cAMP response element binding protein (CREB) and an equally localized expression of the immediate early gene product c-Fos in cochlear nucleus and superior olive. As P-CREB is thought to act as transcription factor on one promoter site of the c-fos gene, we compared immunolabeling for P-CREB and c-fos in adjacent brain sections. Following 2h sustained stimulation in previously deafened animals, labelling for P-CREB declined in regions where c-Fos labelling increased. This suggests that the level or state of P-CREB (e.g. whether it is phosphorylated or not) are affected by intracochlear stimulation in a process that appears to be linked to the stimulation-dependent expression of c-Fos in auditory brain stem nuclei.

Activity-dependent plasticity in the adult auditory brainstem.

Over the past few years we have studied the plasticity of the adult auditory brainstem in the rat following unilateral changes to the pattern of sensory activation, either by intracochlear electrical stimulation or by deafening. We discovered that modifications to afferent activity induced changes in the molecular composition and cellular morphology throughout the auditory brainstem, including its major centers: the cochlear nucleus complex, the superior olivary complex, and the inferior colliculus. The time window studied ranged from 2 h to over 1 year following induction of changes to afferent activity. The molecular markers employed include the NMDA receptor subunit type 1, the cAMP response element binding protein (CREB), the immediate early gene products c-Fos, c-Jun and Egr-1, the growth and plasticity-associated protein GAP-43 and its mRNA, the calcium binding protein calbindin, the cell adhesion molecule integrin-alpha(1), the microtubule-associated protein MAP-1b, and the neurofilament light chain (NF-L). As a consequence of the specific electrical stimulation of the auditory afferents or the loss of hearing, a cascade of events is triggered that apparently modifies the integrative action and computational abilities of the central auditory system. An attempt is made to relate the diverse phenomena observed to a common molecular signaling network that is suspected to bridge sensory experience to changes in the structure and function of the brain. Eventually, a thorough understanding of these events will be essential for the specific diagnosis of patients, optimal timing for implantation, and suitable parameters for running of a cochlear implant or an auditory brainstem implant in humans. In this report an overview of the results obtained in the past years in our lab is presented, flanked by an introduction into the history of plasticity research and a model proposed for intracellular signal cascades related to activity-dependent plasticity.

Plasticity of the superior olivary complex.

The superior olivary complex (SOC) is part of the auditory brainstem of the vertebrate brain. Residing ventrally in the rhombencephalon, it receives sensory signals from both cochleae through multisynaptic pathways. Neurons of the SOC are also a target of bilateral descending projections. Ascending and descending efferents of the SOC affect the processing of auditory signals on both sides of the brainstem and in both organs of Corti. The pattern of connectivity indicates that the SOC fulfills functions of binaural signal integration serving sound localization. But whereas many of these connectional features are shared with the inferior colliculus (with the important exception of a projection to the inner ear), cellular and molecular investigations have shown that cells residing in SOC are unique in several respects. Unlike those of other auditory brainstem nuclei, they specifically express molecules known to be involved in development, plasticity, and learning (e.g., GAP-43 mRNA, specific subunits of integrin). Moreover, neurons of the SOC in adult mammals respond to various kinds of hearing impairment with the expression of plasticity-related substances (e.g., GAP-43, c-Jun, c-Fos, cytoskeletal elements), indicative of a restructuring of auditory connectivity. These observations suggest that the SOC is pivotal in the developmental and adaptive tuning of binaural processing in young and adult vertebrates.

Olivocochlear neurons sending axon collaterals into the ventral cochlear nucleus of the rat.

The olivocochlear projection constitutes the last stage of the descending auditory system in the mammalian brain. Its neurons reside in the superior olivary complex (SOC) and project to the inner and outer hair cell receptors in the cochlea. Olivocochlear neurons were also reported to send axon collaterals into the cochlear nucleus, but controversies about their number and about species differences persist. By injecting the fluorescent retrograde axonal tracers diamidino yellow and fast blue into the cochlea and the ventral cochlear nucleus (VCN), we studied the distribution and number of olivocochlear neurons with and without axon collaterals into the VCN of the rat. We found that olivocochlear neurons residing in the lateral superior olive (LSO), the intrinsic lateral olivocochlear cells (intrinsic LOCs), do not send axon collaterals into the VCN. By contrast, a majority, and possibly all, olivocochlear neurons residing in the ventral nucleus of the trapezoid body (VNTB), the medial olivocochlear cells (MOCs), do have such axon collaterals. These cells may thus affect processing in the ascending auditory pathway at the level of the receptors and concurrently at the level of the secondary sensory neurons in the cochlear nucleus. Belonging to the lateral olivocochlear system, shell neurons reside around the LSO and form a third group of olivocochlear cells (shell LOCs). Like intrinsic LOCs, they innervate the inner hair cells, but like MOCs they do, by means of axon collaterals, project into the VCN. These findings have implications for understanding both auditory signal processing and the plasticity responses that occur following loss of cochlear function.

