K. Elisevich

Biotinylated dextran : a versatile anterograde and retrograde neuronal tracer

A powerful and versatile axonal tracing method using biotinylated dextran, a novel analogue of biotin, is described. Pressure injection of varying volumes of 5% biotinylated dextran into various parts of the brain and spinal cord resulted in Golgi-like retrograde labeling and PHA-L-like anterograde labeling. The tracer filled the finest processes, revealing terminal axonal ramifications, distal dendrites and dendritic spines and excrescences. Extensive anterograde and retrograde labeling occurred in all pathways studied and in animals of all ages. Labeling appeared as early as 48 h and remained unchanged up to 14 days following injection. Biotinylated dextran can be detected easily with any avidin-conjugated marker for light and electron microscopic study. The resultant labeling can be combined readily with other morphological methods, such as tract tracing and/or immunocytochemical demonstration of endogenous substances. Biotinylated dextran is thus an efficient anterograde and retrograde tracer that can be combined with other neuroanatomical techniques to study details of synaptic interaction at all levels of dendritic organization.

The pallidostriatal projection in the rat: a recurrent inhibitory loop?

The pallidostriatal projection in the rat was investigated employing the PHA-L tracing technique. Following inotophoretic injections into the lateral aspect of the globus pallidus external segment, the ipsilateral striatum showed patches of dense anterograde labeling separated by areas containing sparse anterograde labeling and isolated retrogradely labeled neurons. The densely labeled patches did not correspond to any known compartments of the striatum. The retrogradely labeled neurons consistently showed similar distribution and morphological features reminiscent of striatal type II projection neurons. As all projection neurons of the striatum and all pallidal neurons are GABAergic, the complementary pattern of anterogradely and retrogradely labeled profiles from the globus pallidus suggest a possible mechanism whereby a horizontal inhibition may be exerted on groups of striatal neurons via the striato-pallido-striatal pathway.

GABAergic synaptic interactions in the substantia nigra

The substantia nigra receives a strong GABAergic input from the ipsilateral striatum and globus pallidus. Nigral GABAergic synaptic interactions have been described in the pars compacta (SNC) and pars reticulata (SNR) but not in the pars lateralis (SNL). The SNR and particularly the SNL are the nodal points of the GABAergic nigrotectal pathway. The present study analyzes the synaptic connections of GABAergic and dopaminergic neurons in each of the divisions of the substantia nigra by employing a double-labeling immunocytochemical technique at the light and electron microscope levels. Glutamic acid decarboxylase (GAD)-containing terminals make symmetrical synaptic contacts with dopaminergic neurons in the SNC and SNR. Neurons that contain GAD also receive a GABAergic input in the SNR and SNL. The proportion of GAD-GAD contacts appears to be highest in the SNL where virtually all GAD-positive terminals are found to be in synaptic contact with or apposed to GAD positive profiles. This study demonstrates a strong GABAergic input onto nigral dopaminergic neurons and GABAergic neurons in the SNR and SNL. This GABAergic influence which is ostensibly striatal or pallidal in origin is particularly prominent in relation to the SNL-mediated nigro-collicular pathway.

Normalization of Striatal Proenkephalin and Preprotachykinin mRNA Expression by Fetal Substantia Nigra Grafts

The expression of striatal proenkephalin and preprotachykinin mRNA was studied in Wistar rats following unilateral injections of 6-hydroxydopamine into the nigrostriatal pathway and after grafting dopaminergic fetal cell suspensions into the dopamine-depleted striatum. Striatal dopaminergic deafferentation resulted in an amphetamine-induced rotational asymmetry, an increase in ipsilateral striatal proenkephalin mRNA, and a decrease in preprotachykinin mRNA expression. Twelve months following grafting, proenkephalin and preprotachykinin mRNA returned to near-normal levels in contrast to control nongrafted lesioned animals. In addition, reversal of the rotational asymmetry was routinely observed. This study has demonstrated long-term graft-induced functional recovery coincident with normalization of striatal proenkephalin and preprotachykinin mRNA expression.

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.

