The influence of chronic lithium administration on deafferentation-induced cellular changes in the chick cochlear nucleus

The avian brainstem serves as a useful model system to address the question of how afferent activity influences viability of target neurons. Approximately 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e. deafness produced by cochlea removal). Previous studies have identified cellular events that occur within hours following cochlea removal, which are thought to lead to the ultimate death of NM neurons. We have recently shown that chronic lithium treatment increases neuronal survival following deafferentation. To assess where in the cell death cascade lithium is having its effect, we evaluated some of the early deafferentation-induced cellular changes in NM neurons. Lithium did not affect deafferentation-induced changes that occur across the entire population of NM neurons. There were still deafferentation-induced increases in intracellular calcium concentrations and early changes in the ribosomes, as indicated by Y10b immunolabeling. Lithium did, however, affect changes that are believed to be indicative of the subpopulation of NM neurons that will eventually die. Ribosomes recovered in all of the deafferented NM neurons (as assessed by Y10b labeling) by 10 h following cochlea removal in subjects pretreated with lithium, while a subpopulation of the NM neurons in saline-treated subjects showed dramatic reduction in Y10b labeling at that time. Lithium treatment also prevented the robust upregulation of b cell leukemia/lymphoma-2 (Bcl-2) mRNA that is observed in a subpopulation of deafferented NM neurons 6 h following cochlea removal.     

Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3–5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.     

Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.     

Deafferentation-induced caspase-3 activation and DNA fragmentation in chick cochlear nucleus neurons

Cochlea removal severs peripheral processes of cochlear ganglion cells and permanently abolishes afferent input to nucleus magnocellularis (NM) neurons. Deafferented chick NM neurons undergo a series of morphologic and metabolic changes, which ultimately trigger the death of 20%–40% of neurons. Previous studies suggested that this cell specific death involves activation of the intrinsic apoptotic pathway, including increased presence of cytochrome c and active caspase-9 in the cytoplasm of deafferented NM neurons. Interestingly, however, both markers were detected pan-neuronally, in both degenerating and surviving NM neurons [Wilkinson BL, Elam JS, Fadool DA, Hyson RL (2003) Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus. Neuroscience 120:1071–1079]. Here, we provide evidence for the increased appearance of late apoptotic indicators and describe novel characteristics of cell death in deafferented auditory neurons. Young broiler chickens were subjected to unilateral cochlea removal, and brainstem sections through NM were reacted for active caspase-3 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL). Caspase-3 activation is observed in the cytoplasm of both dying and surviving deafferented NM neurons 24 h to 7 days following cochlea removal, suggesting that caspase-3, usually considered an “executioner” of apoptotic death, may also function as a “modulator” of death. In addition, we find that TUNEL labeling of degraded DNA is observed in deafferented NM. In contrast to upstream apoptotic markers, however, TUNEL labeling is restricted to a subpopulation of deafferented neurons. Twelve hours following cochlea removal, TUNEL labeling is observed as punctate accumulations within nuclei. Twenty-four hours following cochlea removal, TUNEL accumulates diffusely throughout neuronal cytoplasm in those neurons likely to die. This cytoplasmic TUNEL labeling may implicate mitochondrial nucleic acid degradation in the selective death of some deafferented NM neurons. Our study examines the subcellular distributions of two prominent apoptotic mediators, active caspase-3 and TUNEL, relative to known histochemical markers, in deafferented NM; provides new insight into the apoptotic mechanism of cell death; and proposes a role for mitochondrial DNA in deafferentation-induced cell death.     

Neuronal architecture in nucleus magnocellularis of the chicken auditory system with observations on nucleus laminaris: A light and electron microscope study