Plasticity of the auditory brainstem: cochleotomy-induced changes of calbindin-D28k expression in the rat.

Calbindin is a calcium binding protein that is characteristically expressed in several auditory brainstem nuclei during ontogeny and is thought to serve as a buffer, protecting cells against toxic levels of calcium. Upon maturation, calbindin is drastically reduced or entirely lost in many auditory nuclei. We made cochleotomies in mature rats to study effects of deafening and deafferentation on the expression of calbindin in the auditory brainstem. Following unilateral cochleotomy, we observed a substantial increase in the number of calbindin-immunoreactive fibers and boutons in the ventral subdivisions of the ipsilateral cochlear nucleus. At the same time, calbindin-positive astrocytes emerged in the dorsal and ventral cochlear nucleus. Beyond the immediately affected ipsilateral cochlear nucleus, we found calbindin-positive neurons in the lateral superior olive and in the central inferior colliculus, both contralateral to the operation. The loss of one cochlea reduces auditory input and puts the flow of neuronal activity originating in the two ears out of balance. Our findings indicate that the need for the neuronal networks in the auditory brainstem to adjust to this drastically changed pattern of sensory signals invokes the expression of calbindin in glial cells as well as in directly and indirectly affected neuronal cell populations.

On a novel type of neuron with proposed mechanoreceptor function in the human round window membrane--an immunohistochemical study.

An immunohistochemical study was performed on surgically obtained human fresh cochlear tissue, using synaptophysin antibodies. After immediate aldehyde fixation and decalcification in Na-EDTA serial cryosections were made of the cochlea including the round window membrane (RWM). Apart from highly specific immunostaining of spiral ganglion cells and unmyelinated nerve fibers an immunoreactive neuroreceptor could be demonstrated at the postero-medial insertion of the RWM. The perikaryon showed intense synaptophysin immunoreativity with a distal process projecting into the fibrous stroma of the RWM displaying structural specializations suggestive of a mechanoreceptor function. It is speculated whether the neuroreceptor may be involved in the proprioception and/or mechanoreception of tensile forces generated within the lamina propria during displacement of the yielding RWM in the bony labyrinth. Such a function could be important for the regulation of perilymph pressure.

Auditory brainstem: development and plasticity of GAP-43 mRNA expression in the rat.

Expression of the growth and plasticity associated protein GAP-43 is closely related to synaptogenesis and synaptic remodeling in the developing as well as in the mature nervous system. We have studied the postnatal development of GAP-43 mRNA expression in the auditory brainstem and determined the time course of its reexpression following deafening through cochlear ablation using a digoxigenin-coupled mRNA probe. By the first postnatal day, GAP-43 mRNA was expressed at high levels in all auditory brainstem nuclei. But whereas GAP-43 mRNA is almost entirely lost in most of these nuclei in the adult animal, significant levels of this molecule are retained in the inferior colliculus and, most notably, in the lateral and medial superior olivary nucleus. As a consequence of unilateral cochleotomy, GAP-43 mRNA rose dramatically in some neurons of the ipsilateral lateral superior olive, whereas the hybridization signal decreased in others. Using double staining protocols, we found that those olivary neurons that increase their level of GAP-43 mRNA appear to be identical with the cells developing strong GAP-43 immunoreactivity after cochleotomy. By combining axonal tracing with in situ hybridization, we proved that at least some of the cells with increased levels of GAP-43 mRNA and protein are the cells of origin of olivocochlear projections. A substantial decrease of the level of GAP-43 mRNA took place in the inferior colliculus contralateral to the lesioned cochlea. Our results led us to suggest that neurons in the superior olivary complex may play a crucial role in orchestrating auditory brainstem plasticity.

Redistribution of NMDA receptors in the cochlear nucleus following cochleotomy.

The major input to neurons of the cochlear nucleus comes from the glutamatergic cells of the spiral ganglion. We have studied the effect of unilateral destruction of the inner ear, including the spiral ganglion, with two antibodies against different types of NMDA receptor subunits, NMDAR1 and NMDAR2A/B, in the cochlear nucleus of the rat. Following cochleotomy, a dramatic redistribution of the receptor subunits was observed from a mostly perikaryal to a predominantly dendritic localization. Moreover, distinct changes in the composition of NMDA receptor complexes occurred. These effects were interpreted as compensatory responses to the massive loss of presynaptic release of the transmitter glutamate.