Cerebellar, medullary and spinal afferent connections of the paramedian reticular nucleus in the cat

The topographic organization of afferent projections from the deep cerebellar nuclei, medulla oblongata and spinal cord to the paramedian reticular nucleus (PRN) of the cat was studied using the horseradish peroxidase (HRP) method of retrograde labelling. Discrete placements of HRP within each of the dorsal (dPRN) and ventral (vPRN) regions of the PRN showed some segregation of input. The deep cerebellar nuclei project in a predominantly contralateral fashion upon the PRN. A small but significant ipsilateral fastigial afferent component is also present. The fastigial and dentate nuclei contribute the majority of fibers to the dPRN whereas the interposed nucleus provides very little. The vPRN receives a relatively uniform input from all 3 cerebellar nuclei. Both lateral vestibular nuclei contribute the majority of fibers from the vestibular nuclear complex largely from their dorsal division. Additional input arises from bilateral medial and inferior vestibular nuclei. The vPRN receives relatively more fibers from the inferior vestibular nuclei than does the dPRN while inputs from the medial vestibular nuclei are comparably sparse. The PRN receives bilateral projections from the nucleus intercalatus (of Staderini). A significant projection to the contralateral PRN occurs from the ventrolateral subnucleus of the solitary complex and its immediate vicinity. Additional sources of medullary afferent input include the lateral, gigantocellular and magnocellular tegmental fields, the contralateral PRN and the raphe nuclei. Sites of origin of spinal afferents to the dPRN are bilaterally distributed mainly within Rexeds laminae VII and VIII of the cervical cord whereas those to the vPRN are confined largely to the medial portion of the contralateral lamina VI in the C1 segment. A few labelled cells are found in the thoracolumbar cord with those to the vPRN being more caudal. These data provide the neuroanatomical substrate for a better understanding of the functional role of the PRN in mediating cardiovascular responses appropriate to postural changes.

Cardiovascular afferent and fastigial nucleus inputs to paramedian reticulospinal neurons

In chloralose anesthetized, paralyzed and artificially ventilated cats, the region of the paramedian reticular nucleus (PRN) was systematically explored for single units antidromically activated by electrical stimulation of histologically verified sites in the intermediate gray region of the upper thoracic cord (T2). These antidromically identified units were then tested for their orthodromic responses to electrical stimulation of ipsilateral carotid sinus nerve (CSN) and of pressor sites in the contralateral fastigial nucleus (FN). Sixty-two histologically verified single units, located predominantly in the caudal half of the ventral PRN, were antidromically activated with latencies corresponding to a mean conduction velocity of 36.4 +/- 2.1 m/s. Of these units 25 (40%) were excited orthodromically by stimulation of the CSN and/or FN: 5 to stimulation of the CSN only (mean latency, 18.3 +/- 9.9 ms), 6 to stimulation of the FN only (mean latency, 7 +/- 1.7 ms), and 14 to stimulation of both the CSN and FN (mean latencies, 12.3 +/- 2.9 ms and 8.4 +/- 1 ms, respectively). These data provide electrophysiological evidence for the existence of PRN reticulo-spinal neurons that integrate and relay cardiovascular afferent information from the CSN and FN to spinal autonomic neurons.

Axonal branching in the projections from the paramedian reticular nucleus to the cerebellar cortex

Injections of fluorescent tracers into cat cerebellar cortex gave evidence of collateral axonal branching of neurons situated in the paramedian reticular nucleus. These branched reticulocerebellar projections were distributed to opposing sides of the cerebellum, in particular the anterior lobe and the ansiform lobule. No topographical organization was observed in the PRN. Less than 30% of ipsilaterally projecting reticulocerebellar fibers had contralaterally directed collateral branches. These results are in keeping with a bilateral fastigial projection to the PRN forming a feedback loop circuit through which orthostatic reflexes may be mediated.

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons.

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T4 or T7 levels centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T2 level were observed throughout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.

Vestibular nucleus inputs to paramedian reticulospinal neurons in the cat

Experiments were done in chloralose-anesthetized cats to identify single units in the paramedian reticular nucleus (PRN) that responded to stimulation of pressor sites in the vestibular nucleus complex (VNC) and that projected directly to the intermediate gray (IG) region of the upper thoracic cord. Forty-seven units responded orthodromically to stimulation of the ipsilateral VNC with a mean latency of 6.3 +/- 0.6 ms: 44 were excited and 3 were inhibited. Of these 47 units, 29 (62%) also were antidromically activated by stimulation of the ipsilateral IG at the level of T2. These data provide electrophysiological evidence for the existence of neurons in PRN that receive VNC inputs and project directly to spinal autonomic areas, and suggest that this pathway may be involved in mediating vestibulosympathetic reflex responses associated with postural adjustments.