Comparison of growth and reinnervation properties of cholinergic neurons from different brain regions grafted to the hippocampus.

Grafts of five different types of central cholinergic neurons, from the septal-diagonal band region, the nucleus basalis magnocellularis region (NBM), the striatum, the pontomesencephalic tegmentum of the brainstem, and the spinal cord, were compared with respect to their ability to grow and to reinnervate the cholinergically denervated hippocampal formation of adult rats. The areas were dissected from 14 to 15-day-old rat fetuses, and the same number of viable cells (35 X 10(4) from each of the different regions were stereotaxically injected as cell suspensions into the hippocampus of rats subjected to a transection of the intrinsic septo-hippocampal cholinergic pathways. At 17-19 weeks after transplantation, the various graft types differed considerably in their volume, the total amount of acetylcholinesterase (AChE)-positive fiber outgrowth, and the innervation pattern and morphology of the AChE-positive fibers growing into the host hippocampus. On average the NBM and spinal cord grafts had grown to become three to four times larger than the septal and the brainstem grafts, and 15-20 times larger than the striatal grafts. By contrast, the total ingrowth score of AChE-positive fibers in the host hippocampus from the septal grafts was about twice that of the NBM and brainstem grafts, about five times greater than the striatal grafts, and about six times greater than that of the spinal cord grafts. The large NBM grafts thus exhibited similar fiber outgrowth to the much smaller brainstem grafts, and the AChE-positive neurons of the grafted spinal cord grew very poorly into the hippocampus despite the fact that they survived very well. The innervation pattern and morphological features of the ingrowing AChE-positive fibers in the host hippocampus proper and in the dentate gyrus resembled those of normal rats in animals with grafts from any of the three forebrain regions (i.e., septum, NBM, or striatum), whereas ingrowth from the brainstem and spinal cord grafts were markedly abnormal with respect to both innervation pattern and fiber morphology. These results provide further evidence that the overall survival, growth, and fiber outgrowth of intracerebral neural grafts depend on interactions with the surrounding host tissue. Since the ability to reinnervate the previously denervated host target was greatest for the neuron type normally innervating that area, i.e., the septal-diagonal band neurons, we conclude that neuronal properties beyond the transmitter type are essential for the optimal performance of implanted neurons in intracerebral grafting experiments.

NGF effects on developing forebrain cholinergic neurons are regionally specific.

Nerve growth factor (NGF) has been shown to have an effect on neurons in the central nervous system (CNS). A number of observations suggest that NGF acts as a trophic factor for cholinergic neurons of the basal forebrain and the caudate-putamen. We sought to further characterize the CNS actions of NGF by examining its effect on choline acetyltransferase (ChAT) activity in the cell bodies and fibers of developing neurons of the septum and caudate-putamen. ChAT activity was increased after even a single NGF injection. Interestingly, the magnitude of the effect of multiple NGF injections suggested that repeated treatments may augment NGF actions on these neurons. The time-course of the response to NGF was followed after a single injection on postnatal day (PD) 2. NGF treatment produced long-lasting increases in ChAT activity in septum, hippocampus and caudate-putamen. The response in cell body regions (septum, caudate-putamen) was characterized by an initial lag period of approximately 24 hr, a rapid rise to maximum values, a plateau phase and a return to baseline. The response in hippocampus was delayed by 48 hr relative to that in septum, indicating that NGF actions on ChAT were first registered in septal cell bodies. Finally, developmental events were shown to have a regionally specific influence on the response of neurons to NGF. For though the septal response to a single NGF injection was undiminished well into the third postnatal week, little or no response was detected in caudate-putamen at that time. In highlighting the potency and regional specificity of NGF effects, these observations provide additional, support for the hypothesis that NGF is a trophic factor for CNS cholinergic neurons.

Nerve growth factor promotes development of the rat septo-hippocampal cholinergic projection in vitro.