This report presents the major structural features of neurons and their afferent input in nucleus magnocellularis, the avian homologue of the mammalian anteroventral cochlear nucleus. Results of light-microscope observations, as seen in Golgi, Nissl, and normal fiber preparations, as well as ultrastructural morphology are reported. In addition, cells and axons in nucleus laminaris, the presumed homologue of the mammalian medial superior olivary nucleus, are also described.In Golgi-impregnated material, the mature principal cell in nucleus magnocellularis has an ovoid soma encrusted with somatic spines. A dendrite, when present, emerges from the cell soma, travels for a short distance and breaks into a tuft of stubby terminal branches. Foremost among the afferents to nucleus magnocellularis are auditory nerve axons that terminate in large, axosomatic endings, or endbulbs, covering a large portion of the somatic surface. Other afferents, which also end in relation to the perikaryon, are of undetermined and perhaps multiple origins. The neurons resemble the bushy cells of the mammalian anteroventral cochlear nucleus. Evidence is presented that individual axons from the nucleus magnocellularis bifurcate and send branches to the nucleus laminaris bilaterally, thus placing constraints on the binaural interactions possibly involved in lateralization functions.In electron micrographs, the end-bulbs appear as large, elongate structures which can cover a third of the cell soma. Multiple sites of synaptic specialization occur along these terminals. The synaptic membrane complexes may form directly on the cell body or on the sides or crests of somatic spines. These complexes are characterized by asymmetric membrane densities with a cluster of clear, spherical vesicles on the axonal side. Other small terminal profiles are also present on the somata receiving the end-bulbs. Dendritic profiles are scarce, in agreement with observations in Golgi impregnations.The structural findings indicate that the medial part of the nucleus magnocellularis is homologous to the anterior part of the mammalian anteroventral cochlear nucleus in that the neurons of nucleus magnocellularis are homologous to the bushy cells of the cat. On this basis, the cells in nucleus magnocellularis could faithfully preserve the acoustic response patterns generated in the auditory nerve. This should, in turn, allow a secure relay of bilateral latency differences essential for binaural interactions in the nucleus laminaris.     

Neuroprotective and neurotrophic actions of the mood stabilizer lithium: can it be used to treat neurodegenerative diseases?

The mood stabilizing drug lithium has emerged as a robust neuroprotective agent in preventing apoptosis of neurons. Long-term treatment with lithium effectively protects primary cultures of rat brain neurons from glutamate-induced, NMDA receptor-mediated excitotoxicity. This neuroprotection is accompanied by an inhibition of NMDA-receptor-mediated calcium influx, upregulation of anti-apoptotic Bcl-2, downregulation of pro-apoptotic p53 and Bax, and activation of cell survival factors. Lithium treatment antagonizes glutamate-induced activation of c-Jun-N-terminal kinase (JNK), p38 kinase, and AP-1 binding, which has a major role in cytotoxicity, and suppresses glutamate-induced loss of phosphorylated cAMP responsive element binding protein (CREB). Lithium also induces the expression of brain-derived neurotrophic factor (BDNF) and subsequent activation TrkB, the receptor for BDNF, in cortical neurons. The activation of BDNF/TrkB signaling is essential for the neuroprotective effects of this drug. In addition, lithium stimulates the proliferation of neuroblasts in primary cultures of CNS neurons. Lithium also shows neuroprotective effects in rodent models of diseases. In a rat model of stroke, post-insult treatment with lithium or valproate, another mood stabilizer, at therapeutic doses markedly reduces brain infarction and neurological deficits. This neuroprotection is associated with suppression of caspase-3 activation and induction of chaperone proteins such as heat shock protein 70. In a rat model of Huntington's disease (HD) in which an excitotoxin is unilaterally infused into the striatum, both long- and short-term pretreatment with lithium reduces DNA damage, caspase-3 activation, and loss of striatal neurons. This neuroprotection is associated with upregulation of Bcl-2. Lithium also induces cell proliferation near the injury site with a concomitant loss of proliferating cells in the subventricular zone. Some of these proliferating cells display neuronal or astroglial phenotypes. These results corroborate our findings obtained in primary neuronal cultures. The neuroprotective and neurotrophic actions of lithium have profound clinical implications. In addition to its present use in bipolar patients, lithium could be used to treat acute brain injuries such as stroke and chronic progressive neurodegenerative diseases.     

