Some neurons of the rat central nervous system contain aromatic-L-amino-acid decarboxylase but not monoamines.

Neurons containing the enzyme aromatic-L-amino-acid decarboxylase (AADC) but lacking either tyrosine hydroxylase or serotonin were found in the spinal cord of neonatal and adult rats by light and electron microscopic immunocytochemistry. The majority of these neurons localized to area X of Rexed contact ependyma. Thus, spinal AADC neurons have the enzymatic capacity to catalyze directly the conversion of the amino acids tyrosine, tryptophan, or phenylalanine to their respective amines tyramine, tryptamine, or phenylethylamine. These amines normally present in the central nervous system may be of potential clinical significance as endogenous psychotomimetics.

Cytoarchitectonics of substantia nigra grafts: a light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes.

Maturation of dopaminergic (DA) neurons and astroglia was studied in transplants of the substantia nigra grown for up to 7 months in the brain of rats. The investigation had three specific aims. The first was to observe effects of different transplant positions on the longevity of DA neurons. Second, the grafts were examined for changes of synaptic interactions and associations between DA neurons and astroglia. Third, an answer was sought to the question whether transplanted DA neurons migrate into the adjacent host brain. The grafts were taken from the ventral mesencephalon of rat embryos of different ages (day 14 to 18 of gestation) and placed into the cerebral cortex, tectum, cerebellum, or ventricles of newborn host animals. Following different times of survival the immunocytochemical localization of tyrosine hydroxylase (TH) and of glia filament protein (GFA) in the transplants were observed. In all of the transplantation sites, except for one, neurons of different morphologies that contained TH were found in the grafts. The cerebellar white matter of the host brain failed to support the long-term survival of DA neurons. The overall structure of mature substantia nigra grafts had some resemblance to intact substantia nigra (SN). On the ultrastructural level, it was found that morphological expression of some immature features of DA neurons, such as glial sheaths, somatic spines, and lack of oligodendroglia, persisted in mature grafts. Specific associations of DA neurons and astroglia in the grafts suggested that the cytoarchitectonic appearance of a given brain region may be related to the existence of particular neuron glia relationships. In contrast to intact SN, transplants revealed deficiencies in unlabeled pleomorphic boutons and contained some TH-immunoreactive terminals. Migration of DA neurons and their processes into the adjacent host brain was rarely observed.

Transplantation of embryonic occipital cortex to the brain of newborn rats: a Golgi study of mature and developing transplants.

Segments of the occipital cortex were taken from rat embryos (E16-E19) and transplanted to the cerebral cortex or the tectal region of a newborn rat host. With the aid of Golgi impregnation techniques, neuron morphology was studied in cortical transplants which had survived for 1 week or more in the host brain. In mature transplants (greater than 4 weeks) three main groups of neurons, termed groups I-III, were identified. Group I neurons resembled pyramidal neurons of the intact cerebral cortex. No preferential orientation of either soma or dendrites of group I neurons was observed in the transplants, and some group I neurons had curved apical dendrites. Group II neurons had predominantly stellate form and their dendrites were densely covered with spines. Paucity or absence of dendritic spines characterized group III neurons which exhibited various dendritic topologies. Different neuron types were also recognized in immature transplants growing for 1 and 2 weeks in the host brain. The sequence of dendritic maturation of transplanted cortical neurons is similar to that seen in intact cortex, although the stage reached related more to the actual age of the transplant than to that of the host. Thus, group I neurons in the 1-week-old transplants taken from E16 embryos had not attained the same complexity of branching as pyramidal neurons in the surrounding host cortex, but rather resembled slightly younger cells more like those found in the cerebral cortex of the newborn rat. These results show, therefore, that at least the basic cell classes identified in intact visual cortex can also be recognized in the cortical transplants. This will provide a foundation for studies defining which cells project to the host brain and which are involved in particular intrinsic connections.

Transplantation of embryonic occipital cortex to the tectal region of newborn rats: a light microscopic study of organization and connectivity of the transplants.