There is now widespread evidence indicating that nerve growth factor (NGF) is involved in the function of central cholinergic neurons. Its possible role in the establishment of cholinergic fiber connectivities was studied in co-cultures of rat septum and hippocampus. Application of 100 ng/ml NGF greatly increased the number of acetylcholinesterase (AChE)-positive fibers which invaded the hippocampal slices, an effect which was accompanied by a more than 6-fold elevation of the two major cholinergic enzymes, choline acetyltransferase and AChE. In contrast, application of anti-NGF antiserum, but not a non-immune serum, reduced the number of AChE-positive fibers which grew into and remained within the hippocampal slices. Since no diffuse outgrowth of AChE-positive fibers from the septum was observed following application of NGF, these results suggest that NGF plays a role in the stabilization and long-term maintenance of the cholinergic septo-hippocampal projection in vitro.

Biochemical and functional evidence for nerve sprouting in the decentralized, hypertrophied rat urinary bladder.

The preganglionic nerves of the rat urinary bladder were cut. The decentralized bladders gained markedly in weight. Both one and four weeks post-operatively, transmural nerve stimulation evoked contractile responses of the muscle strips of the decentralized bladders, that were of the same magnitude as those of the control bladders. However, four weeks post-operatively, the atropine resistant fraction of the response was decreased; consequently, the atropine sensitive (cholinergic) fraction was increased. The activity of the acetylcholine synthesizing enzyme, choline acetyltransferase, was increased at the late time of observation. The present biochemical and functional findings may suggest outgrowth of branches from the decentralized post-ganglionic cholinergic nerves.

Is there regeneration of cholinergic axons through traumatic scar tissue in adult mammalian brain?

After a 2-mm coronal incision was made in the cerebral cortex of adult rats, cholinergic axonal reaction and growth were assessed by acetylcholinesterase histochemistry between 3 and 56 days postinjury. Early axonal reaction in the proximal stumps consisted of hypertrophy and terminal swellings, many with extended processes. Axonal fiber density rostral to the lesion remained constant throughout the observation period; caudally it was reduced but increased with time. Cholinergic fibers appeared to cross the lesion, although definitive tracing of individual fibers was not possible.

Prenatal development of nucleus basalis complex and related fiber systems in man: a histochemical study.

To provide parameters for study of the "cholinergic" innervation of a human fetal cerebrum, we have analyzed the prenatal development of histochemical reactivity in the nucleus basalis complex (a magnocellular complex known to contain a high concentration of cholinergic perikarya). Brains from fetuses and premature infants ranging between 8 and 35 weeks of gestation were frozen cut and processed by the thiocholine method for the demonstration of acetylcholinesterase activity. Since no consistent results were obtained with inhibitors on the material younger than 15 weeks, the histochemical reactivity for early stages was expressed as the total cholinesterase reactivity. The first sign of histochemical differentiation of the basal telencephalon is the appearance of a dark cholinesterase reactive "spot" situated between the developing lenticular nucleus and basal telencephalon surface as early as 9 weeks of gestation. The first cholinesterase reactive bundle connects this reactive area (nucleus basalis complex anlage) with the strongly reactive fiber system situated along the dorsal side of the optic tract. During the next "stage" (10.5 weeks), there is a significant increase in the size of the nucleus basalis complex and strongly cholinesterase reactive neuropil occupies the sublenticular, diagonal and septal areas. At this stage we have seen two new cholinesterase-reactive bundles: one well developed cholinesterase reactive fiber stratum approaching (but not penetrating) the neocortical anlage through the external capsule and another minute bundle running towards the medial limbic cortex through the precommissural septum. The supraoptic fiber system can be traced now to the pregeniculate area and the tegmentum. At 15 weeks, the first acetylcholinesterase reactive perikarya appear and the nucleus basalis complex anlage becomes segregated into several strongly reactive territories, corresponding in position to the medial septal, diagonal and basal nuclei as defined on adjacent Nissl stained sections. At this stage, fibers from the nucleus basalis complex enter the "white" matter of frontal, temporal, parietal and occipital parts of the cerebral hemisphere via the external capsule. Between 15 and 18 weeks, acetylcholinesterase fibers spread throughout the "white" matter of the cerebral hemisphere. In the next "stage" (18-22 weeks), strongly reactive fibers can be followed from the nucleus basalis below the putamen and through the external capsule to the transient, synapse-rich subplate zone of frontal, temporal, parietal and occipital cortices.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of electrical activity and trophic factors during cholinergic development in dissociated cultures.