Basal forebrain neurons modulate the synthesis and expression of neuropeptides in the rat suprachiasmatic nucleus

We tested the hypothesis that efferents from the nucleus basalis magnocellularis (NBM) play a direct role in the regulation of neuropeptide synthesis and expression by neurons of the rat suprachiasmatic nucleus (SCN). Adult male rats in which the NBM was destroyed with quinolinic acid, either unilaterally or bilaterally, were compared with rats injected with physiological saline and with control rats. The estimators used to assess the effects of cholinergic deafferentation on the neuroanatomy and neurochemistry of the SCN were the total number of SCN neurons, the total number and somatic size of SCN neurons producing vasopressin (VP) and vasoactive intestinal polypeptide (VIP), and the respective mRNA levels. Bilateral destruction of the NBM did not produce cell death in the SCN, but caused a marked reduction in the number and somatic size of SCN neurons expressing VP and VIP, and in the mRNA levels of these peptides. The decrease in the number of VP- and VIP-producing neurons provoked by unilateral lesions was less striking than that resulting from bilateral lesions. It was, however, statistically significant in the ipsilateral hemisphere, but not in the contralateral hemisphere. The results show that the reduction of cholinergic inputs to the SCN impairs the synthesis, and thereby decreases the expression of neuropeptides by SCN neurons, and that the extent of the decline correlates with the amount of cholinergic afferents destroyed. This supports the notion that acetylcholine plays an important, and direct role in the regulation of the metabolic activity of SCN neurons.     

Alterations in neuronal survival and glial reactions after axotomy by ceftriaxone and minocycline in the mouse hypoglossal nucleus

Some antibiotics are suggested to exert neuroprotective effects via regulation of glial responses. Attenuation of microglial activation by minocycline prevents neuronal death in a variety of experimental models for neurological diseases, such as cerebral ischemia, Parkinson's and Huntington's disease. Ceftriaxone delays loss of neurons in genetic animal models of amyotrophic lateral sclerosis through upregulation of astrocytic glutamate transporter expression (GLT-1). However, it remains largely unknown whether these antibiotics are able to protect neurons in axotomy models for progressive motor neuron diseases. Recent studies have shown that the axotomized motoneurons of the adult rat can survive, whereas those of the adult mouse undergo neuronal degeneration. We thus examined the possible effects of ceftriaxone and minocycline on neuronal loss and glial reactions in the mouse hypoglossal nucleus after axotomy. The survival rate of lesioned motoneurons at 28 days after axotomy (D28) was significantly improved by ceftriaxone and minocycline treatment. There were no significant differences in the cellular densities of astrocytes between ceftriaxone-treated and saline-treated animals. Ceftriaxone administration increased the expression of GLT-1 in the hypoglossal nucleus, while it suppressed the reactive increase of glial fibrillary acidic protein (GFAP) expression to control level. The cellular densities of microglia at D28 were significantly lower in minocycline-treated mice than in saline-treated mice. The time course analysis showed that immediate increase in microglia at D3 and D7 was not suppressed by minocycline. The present observations show that minocycline and ceftriaxone promote survival of lesioned motoneurons in the mouse hypoglossal nucleus, and also suggest that alterations in glial responses might be involved in neuroprotective actions of antibiotics.     

Effects of tooth pulp deafferentation on nociceptive and nonnociceptive neurons of the feline trigeminal subnucleus caudalis (medullary dorsal horn)