Occipital cortex was taken from fetal rats and transplanted to the tectal region of newborn rats, where it developed a specific structural identity reflecting in part its cortical origin. The implants showed locally distributing intratransplant connections, and the majority formed connections with defined regions of te host cerebral cortex and the brainstem. A sparse afferent projection from the host had its origin in visual, somatosensory, and cingulate areas of te cortex, pretectum, superior colliculus, central gray, hypothalamus, pontine reticular formation, raphe nuclei, and the locus coeruleus. No input was identified from either the retina or the dorsal thalamus. Efferent fibers were observed in normal fiber preparations as compact bundles running through the host brainstem along two main routes, one group of bundles in a dorsal position and a second group more ventral. Efferent fibers traveling rostrally along the first pathway distributed in the lower part of the stratum griseum superficiale and in the intermediate laminae of the superior colliculus, and in some cases they reached the pretectum and the lateral posterior thalamic nucleus. Deep efferent fibers ran rostrally and caudally in the central gray, and in some cases laterally directed fibers were seen to distribute in the midbrain tegmentum and reticular formation, in one case reaching the pontine gray. The finding that most afferent and many efferent connections of cortical transplants are uncharacteristic of normal cortex stands in marked contrast to retina and tectum, which, when transplanted to the same region, make relatively normal connections.

The effect of forebrain lesions in the neonatal rat: survival of midbrain dopaminergic neurons and the crossed nigrostriatal projection.

Lesions were placed in the striatum and the olfactory tubercle of 1-day-old rat pups. Control and experimental animals were raised to adulthood. Efferent projections of mesencephalic neurons were examined by injecting the retrograde tracers horseradish peroxidase or Fast Blue into the undamaged striata of some experimental animals. The survival of the mesencephalic dopaminergic neurons was monitored by using immunocytochemical localization of tyrosine hydroxylase. Small lesions in the caudate-putamen had no appreciable effect on the survival of tyrosine hydroxylase-containing neurons in the mesencephalon, but the density of dopaminergic terminals adjacent to the lesion increased in the remaining caudate-putamen. Striatal lesions that involved an estimated area of more than one-third resulted in loss of dopamine neurons of the substantia nigra compacta. Rostral lesions in the striatum affected mostly rostrally positioned neurons in the substantia nigra. Dorsal lesions of the caudate-putamen resulted in disappearance of dorsal A9 neurons. Reduction of the A10 and A8 dopamine neuron groups occurred if the neonatal lesions involved the olfactory tubercle and nucleus accumbens. Some tyrosine hydroxylase-containing neurons persisted even after the largest lesions. These dopaminergic neurons formed a crossed nigrostriatal pathway which was confirmed by retrogradely transported tracers. The density of this crossed projection in the adult appeared unaffected by the neonatal lesion. We concluded that dopaminergic neurons form topographically ordered projections with their targets in the newborn rat. Rearrangement of these fibers appeared limited, but compensatory increase of axon terminal density was evident in partially lesioned target areas.

Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization during prenatal development.

Immunocytochemically labeled cells containing the enzyme aromatic L-amino acid decarboxylase were localized in the brain of rat embryos at gestational age E15-E19. Cell groups that contained aromatic L-amino acid decarboxylase but lacked either the enzyme tyrosine hydroxylase or the indolamine serotonin were referred to as "D" groups. Anatomical landmarks, cytoarchitectonic structure and histochemical staining for acetylcholinesterase were used to delineate the position of "D" groups. In the E15 embryo three "D" groups existed. The first to appear, named D1, was located in the spinal cord and had been demonstrated before. A large "D" cell cluster was found in the walls of the central forebrain deep to the hypothalamic sulcus. This group distributed dorsally in the ventral dorsal thalamic region and ventrally in the dorsal hypothalamus. The rostral-most "D" group, D14, occurred in the ventral telencephalon just medial to fibers of the nigrostriatal projection. D14 was the smallest of the early groups. In E16 and E17 embryos dorsal di- and mesencephalic "D" groups were first detected. During the course of ontogeny a considerable increase of immunoreactive cells occurred and segregation of the large central forebrain cluster into several rostrally and laterally distributed "D" groups took place. Some "D" groups that occur in the adult brain were not present in the E19 embryo. This study provides a first report of the localization of several unique cell groups in the brain of rat embryos and their appearance at different stages of gestation. It also gives further support to the notion that variations of aromatic L-amino acid decarboxylase staining intensities may be characteristic of different monoamine neurons.

Influence of grafted glia cells and host mossy fibers on anomalously migrated host granule cells surviving in cortical transplants.

