Relationship of intrinsic connections to forelimb movement representations in monkey motor cortex: a correlative anatomic and physiological study

1. Intracortical microstimulation (ICMS) and horseradish peroxidase (HRP) histochemistry were combined to examine the relationship between intrinsic connections and intracortical microstimulation sites eliciting evoked movements in the forelimb representation of adult macaque monkey motor cortex. 2. The distribution of sites from which stimulation-evoked movements about individual forelimb joints were elicited under anesthesia varied considerably among animals. Identical movements could often be elicited from multiple, noncontiguous sites. 3. After single, small extracellular HRP injections at sites from which thumb movement was evoked, small groups of retrogradely labeled cells and dense patches of axon terminations were found scattered across a wide area of the forelimb representation. Terminal patches were discontinuous and arose from horizontal, intracortical axons. 4. Correlating the HRP labeling with the physiologically defined movement maps revealed a profuse set of intrinsic, bidirectional connections that connect digit representations and representations of movements about the wrist, elbow, and shoulder. 5. HRP injections placed in the forelimb representation close to the physiologically defined face representation resulted in virtually no retrogradely labeled cells or terminal fiber labeling that crossed into the face representation. A patch of anterolaterally placed label that was present may be the dissociated rostrolateral arm area of other authors. 6. Taken together, these data suggest that extensive, horizontally oriented, intrinsic axon collaterals provide inputs to many different forelimb movement representations and may be recruited during complex movements to coordinate the activity of motor cortical zones whose predominant output is to forelimb muscle groups acting synchronously.

A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining.

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.

The effects of tectal lesion on the survival of isthmic neurones in Xenopus

The isthmic nucleus (IN) is a visual relay centre of the frog brain. It receives afferent projection from the optic tectum of the same side and projects bilaterally to both tecta. In young postmetamorphic Xenopus frogs, the survival of neurones in the IN on both sides was studied following the complete removal of the right tectum. In 6- to 8-week-old frogs, the right tectum was surgically removed and the operated animals allowed to survive for 1 to 13 weeks after operation. In selected animals, 3 days before the intended sacrifice, the postoptic commissure was transected and the cut isthmotectal fibres filled with horseradish peroxidase (HRP). In serial paraffin sections of the midbrain, the numbers of surviving and dying (pyknotic) neurones in the left and right IN were counted. The soma size of viable isthmic neurones and the volume of both IN were measured. Pyknotic neurones were seen between 1 and 6 weeks after operation in both the left and right IN, although the rate of cell loss was much greater in the latter. Virtually all the neurones of the right IN degenerated by 6 weeks after tectal ablation. In contrast, approximately 60% of neurones of the left IN survived. HRP histochemistry showed labelled isthmic neurones both in the left and right IN up to 3 weeks after operation. Thereafter, HRP-labelled neurones appeared only in the left IN. These observations indicate that the removal of the natural target of isthmic neurones brings about severe neurone death.(ABSTRACT TRUNCATED AT 250 WORDS)

The characteristics of cardiac vagal preganglionic motoneurones in the dogfish

Preganglionic vagal motoneurones supplying the heart of the dogfish have been located in the medulla by antidromic stimulation of the central cut end of the branchial cardiac branch of the vagus. They supplied axons with conduction velocities between 4.75 and 16.3 m s-1, which is similar to mammalian B fibres. Motoneurones were found in two locations: the rostromedial (N = 5) and lateral (N = 12) divisions of the vagal motor column. Their measured depths and rostrocaudal distributions with respect to obex corresponded with the location of branchial cardiac motoneurones determined by horseradish peroxidase (HRP) histochemistry. All the neurones located in the rostromedial division of the vagal motor column were spontaneously rhythmically active. Their activity contributed to the rhythmic, respiratory-related bursts in peripheral recordings of efferent activity from the branchial cardiac vagus. They could be induced to fire in a prolonged burst by mechanical stimulation of the gill arches. The neurones located lateral to the rostromedial division of the vagal motor column could be divided into three categories: (1) spontaneously, continuously active cells which could be induced to fire more frequently by mechanoreceptor stimulation, (2) silent cells which could be induced to fire by mechanoreceptor stimulation, (3) silent cells which did not respond to mechanoreceptor stimulation. It is concluded, from the response of the medial and two categories of lateral cells to mechanoreceptor stimulation (which results in a transient bradycardia), that branchial cardiac motoneurones from both these central locations exert a chronotropic influence on the heart.

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry.

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.