1. Effects of deafferentation of the tooth pulps of the posterior mandibular teeth were studied in single neurons recorded in the ipsilateral subnucleus caudalis of the trigeminal (V) spinal tract nucleus of adult cats and kittens. The functional properties of neurons in each anesthetized animal were determined electro-physiologically in a series of microelectrode penetrations of the subnucleus. 2. The more than 800 neurons investigated could be subdivided on the basis of their cutaneous mechanoreceptive field properties into low-threshold mechanoreceptive (LTM) neurons, wide dynamic range (WDR) neurons, or nociceptive-specific (NS) neurons. Comparisons of neuronal properties were made between control (intact) cats and 7-15 day deafferented cats studied in a blind design, as well as groups of longer term deafferented cats, and kittens undergoing a "natural" deafferentation as a result of exfoliation of primary teeth. 3. There was no apparent change in the somatotopic pattern of organization of the subnucleus in the kittens and pulp-deafferented cats and no statistically significant differences were noted between kittens and control cats in any property except for alterations in the incidence of spontaneously active neurons. 4. Limited but statistically significant alterations were noted in some of the neuronal properties in the deafferented cats. These changes were especially apparent in the LTM neurons. The incidence of spontaneous activity was significantly decreased in the neurons of most long-term deafferented groups of cats. In the 7-15 day deafferented group, significantly more LTM neurons had a mechanoreceptive field involving all three divisions of the V nerve, and there was a significant increase in the incidence of LTM neurons activated by electrical stimulation of intraoral sites. Mechanosensitive neurons responsive only to tap stimuli were found only in the deafferented groups of cats. 5. These alterations in caudalis contrast with previous reports claiming marked hyperexcitability of caudal V brain stem neurons as a consequence of deafferentation and implicating such effects in the development of pain. However, some of the changes are in general not inconsistent with deafferentation-induced changes reported in spinal somatosensory neurons and with the pulp deafferentation-induced changes that we have recently documented in LTM neurons of subnucleus oralis of the V spinal tract nucleus of adult cats.(ABSTRACT TRUNCATED AT 400 WORDS)     

The hypoglossal nucleus of the primate: a Golgi study

The neurons of the hypoglossal nucleus were examined in Golgi preparations of adult macaque and squirrel monkeys. Two distinct types of neurons were found. The first type was a large multipolar cell which was typical of the majority of the neurons of the nucleus. Its soma ranged in size from 18 to 50 micrometers and numerous dendrites emerged from it. The dendritic spread of these neurons was within the ipsilateral nucleus, across the midline into the contralateral hypoglossal nucleus, or out into the adjacent reticular formation. The second type of neuron was a small cell. The soma of this neuron was oval in shape, measured less than 20 micrometers in its largest diameter, and gave off only two or three dendrites which remained within the nucleus.     

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an "L-cut" lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the "L-cut". Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex. The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

Synaptic excitation of the second and third order auditory neurons in the avian brain stem

Synaptic potentials were examined in the second- and third-order auditory neurons of nucleus magnocellularis and nucleus laminaris in the chick. Brain stems of mature chick embryos were explanted and maintained in vitro for 4 to 8 h. Field potentials, extracellular spike potentials and intracellular potentials evoked by 8th-nerve stimulation were examined. Eighth-nerve stimulation reliability elicited four identifiable field potentials which could be attributed to: (i) the afferent volley of the 8th-nerve axons, (ii) postsynaptic responses of n. magnocellularis neurons, and (iii) ipsilaterally and, (iv) contralaterally-evoked n. laminaris postsynaptic responses. Intracellular-recorded postsynaptic potentials were characterized by a rapid rise time and short duration. They were apparently monosynaptic with a synaptic delay of 0.4 ms. In each n. magnocellularis neuron the 'fast' excitatory postsynaptic potentials were composed of 1 to 3 all-or-none components. 'Slow' excitatory postsynaptic potentials were characterized by a longer latency, a longer duration and graded amplitude variation in proportion to the intensity of 8th-nerve stimulation. Both 'fast' and 'slow' excitatory postsynaptic potentials had similar reversal potentials. Since the 8th nerve makes monosynaptic connection with n. magnocellularis neurons, it is likely that at this synapse the 'fast' excitatory postsynaptic potentials were produced, while the 'slow' potential may be attributable to the convergence of many boutonal synapses of unknown origin. Intracellular injections of horseradish peroxidase into n. magnocellularis revealed that its efferents bifurcate below the nucleus and send one axon to the contralateral n. laminaris while the other axon forms a highly divergent projection to the ipsilateral laminar nucleus. The intracellular records obtained from n. laminaris are consistent with this anatomical finding in that graded excitatory postsynaptic potentials were elicited by 8th-nerve stimulation.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x(L) in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7-14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3-7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10-14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an “L-cut” lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the “L-cut”. Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex.The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

Bcl-w expression is increased in brain regions affected by focal cerebral ischemia in the rat

Proteins of the bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult and may play a role in cell death following stroke. The recently isolated bcl-w gene encodes a pro-survival member of the bcl-2 family, which is widely expressed. However, it is not known whether bcl-w plays a role in determining cell survival after cerebral ischemia. Using Western blot analysis and immunocytochemistry, regional bcl-w protein expression was studied in rat brain 2, 6, 24 and 72 h following 20 min temporary middle cerebral artery occlusion (MCAO). Focal cerebral ischemia increased bcl-w protein expression within the caudate putamen and parietal cortex, as well as causing milder increases within frontal cortex. Immunocytochemically bcl-w was expressed within neurons (frontal and parietal cortex) and glia (caudate putamen) 24 h after MCAO. These data suggest that bcl-w could play a role in determining cell survival after cerebral ischemia.     