Embryonic cerebral cortex was transplanted over the cerebellum of one- to two-day-old rats. In mature rats, clusters of granule cells that failed to migrate into the internal granule cell layer were now found within the graft tissue. Immunocytochemical staining of astrocytic glial cells in the cortical transplants revealed that glial processes were distributed in an unusual polarized orientation in those regions that contained host granule cells. Other areas of the graft exhibited glia cells with processes that projected radially from their cell body, thus resembling fibrous astrocytes. Fibrous astrocytes in transplants, however, were more heavily stained than similar glial cells in the intact cerebral cortex of the host, indicating a quantitative difference in the glia fibrillary acidic protein. Acetylcholinesterase-positive fibers were observed between the clusters of granule cells in the cortical grafts. Such fibers traversed the molecular layer and, since they could be traced from the white matter of the host cerebellum, they were presumed to be mossy fibers. It is concluded that migration of external granule cells in the cerebellum can altered by placing embryonic cerebral cortex next to the developing cerebellum. Granule cells that have migrated into the grafted cerebral cortical tissue nevertheless receive afferent fibres.

Electrophysiology of the mammalian cerebellar cortex in organ culture.

A direct comparison was made between the electrical properties of rat Purkinje cells in cerebellar organotype cultures and those in acute slices from age-matched animals. Cultures were prepared from 9-11-day-old animals. Intracellular recordings were made 5-12 days later, at which time the folia architecture of the cerebellum was still well preserved. The resting membrane potentials and input resistances of Purkinje cells in cultured and acute slice preparations from young animals were comparable to those of mature Purkinje cells in slices. Neurons from animals younger than 14 days differed from mature Purkinje cells in that they fired at low frequencies in response to outward current pulses. The latter property was found in all cultured neurons studied, independent of their time in culture. These action potentials were generated by Na+ and Ca2+ conductances as shown by the application of selective channel blockers. Cultured or acute slice preparations from animals younger than 11 days shared other immature electroresponsive features. In both groups, Na+-dependent plateau depolarizations were observed in less than 10% of Purkinje cells unless K-conductances were blocked, and considerable membrane depolarization was often required to elicit Ca2+-dependent action potentials. These findings are compatible with the relative prominence of voltage-dependent outward currents in immature Purkinje cells, a property which may be enhanced in culture. The injection of hyperpolarizing current pulses revealed a marked time-dependent anomalous rectification in all Purkinje cells. At the breaks of such pulses, several events were observed. In all cells, a rebound conductance was identified which could generate post-anodal spike bursts. In cultured neurons, however, hyperpolarizing pulses were also followed by a slow return to resting potential. This membrane potential profile was similar to that produced by the activation of an A conductance. Experiments on acute slices from animals of different ages (P9-P17) showed that this A-like conductance was expressed only during a brief period in Purkinje cell development. A higher level of spontaneous synaptic activity was observed in cultured than in acute slice preparations. Both unitary excitatory postsynaptic potentials and inhibitory postsynaptic potentials could be elicited in the former by parallel fiber stimulation, and could be fully reversed by outward or inward transmembrane current injections, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytology and organization of rat cerebellar organ cultures.

Roller tube cultures of parasagittal cerebellar slices were taken from young rats aged 9-11 days, and maintained in vitro for 1-2 weeks. Morphological aspects of cell types and synaptic relationships in such organ cultures were examined at light and electron microscopic levels. Some neurons were marked by intracellular injections of horseradish peroxidase for subsequent identification of their connection patterns. Cytoarchitecture of the cerebellar cortex was largely preserved in the organ cultures. Dendritic trees of Purkinje cells exhibited isoplanar organizations that often resembled their orientation at the time of explanation. Other cerebellar neurons, namely granule cells, Golgi cells, basket cells, stellate cells, all differentiated within the organ cultures. In addition, some neurons of the deep cerebellar nuclei remained viable during the period of culture. Mossy fibers most probably of cerebellar nuclear origin were found terminating on the dendrites of granule cells and Golgi cells. Quite unexpected were certain types of direct synapses of afferent fibers on short necked spines arising from Purkinje cell smooth dendrites and somata. Such terminals resembled climbing fibers. They were most likely modified mossy fiber afferents, since the organ cultures did not include neurons of the inferior olive which are well spearated from the cerebellar mass at postnatal stages. These "ascending" mossy fibers presumably occupied postsynaptic surfaces that were either vacated by deafferentation or induced by the afferent fibers themselves. Intracellularly labeled Purkinje cells had widely distributed axonal collateral branches. Labeled axons were distributed within the Purkinje cell layer. Several recurrent Purkinje cell axon collaterals stained with reaction products of horseradish peroxidase tracer were followed at the ultrastructural level. In one case, labeled terminals were examined in an area of approximately 2 mm2. Terminals of Purkinje cell collaterals formed symmetric synapses with somata of basket cells and dendrites of Golgi cells, but not Purkinje cell somata. Some large boutons of serially traced Purkinje cell axon collaterals formed asymmetric contacts with profiles interpreted as Golgi cell dendrites. In contrast to the apparent axonal sprouting in cerebellar organ cultures, maturation of dendritic processes remained static. Astroglia cells of diverse shapes were observed following immunocytochemical staining with antisera to glia filament proteins. The distribution patterns of immunoreactive astrocytes changed dramatically in cerebellar slice cultures maintained for 3-6 weeks in vitro.

Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia.

We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models. Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients. A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma. Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum. Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation. Transplants were examined at three, four, six, and eight weeks after surgery. Some of the donor tissue was grown in vitro, in order to determine its cellular composition. These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy. At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system. They had well-defined boundaries. Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts. Likewise, macrophages were located in areas abutting the graft. Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels. Grafts grown for at least eight weeks were characterized by interdigitating boundaries. Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord. Such transplants also had reactive astrocytes and ED1-positive macrophages. At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated. We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia. Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats.

Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization in neurons of the brain stem.

Neurons containing the enzyme aromatic L-amino acid decarboxylase were immunocytochemically localized in the brain stem of the rat. The enzyme occurred as expected in previously well characterized monoaminergic cell groups, and in addition in some nuclei with unknown neurotransmitters. Major aggregates of neurons that were immunoreactive for aromatic L-amino acid decarboxylase but contained neither tyrosine hydroxylase nor serotonin, were found in the pretectal nuclei, the lateral parabrachial nucleus, and the dorsolateral subdivision of the nucleus tractus solitarius. Aromatic L-amino acid decarboxylase was also present in serotonin neurons and the majority of catecholamine cell groups. Dopamine, noradrenaline, and adrenaline cells exhibited characteristic staining intensities to anti-aromatic L-amino acid decarboxylase reflective of relative enzyme levels in the different groups. Some cells in the dorsal motor nucleus of the vagus that were previously classified as dopaminergic lacked immunoreactivity to aromatic L-amino acid decarboxylase.

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymer-encapsulated PC12 cells: long-term survival and associated reduction in lesion-induced rotational behavior.

Intrastriatal implantation of a dopaminergic cell line surrounded by a permeable, thermoplastic membrane was investigated as a method of long-term dopamine (DA) delivery within the central nervous system (CNS). An increase in DA release from PC12 cell-loaded capsules maintained in vitro was associated with an increase in mitotic activity of the encapsulated cell line. A significant reduction in apomorphine-induced rotational behavior was observed after PC12 cell-containing capsules were implanted into unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, which was sustained for 24 wk. Four wk after implantation, microdialysis studies revealed the presence of DA near PC12 cell-containing capsules, which was comparable to extracellular striatal levels of unlesioned controls. Extracellular striatal DA was undetectable by microdialysis in lesioned animals near empty polymer capsules. Histological analysis after 24 wk in vivo demonstrated that encapsulated PC12 cells survived, continued to express tyrosine hydroxylase, and that encapsulation prevented tumorigenesis. The data suggested that the release of a diffusible substance, most likely DA, from an implant is sufficient to exert a long-term functional influence upon 6-OHDA unilaterally lesioned rats and that capsules containing DA-secreting cells may be an effective method of long-term DA delivery in the CNS.

Axon terminal clustering in nigrostriatal double grafts.

Embryonic dopaminergic terminals of the ventral mesencephalon were cotransplanted with other immature neural tissues to the brain of newborn rats. Immunocytochemical localization of tyrosine hydroxylase in mature cografts with light and electron microscopy, demonstrated the existence of different axon terminal distributions in different grafts. These findings implicate the influence of local cell interactions in determining axon arborization.

Transient expression of tyrosine hydroxylase in some neurons of the developing inferior colliculus of the rat.

Using immunocytochemistry, we detected tyrosine hydroxylase (TH)-containing cells in the developing inferior colliculus. These cells lacked other catecholamine-synthesizing enzymes and they occurred in newborn and immature rats up to 21 days of age. TH-positive cells of the inferior colliculus were contacted by synapses that contained pleomorphic vesicles.

Neo-pathway formation of dissociated neural grafts demonstrated by immunocytochemistry, fluorescent microspheres, and retrograde transport.