The role of electrical activity in the developmental regulation of cholinergic neurons was investigated in dissociated spinal cord--dorsal root ganglion (SC-DRG) cultures. Application of tetrodotoxin (TTX) during the first 6 days after plating had no effect on choline acetyltransferase (CAT) activity. Suppression of electrical activity during the 7th day decreased CAT to 68% of control. These decreases in CAT activity were still apparent 2 weeks after removal of the TTX. GABAergic neurons, as indicated by glutamic acid decarboxylase activity and high affinity [3H]GABA uptake, were not affected by TTX treatment. Addition of 8-bromo-cAMP or conditioned medium obtained from SC-DRG cultures at certain developmental periods produced dose-dependent increases in CAT levels on TTX-treated cultures as compared with those treated with TTX alone. Similar studies with 8-bromo-cGMP revealed no significant effects on CAT activity. Vasoactive intestinal peptide (VIP) produced a dose-dependent increase in CAT activity when added to cultures between days 12 and 14. Similar studies conducted on younger cultures (days 5-7) or older cultures (days 21-23) revealed no increases in CAT activity. Addition of 0.1 nM VIP to TTX-treated cultures resulted in CAT levels which were not significantly different from those of electrically active controls. These data suggest that cyclic AMP, VIP, and trophic substances in conditioned medium may have roles in the mechanism of cholinergic toxicity produced by electrical blockade of developing spinal cord neurons.

Evidence for developmental synaptic regression of cholinergic afferents to the rat main olfactory bulb.

Choline acetyltransferase (ChAT) activity, acetylcholinesterase (AChE) activity and muscarinic cholinergic receptor binding were determined in homogenates of olfactory bulbs from rats killed at intervals from 4 days before through 60 days after birth. In addition, the localization of muscarinic receptors was determined using an in vitro autoradiographic technique in 6-millimicrons thick coronal sections of olfactory bulbs from rats killed at similar intervals after birth. All 3 cholinergic parameters were present in measurable quantities at birth and showed substantial increases between 1 and 20 days after birth. The most rapid increase in cholinergic parameters occurred between days 10 and 20 after birth. ChAT activity and muscarinic receptor binding decreased between days 20 and 35 and increased again between postnatal days 35 and 60. A similar developmental pattern was observed for autoradiographic grain density overlying the granule cell layer of the neonatal bulb. These data suggest that (1) centrifugal cholinergic afferents are present in the rat olfactory bulb at birth, (2) during the early postnatal period (between 10 and 20 days) synaptogenesis occurs resulting in an overproduction of cholinergic synapses and (3) between postnatal days 20 and 35, a period of synaptic reorganization occurs characterized by substantial regression.

Perinatal dietary exposure to soy lecithin: altered sensitivity to central cholinergic stimulation.

The effects of perinatal exposure to soy lecithin preparation (SLP) on the development of cholinergic responses in the rat brain were examined by assessing the ability of intracisternally administered carbachol to stimulate 33Pi incorporation into phospholipids in vivo, an effect of carbachol mediated by muscarinic cholinergic receptors. Maternal intake of SLP produced a suppression of the cholinergic response in the offspring, an effect which was specific in that basal (unstimulated) incorporation rates were not reduced (in fact, they eventually became elevated), nor was the response to another neurotransmitter (dopamine) compromised. The effect occurred early in the preweanling stage, a period in which SLP exposure also enhances development of cholinergic nerve terminals. These results suggest that SLP exposure has a major effect on cholinergic synaptic development and reactivity, followed by secondary changes in other neurotransmitter pathways and by more generalized effects on basal membrane phospholipid turnover.

Neonatal treatment with nerve growth factor antiserum eliminates cholinergic sympathetic innervation of rat sweat glands.

Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.

Low-molecular-weight peptide stimulates cholinergic development in ventral spinal cord cultures.