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya.The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus.Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar?ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.     

Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: An electron microscope study

The development of the auditory nerve endings and their target cells in nucleus magnocellularis was studied by electron microscopy of perfusion-fixed brains from embryonic day 12 to hatching. Embryonic days 12–13: somatic processes extend from the perikaryon. The cytoplasm of the soma and processes contains free ribosomes, mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus and an eccentric, heterochromatic nucleus. Small profiles of auditory nerve fibers containing round, clear vesicles make specialized contacts, including some synapses, on distal somatic processes but rarely on proximal somatic processes or on the soma. The postsynaptic zones contain a flocculent matrix. Days 15–17: somatic processes disappear and occasional attachment plaques are seen between cell bodies. The nucleus appears euchromatic. Cytoplasmic organelles form a dense matrix indicative of intense metabolic activity. Somatic spines are evident. The afferent axons form large, vesiculated profiles located, increasingly, on the cell body and somatic spines, with many points of synaptic contact. Opposite each ending a band of amorphous, flocculent material fills the postsynaptic cytoplasm. Embryonic day 18-hatching: the somatic cytoplasm becomes less dense; stacks of rough endoplasmic reticulum start to condense. Afferent axon terminals mature, especially the synaptic membrane complex and associated densities. The postsynaptic flocculent material diminishes in extent until it is found associated only with somatic spines.The ultrastructural observations on the maturation of nucleus magnocellularis closely corroborate and extend previous results with the Golgi methods. Developing auditory nerve fibers initially synapse on the distal parts of the somatic processes of the immature cells. As the somatic processes disappear or retract, axonal endings move to the soma and develop into large axosomatic end-bulbs. Possibly, the somatic processes as they retract drag the auditory nerve endings to the cell body. The findings also suggest a role of the transiently appearing, flocculent material of the postsynaptic regions in the formation of synapses.     

Large diameter primary afferent input is required for expression of the Cat-301 proteoglycan on the surface of motor neurons

The expression of a cell surface proteoglycan, recognized by monoclonal antibody Cat-301, is regulated by neuronal activity in early life. Here we report that the expression of the Cat-301 proteoglycan on motor neurons depends on primary afferent input in the early postnatal period. Previously we showed that in two different systems, Y-cells in the cat lateral geniculate nucleus and motor neurons in the hamster spinal cord, the expression of the Cat-301 antigen requires neuronal activity during a circumscribed period in development. Disrupting the activity of Y-cells (by dark rearing or by monocular lid suture) or of motor neurons (by sciatic nerve crush or by spinal cord lesion) during the early postnatal period prevents Cat-301 expression. Disrupting neuronal activity in adults has no effect on Cat-301 expression. The onset of Cat-301 expression corresponds to the end of the period of activity-dependent development. In order to further dissect the components of the segmental reflex are required for the expression of Cat-301 on motor neurons, here we evaluated the effect of deafferentation by dorsal rhizotomy. In adult animals two weeks after deafferentation all sciatic motor neurons continue to express Cat-301. In contrast, in neonates two weeks after deafferentation the normal developmental expression of Cat-301 is reduced and less than 50% of sciatic motor neurons express Cat-301. We next selectively lesioned the small diameter afferents using the neurotoxin capsaicin. In contrast to rhizotomy, neonatal deletion of small diameter afferents has no effect on the development of Cat-301 expression on motor neurons. These results imply that input relayed by large diameter primary afferents (probably those conveying muscle and/or joint information) is required for normal maturation of motor neuronal properties during early life. They also provide further evidence for activity-dependent maturation of motor neurons.