Embryonic neural tissues were taken from the ventral mesencephalon, the striatal anlage, the cerebellum, or the dorsal telencephalon. Prior to grafting these tissues were dissociated. This provided the opportunity to generate experimental cografts and allowed the introduction of neuronal tracers. Projections from grafts that contained dopaminergic neurons of the mesencephalon were identified immunocytochemically with antisera to tyrosine hydroxylase. Acetylcholinesterase histochemistry was employed to both stain the graft and to visualize graft efferent fibers. Moreover, dissociated tissues, with the exception of the ventral mesencephalon, were treated in vitro with rhodamine-conjugated microspheres. The immature cells incorporated the tracer in vitro. Labeled grafts were placed adjacent to large fiber tracts of the recipient brain or into the lateral ventricle and grown for one to two months. Different types of neurons within the grafts retained the rhodamine tracer. Some of the labeled neurons were positive for acetylcholinesterase. Efferent fibers from the different neural grafts followed similar routes within the host brain. Fibers containing microspheres projected commonly along the nearest host pathway or the labeled fibers followed the reactive glia along the original stab wound. Grafts that were located within the lateral ventricle projected their efferent fibers adjacent to the ventricular surface. Similar routes were followed by efferent fibers from transplanted dopaminergic neurons. One of the projection routes along the hosts corpus callosum was confirmed by horseradish peroxidase tracer transport. We conclude that elongation of graft pathways may occur along existing fiber tracts of the host brain, near structures of the ventricular surface, and alongside glia scars of a graft placement tract.

Repair of the blood-brain barrier following implantation of polymer capsules.

Past studies of polymer-encapsulated cell lines implanted in the brain indicated their usefulness for transmitter replacement therapy in animal models. Such grafts may have potentially important clinical applications, but their placement into neural parenchyma may cause a traumatic injury resulting in a leaky blood-brain barrier around the implant. This study investigated whether or not injury repair and reformation of the barrier takes place near a polymer capsule implanted in the brain of Sprague-Dawley rats. The two methods used for detection of a leaky barrier were immunocytochemical localization of extravasated serum albumin and circulating Evans blue that binds to serum albumin. Immunocytochemical staining for glial filament protein provided a measure for evaluating injury associated gliosis. Polymer capsules implanted for 10, 16 and 18 days were surrounded by microvessels that leaked detectable quantities of serum albumin into interstitial spaces and, by secondary uptake, into some nearby neurons and reactive astrocytes. Reactive astroglia were observed within the outer regions of the capsule wall and in the near vicinity of the implant after these early survival times. In contrast, at post-implantation times of 46 and 54 days, serum albumin was no longer detected in the neural parenchyma near the macrocapsules and only few reactive astrocytes remained. These findings show that polymer capsules implanted within the cerebrum permit (a) reformation of the blood-brain barrier and (b) occurrence of repair processes that lead to minimal deposition of reactive astroglia near the implanted polymer capsule.

Aromatic L-amino acid decarboxylase in the rat brain: coexistence with vasopressin in small neurons of the suprachiasmatic nucleus.

We demonstrated the coexistence of aromatic L-amino acid decarboxylase (AADC) and arginine-vasopressin in neurons of the hypothalamic suprachiasmatic nucleus of Sprague-Dawley rats. Neurons that lacked monoamines but expressed immunoreactivity to the enzyme AADC occupied the rostral and caudal poles of the suprachiasmatic nucleus and mediodorsal and dorsolateral positions along the entire extent of the nucleus. AADC was also localized in similar neurons of the suprachiasmatic nucleus of rats from other strains including the homozygous Brattleboro rat.

Hypothalamic projections of medullary catecholamine neurons in the rabbit: a combined catecholamine fluorescence and HRP transport study.

Injections of horseradish peroxidase into the rabbit hypothalamus, centered on the paraventricular nucleus, result in retrograde transport to A1 and A2 neurons in the medulla oblongata, in a bilateral distribution with ipsilateral predominance. Some HRP labeled cells in both A1 and A2 areas do not exhibit a positive histochemical reaction for catecholamines, but, in the A1 area, nearly all HRP-positive neurons are catecholamine-containing A1 cells. Injection of radioisotope into the A1 area of the rabbit results in terminal labeling within the paraventricular and supraoptic nuclei. This study complements previous work showing that rabbit A1 and A2 cells have minimal projections to the spinal cord.