Skeletal muscle extract contains a previously undocumented 1300- to 1500-Da neurotrophic factor. Incubation of ventral spinal cord neurons in the presence of this factor enhances the rate of de novo acetylcholine synthesis two- to threefold over control cells, after 6 days in culture. This effect on cholinergic activity appears to be selective, since incubation with the factor results in only slight elevations of lactate dehydrogenase activity and DNA content, and no increase in the acetylcholinesterase activity. The 1300- to 1500-Da factor is acid-stable and partially sensitive to proteolysis by proteinase K, Staphylococcus aureus V8 protease, and subtilisin, but insensitive to trypsin. These results indicate that the active moiety is a peptide. The importance of peptides as neurotransmitters or neuromodulators is well accepted, but their role in the regulation of neuronal development is not widely appreciated. The present cholinergic neurotrophic peptide is distinct from previously characterized cholinergic trophic factors and represents the first example of a small, target-derived peptide which influences cholinergic development.

Development of sympathetic neurones in vivo: an investigation of the possible role of glucocorticosteroids in regulating transmitter type.

In vivo the majority of sympathetic neurones are adrenergic, whereas in vitro they can become cholinergic. This atypical cholinergic development can be suppressed by addition of a glucocorticoid to the culture medium (McLennan, Hill and Hendry, 1980). In this paper we have investigated the possibility that glucocorticoids influence the choice of sympathetic transmitter type in vivo. Reduction in circulatory glucocorticoids by adrenalectomy of 2-day-old rat pups did not result in increased cholinergic development in their superior cervical ganglia. Furthermore, administration of exogenous corticosterone to increase circulatory glucocorticoids in young rats did not prevent the developmental switch in transmitter type of the sympathetic innervation of the sweat gland from adrenergic to cholinergic. It is concluded that the developmental regulation of sympathetic transmitter type in vitro may not be analogous to that in vivo.

Postnatal changes in the distribution of acetylcholinesterase in kitten striate cortex.

We have traced the postnatal development of axons and cells in kitten striate cortex that contain acetylcholinesterase (AChE) by using a modification of Koelle's histochemical method. The maturation of AChE-positive axons was not found to be fully complete until at least 3 months of age, and was characterized by several distinct developmental trends. AChE-positive fibers in layers IVc-VI proliferate rapidly after birth until, by 4 weeks postnatal, they appear to exceed the adult density. They remain at this level as late as 8 weeks and then decrease to the adult density by 13 weeks. In contrast, the AChE-positive fibers in layer I do not show a substantial increase in density until 6 weeks of age and the adult level is not achieved before 3 months postnatal. Finally, the density of AChE-positive fibers in layers II and III appears to increase gradually from birth until the mature pattern is reached at about 6 weeks. AChE could also be localized histochemically to cell bodies whose position and appearance depended on postnatal age. Stained cells first appeared in the white matter subjacent to layer VI shortly after birth. By 2 weeks of age, most cells in layer VI were also AChE positive. The staining of these cells gradually disappears over the next 2 months until, at 3 months of age, there are no AChE-positive cells in cat striate cortex. However, a subpopulation of stained neurons appears in layer V by 1 year of age that persists throughout adulthood. The possible contributions of acetylcholine and AChE to the postnatal development of kitten striate cortex are discussed.

AChE-positive fiber growth after hippocampal fimbria transection and peripheral nerve homogenate implantation.

Gelfoam treated with peripheral nerve homogenate was implanted into a site of hippocampal fimbria transection in the adult rat to assess whether the homogenate might enhance growth of AChE-positive fibers into the lesion site and whether the fiber growth might be directed to the implants. Homogenate was prepared from intact peripheral nerve and in a few cases from degenerated nerve. Some implants were encased in Silastic. Homogenate was also injected into the denervated hippocampus. The major finding was that AChE-positive fiber growth was associated with regions of high astroglial cell content in preference to the relatively hypocellular implants. No clear differences in AChE fiber sprouting into the lesion site, fiber growth into implants, or hippocampal reinnervation were noted between homogenate and saline-treated animals.

Transplanted septal neurons make viable cholinergic synapses with a host hippocampus.

