H.T. Chang

Interneurons in the rat striatum: relationships between parvalbumin neurons and cholinergic neurons

A pre-embedding double-labeling immunocytochemical method was used to examine the synaptic relationships between cholinergic neurons and the parvalbumin immunoreactive (PV+) neurons. The PV+ neurons were labeled by silver-intensified colloidal gold particles, and the cholinergic neurons by immunoperoxidase reaction products. Cholinergic and PV+ axon terminals form synapses with both the somata and dendrites of PV+ neurons, as well as unlabeled medium-sized somata with round un-indented nuclei, a typical characteristic of the medium spiny projection neurons. These observations suggest that the PV+ and the cholinergic neurons have converging influences on both the projection neurons and the PV+ interneurons in the striatum.

Substance P-dopamine relationship in the rat substantia nigra: a light and electron microscopy study of double immunocytochemically labeled materials

The synaptic relationships between substance P-containing terminals and dopaminergic neurons (immunoreactive for tyrosine hydroxylase) in the substantia nigra were studied at both light and electron microscopic levels using a pre-embedding double-labeling immunocytochemical method. Many substance P-containing terminals were found to form synapses directly with dendrites and somata of nigral dopaminergic neurons. Since most of the substance P-containing axon terminals arise from the striatum, this result suggests that striatal substance P neurons can have monosynaptic influence on nigral dopaminergic neurons.

Enkephalinergic-cholinergic interaction in the rat globus pallidus: a pre-embedding double-labeling immunocytochemistry study

The synaptic relationships between leucine-enkephalin containing axon terminals and cholinergic neurons in the rat globus pallidus were studied at both light and electron microscopic levels using a high resolution pre-embedding double-labeling immunocytochemical method. Results indicated that leucine-enkephalin terminals very rarely form monosynaptic connections with cholinergic neurons in the rat globus pallidus, suggesting that enkephalinergic neostriatal efferents probably have little monosynaptic influences on the activities of pallidal cholinergic neurons.

Noradrenergic innervation of the substantia innominata: A light and electron microscopic analysis of dopamine β-hydroxylase immunoreactive elements in the rat

Noradrenergic input to the rat substantia innominata (SI) was analyzed in this study by immunocytochemical localization of dopamine beta-hydroxylase (DBH), the synthetic enzyme for noradrenaline. DBH immunoreactive (DBH+) axons ramified extensively within the SI and appeared to be contiguous with the DBH+ terminal fields within the bed nucleus of stria terminalis and the amygdaloid complex. DBH+ axons in the SI exhibited many large boutons en passant and boutons terminaux. These DBH+ boutons appeared much larger than those in the cerebral cortex, hippocampus, and thalamus. Electron microscopic analysis revealed that DBH+ boutons formed asymmetrical synapses with mainly dendrites, but also somata and spines of SI neurons. Dendrites which were postsynaptic to DBH+ boutons also formed synapses with many other unlabeled axon terminals. Since previous studies have shown that dendrites of SI cholinergic neurons formed few synapses, the present result suggests that the noradrenergic influence of SI cholinergic neurons may be mediated mainly by polysynaptic pathways.

Intracellular recordings from rat nucleus accumbens neurons in vitro

Intracellular recordings were obtained from rat nucleus accumbens (NAC) neurons in brain slice preparations. Local stimulations evoked depolarizing postsynaptic potential (DPSP). Injections of low intensity depolarizing currents decreased the amplitude of the DPSP and reversed a later portion of the DPSP into a hyperpolarizing potential. Superfusion of pentobarbital facilitated the reversal of this later portion of DPSP and bicuculline abolished this polarity reversal. These data suggested that the DPSP evoked by local stimulation consisted of a combination of an excitatory and an inhibitory postsynaptic potential, and that the latter was probably mediated by gamma-aminobutyric acid.

Relationship of calbindin D-28k and cholinergic neurons in the nucleus basalis of Meynert of the monkey and the rat

Double-labeling immunocytochemistry reactions were carried out in the monkey and the rat nucleus basalis of Meynert (NBM) to determine the extent of overlap between cholinergic neurons and neurons immunoreactive for calbindin-D-28k (CaBP), a Vitamin D-dependent calcium binding protein. The results indicate that most, but not all, NBM cholinergic neurons in the monkey are immunoreactive for CaBP. On the other hand, none of the rat NBM cholinergic neurons are immunoreactive for CaBP.