Evaluating the plasticity potential of the auditory brain stem nucleus in the rat.

OBJECTIVE: To study the adaptation of the auditory brainstem to auditory loss. STUDY DESIGN: Growth-associated protein 43 (GAP-43) immunoreactivity was studied in in rats whose cochleas had been removed. RESULTS AND DISCUSSION: Neurons in the lateral superior olive were found to synthesize GAP-43 in a pattern that paralleled the changes in GAP-43 immunoreactivity in the cochlear nucleus after cochlear ablation. These findings suggest that new patterns of synaptic communication can be established after damage to the cochlea.

Postnatal development of GAP-43 immunoreactivity in the auditory brainstem of the rat.

Extensive data link the growth associated protein GAP-43 to axonal elongation and synapse formation during development and in plastic responses of nervous tissue. We have studied the changing levels of GAP-43 expression in the auditory brainstem nuclei of the developing rat by applying immunocytochemical techniques. By the first postnatal day (P1), GAP-43 was expressed at high concentrations in all subdivisions of the cochlear nuclear complex and the superior olivary complex. At this stage, neuropil structures recognized by the antibody did not show any varicosities on cellular processes in all these regions. By P8, the texture of the stain has turned markedly more granular, a pattern likely to reflect the formation of presynaptic endings. A predominantly granular distribution of GAP-43 has developed by P12. At that time, the staining intensity is markedly reduced compared to the levels of the newborn. By P16, the auditory brainstem nuclei have lost most of their GAP-43 immunoreactivity, but a distinct level of staining persisted into adulthood in all of them. This staining was restricted to boutons, which are thought to be presynaptic terminals. We conclude that a moderate but apparently relevant potential for plasticity is retained in these auditory structures. Should the patterns of neural signals, mediated by the inner ear, change during adulthood, the central structures appear to be able to respond with the formation of altered synaptic connectivity.

Plasticity of the auditory brainstem: effects of cochlear ablation on GAP-43 immunoreactivity in the rat.

In the adult brain, expression of the growth associated protein GAP-43 may serve as an indicator of synaptic remodeling. We have studied localization and time course of the re-expression of GAP-43 following deafening through cochlear ablation. As a consequence of unilateral cochlear lesioning, a substantial increase in the expression of GAP-43 was observed in the neuropil of all subnuclei of the ipsilateral cochlear nuclear complex. This expression of GAP-43 occurred in well-defined fibers and boutons. In the ventral cochlear nuclei, boutons immunoreactive for GAP-43 were often localized on cell bodies. However, they were found only on selected subpopulations of cochlear nucleus neurons, i.e., on cell bodies containing glutamate or calretinin immunoreactivity, but apparently not on GABAergic neurons. Olivocochlear neurons must have been axotomized by the operation. Following cochlear ablation, a dramatic re-expression of GAP-43 occurred in cell bodies of the ipsilateral lateral superior olive but not in the ventral nucleus of the trapezoid body. Position and number of these cells suggested that most, if not all, of them serve the lateral olivocochlear bundle. However, although axon collaterals are given off by certain types of olivocochlear neurons, a direct involvement of the immunoreactive cell bodies in the emergence of GAP-43 in the cochlear nucleus is not obvious. A transient rise of GAP-43 immunoreactivity that could not be attributed to axotomized neurons was observed in the contralateral dorsal cochlear nucleus and in the ipsilateral inferior colliculus. Given the functional significance attributed to GAP-43, we conclude that the sudden loss of spiral ganglion cells leads to a reactive synaptogenesis in complex patterns across several auditory brainstem nuclei.

The mosaic architecture of the superior colliculus.

The superior colliculus is a midbrain structure serving visual, multisensory and sensorimotor processing. Throughout various collicular layers, visual afferents are linked together with afferents related to other sensory modalities as well as with afferents from sources not easily subsumed under the term 'sensory'. These inputs are orchestrated in a topographic fashion and led to premotor neurons that are important elements in generating saccadic eye movements and orientation movements of other kinds. Using immunocytochemical techniques to chart the distribution of various substances serving neurotransmission and neuromodulation, it was found that many of them, e.g. acetylcholinesterase (AChE), choline acetyltransferase, the enkephalins, substance P, and parvalbumin, relate to repetitive structural islands, or modules, in the superior colliculus. From studies on the distribution of three further neuroactive substances in rat superior collicular tissue: the calcium binding protein calretinin, the growth and plasticity related protein neuromodulin (GAP-43), and a glutamate receptor of the NMDA-type, we were led to conclude (1) that the intermediate layers of the superior colliculus are composed not of two, but of at least three disjunct types of modules, (2) that not just the intermediate layers but more or less the whole superior colliculus is an assemblage of modules, and (3) that, besides topographic connectivity and laminar structuring, the modules constituting an iterative partitioning represent a third major feature of superior collicular architecture. The origin of the collicular mosaic is considered under an evolutionary perspective, and a hypothesis is presented stating that the pattern of AChE-rich modules on the level of the multimodal collicular layers can be predicted from retinal ganglion cell topography.