Cell suspensions from the fetal septal region were injected stereotaxically into the hippocampus of fornix-fimbria-transected adult rats. The host rats were sacrificed up to 3 months after the operation and the hippocampus sliced into 350 microns transverse slices. Intracellular recording was made from CA1 neurons adjacent to the graft. Electrical stimulation of the graft produced a voltage-dependent depolarization in some recorded neurons. This was associated with an increase in spontaneous and anodal break action potential discharges. In addition, a slow after-hyperpolarization (AHP) which typically follows a burst discharge was blocked during the depolarization indicating that the stimulation may block a Ca2+-dependent K+ current. The effects of the stimulation were antagonized by atropine. A response to the stimulation was seen 2 weeks but not 1 week after grafting. Over time, cells that were located away from the graft became activated by the stimulation. This was correlated with the extent of proliferation of acetylcholinesterase-containing fibers around the graft. These results suggest that grafted septal neurons make viable cholinergic connections with a host hippocampus.

[Development of the neural apparatus of blood vessels in the common frog in ontogenesis]. / Razvitie nervnogo apparata krovenosnykh sosudov travianoi liagushki v ontogeneze.

The neural apparatus of the aorta, abdominal vein, ischiatic, femoral, pulmonary and caudal vessels has been studied histochemically in tadpoles (the 30th-50th stages of development) and in 1-year-old animals. It has been stated for the first time that in the frog, a representative of the Amphibia class, like in mammals and birds, formation of the adrenergic apparatus in various vessels does not take place simultaneously. For instance, the first adrenergic fibers in the hind limb vessels appear much earlier than in other arteries and veins. The process of the adrenergic innervation development and its completion in vessels of various areas is taking its course differently. In the aorta and in the abdominal vein the formation of the adrenergic plexus develops as increasing density and amount of the mediator in the adrenergic fibers and is completed with maturation in an adult animal. In contrast to these vessels, maturation of the adrenergic apparatus in the hind limb arteries and veins takes place during a shorter interval and is completed at the end of metamorphosis AchE-containing fibers are revealed in tadpoles, as well as in a mature frog only in the aortal arc and in the pulmonary artery. In these vessels the development of cholinergic innervation leaves behind that of the adrenergic innervation, as it does in the vessels of Mammalia, and the human subject.

Development of cholinergic sympathetic neurons: evidence for transmitter plasticity in vivo.

Most principal neurons in sympathetic ganglia are noradrenergic. A small population, especially those that innervate sweat glands in rat footpads, are cholinergic. We have characterized the innervation of the glands in adult and developing rats to determine whether sympathetic neurons undergo a transition from noradrenergic to cholinergic during normal development as has been observed in culture. In adult rats, the fibers innervating sweat glands exhibited strong acetylcholinesterase (AChE) staining and vasoactive intestinal peptide (VIP)-like immunoreactivity. None of the axons contained endogenous catecholamines detectable with formaldehyde-induced fluorescence or permanganate fixation. However, like cholinergic sympathetic neurons in culture, all axons could take up and store exogenous catecholamine. The sweat glands and their innervation develop postnatally. At 7 days, the axons innervating sweat glands possessed endogenous catecholamine histofluorescence and small granular vesicles but not AChE or VIP. By 14 days, AChE and VIP staining was pronounced. In contrast, catecholamine fluorescence and the number of small granular vesicles were reduced, and by 21 days they were absent. Further, neonatal treatment with 6-hydroxydopamine, a toxic norepinephrine congener, resulted in the loss of cholinergic as well as noradrenergic sympathetic innervation. These observations are consistent with a transition from noradrenergic to cholinergic function in vivo.

A histochemical study of the cholinergic and adrenergic innervation of the developing teeth and oral tissues in the mouse.

The dental follicle and papilla are innervated at different stages of tooth development. The type of nerves innervating these structures at different stages was investigated using an enzymic method for acetylcholinesterase (AChE) and direct visualization for noradrenaline on fetal and neonatal stages up to 7 days after birth. Glandular and muscular tissue were positive for AChE in 17-day fetuses but no reaction was observed in the teeth or supporting tissues up to 7 days after birth. Noradrenaline was detected in blood vessels at 18 days and in glandular tissue at birth but was not observed in the teeth within the period covered. It is unlikely that AChE plays any role in the innervation of teeth; the adrenergic nerve supply probably develops later.