Synaptic relationships of enkephalinergic and cholinergic neurons in the nucleus accumbens of the rat

Leucine5-enkephalin- and choline acetyltransferase-containing, presumably cholinergic, neurons revealed by dual label immunocytochemistry were found in the shell and core of the rat nucleus accumbens. The perikarya, dendrites and boutons of cholinergic neurons were labeled with the diaminobenzidine precipitate, whereas those of the enkephalinergic neurons were labeled with silver-intensified colloidal gold. Ultrastructural examination revealed that both the enkephalinergic and the cholinergic boutons generally formed symmetric synapses with unlabeled dendrites and, occasionally, with unlabeled dendritic spines. Enkephalin-immunoreactive terminals which were much larger than cholinergic boutons, seldom apposed or formed synapses with cholinergic structures in the nucleus. In the core, cholinergic terminals were frequently found apposed to enkephalin-immunoreactive dendrites and perikarya and were often seen in synaptic contact with enkephalinergic dendrites, whereas in the shell, cholinergic boutons seldom apposed or contacted enkephalinergic targets. These findings show that enkephalinergic and cholinergic neurons differ in their synaptic arrangements within the nucleus accumbens and provide further evidence for differentially organized intrinsic connections of shell and core territories.

Relationship of calbindin D-28k with afferent neurons to the rostral ventrolateral medulla in the rat.

The phenylethanolamine-N-methyltransferase (PNMT)-containing neurons in the rostral ventrolateral medulla (RVLM) (the C1 adrenergic group) have been implicated in the generation of the tonic sympathetic nerve activity. Using a double-labeling immunohistofluorescence technique, we found that 34.6 +/- 11.4% (mean +/- S.D.) of PNMT immunoreactive neurons in the RVLM were immunoreactive for Calbindin D-28k (CaBP), a Vitamin D-dependent calcium binding protein. Since CaBP is probably involved in regulating intracellular calcium concentrations in cells that are metabolically or electrically very active, our results suggest that at least some C1 adrenergic neurons (those containing calbindin) may have calcium mediated high metabolic or electrophysiologic activity that is associated with generating tonic nerve function. The RVLM has wide connections with many different nuclei in the brain which are known to contain clusters of neurons that express immunoreactivity to CaBP. In order to determine whether CaBP could be used as a molecular marker for projection neurons to the RVLM or to identify a subpopulation of projection neurons containing CaBP, we sought to determine the relationships between CaBP and the neurons that project to RVLM. Following injections of the retrograde tracer FluoroGold (FG) into the rat RVLM, sections containing retrogradely labeled neurons in (1) the nucleus tractus solitarii (NTS), (2) the contralateral RVLM, (3) the area postrema, (4) the mesencephalic central gray (mCG), (5) the lateral hypothalamus (LH), (6) the substantia innominata (SI), and (7) the paraventricular hypothalamic nucleus (PV) were tested for CaBP immunoreactivity. Although many retrogradely labeled neurons were found amidst many CaBP immunoreactive neurons in each of these nuclei, only a subpopulation of the retrogradely labeled neurons expressed CaBP immunoreactivity. The NTS demonstrated the higher proportion of double-labeled cells (mean 31.5 +/- 4.3%), whereas the lower proportion corresponded to the contralateral RVLM (mean 9.6 +/- 3.2%). On the other hand, both the retrogradely labeled neurons and the CaBP immunoreactive neurons in each of these nuclei were often found in regions containing a great number of adrenergic axons (i.e. immunoreactive for PNMT). Our results suggest that: (1) Two types of adrenergic RVLM neurons could be found, those containing CaBP and those lacking this calcium binding protein. (2) CaBP is not a common marker for the afferent neurons to the RVLM, but rather is found in selective subsets of them. (3) Both the non-CaBP projection neurons and the CaBP immunoreactive neurons in these nuclei may be innervated by adrenergic fibers.

Adrenergic innervation of the substantia innominata: co-localization of phenylethanolamine N-methyltransferase and tyrosine hydroxylase immunoreactivities within the same axons☆

Adrenergic input to the rat substantia innominata (SI) was studied by immunocytochemical localization of phenylethanolamine N-methyltransferase (PNMT), the synthetic enzyme for adrenaline. Using primary antibodies derived from different species, we demonstrated in double-immunofluorescence labeling experiments that all PNMT-immunoreactive fibers in SI are also immunoreactive for tyrosine hydroxylase, the synthetic enzyme for dopamine. This result indicates that the PNMT immunoreactive axons in SI are indeed adrenergic fibers.

Calcitonin gene-related peptide (CGRP) in the rat substantia innominata and globus pallidus: a light and electron microscopic immunocytochemical study

The substantia innominata (SI) and the caudal part of the globus pallidus (GP) in the rat are densely innervated by calcitonin gene-related peptide-immunoreactive (CGRP+) axons. Electron microscopic analysis revealed that CGRP+ boutons mainly formed asymmetrical synapses with dendrites in GP and SI.