Morphology and connections of neurons in area 17 projecting to the extrastriate areas MT and 19DM and to the superior colliculus in the monkey Callithrix jacchus.

Neurons of area 17, the primary visual cortex, project to various anatomically and physiologically different extrastriate areas and subcortical regions. In the present investigation, we addressed the question of whether the efferent neurons in area 17 can contribute to functional diversity between these regions. We approached this question by analyzing the dendritic morphology of neurons in area 17 projecting to area MT, area 19DM, and the superior colliculus in the new world simian primate Callithrix jacchus, because dendritic morphology is an important factor in determining physiological properties of nerve cells. Retrograde transport of fluorochromes injected into the target regions, and intracellular injections of Lucifer yellow in the prelabelled neurons, revealed the following. 1) Morphologically identical large pyramidal cells in layer VI of area 17 project to all three targets. Some of them possess axon collaterals to two or all three targets, suggesting that they provide common information to all three areas. 2) Pyramidal cells in layer IIIc projecting to area MT form a morphologically homogeneous population. 3) Three small to medium-sized pyramidal cell types in layers IIIa-c, spiny stellate cells in layer IIIc, and another large pyramidal cell type in layer VI project to area 19DM. 4) Pyramidal cells in the lower two-thirds of layer V in area 17 project to the superior colliculus. In conclusion, we have shown that in Callithrix one efferent pathway may originate from several cell types. However, with the exception of the large cells in layer VI, efferent cells projecting to area MT, area 19DM, and the superior colliculus were morphologically distinct. This suggests that functional differences between brain regions could arise in part from morphological heterogeneity between and within the efferent cell populations.

Re-emergence of GAP-43 in cochlear nucleus and superior olive following cochlear ablation in the rat.

The effect of cochlear lesion on the expression of the growth associated protein GAP-43 in superior olive and cochlear nucleus was studied in the rat. In normal development of these auditory brainstem nuclei, GAP-43 immunoreactivity is high perinatally but low 10 days postnatally or thereafter. Removal of one spiral ganglion in grown-up animals caused a substantial re-emergence of GAP-43 immunoreactivity in varicose fibers of the ipsilateral ventral cochlear nucleus and cell bodies of the lateral superior olive. These findings suggest that a reactive synaptogenesis takes place in the cochlear nucleus as a consequence of deafening through spiral ganglion loss.

Enkephalin-positive and acetylcholinesterase-positive patch systems in the superior colliculus have matching distributions but distinct developmental histories.

Histochemical stains for acetylcholinesterase activity and enkephalin-like immunoreactivity both demonstrate a high degree of patterning in the superior colliculus, particularly in the intermediate and deep layers. Both markers occur predominantly in the neuropil of these layers, and both are principally distributed in distinct macroscopic compartments. We report here that patches of heightened acetylcholinesterase activity correspond to patches of high enkephalin-like immunoreactivity. The two markers thus delineate largely the same domain in the intermediate and deep layers. The most prominent zones of staining for enkephalin-like peptide and for acetylcholinesterase also coincided in the dorsolateral periaqueductal gray matter. These findings suggest a close interlocking of one or more acetylcholinesterase-containing systems with one or more pathways related to endogenous opioids in the superior colliculus. As the acetylcholinesterase expression in the patches is known to match in detail choline acetyltransferase expression, our results also suggest the possibility of local cholinergic-opiatergic interactions. In some sections, blood vessels associated with enkephalin-rich and acetylcholinesterase-rich patches extended beyond the colliculus into the periaqueductal gray matter, where they again became surrounded by dense fibrous labeling. This pattern suggests that neurohumoral signal exchange might occur through blood vessels even in a sensory-motor structure such as the colliculus. In a postnatal developmental series of kitten brains we found that enkephalin-like immunoreactivity was already distinctly compartmental in the intermediate layers at birth and continued to show this distribution throughout postnatal development. By contrast, acetylcholinesterase staining was nearly homogeneous at birth and became compartmental gradually during the first postnatal weeks. Thus, despite the eventual near coincidence of the enkephalin-rich and acetylcholinesterase-rich compartments of the superior colliculus, they mark systems that follow distinct programs of neurochemical development.