Lawrence P. Schramm

Peripheral and central pathways regulating the kidney: a study using pseudorabies virus

We used the retrograde transneuronal transport of a neurotropic virus, pseudorabies virus (PRV), to identify the neurons in sympathetic ganglia, spinal cord and brain which regulate renal function and renal circulation. PRV was microinjected into the left kidney of 70, pentobarbital-anesthetized, male rats. After an incubation period of 1-4 days, rats were anesthetized and sacrificed. PRV-infected neurons were located immunocytochemically in pre- and paravertebral sympathetic ganglia, the intermediolateral cell column of the T10-T13 segments and several brainstem cell groups: the medullary raphe nuclei, rostral ventrolateral medulla, rostral ventromedial medulla, A5 cell group, and the paraventricular hypothalamic nucleus. In more heavily infected rats, additional labeling was found in the locus coeruleus, periaqueductal gray matter, lateral hypothalamic area, zona incerta, and anterior hypothalamic area. No infected propriospinal neurons were observed in the lateral spinal nucleus or gray matter of the caudal cervical, lumbosacral or thoracic spinal segments not containing infected putative sympathetic preganglionic neurons. The paucity of infected propriospinal neurons in the presence of infected brainstem neurons, even in lightly infected rats, is discussed in reference to the relative importance of descending vs spinal regulation of the sympathetic outflow to the kidney.

Preganglionic innervation of the adrenal gland of the rat: A study using horseradish peroxidase

A method and an apparatus for joining plastic brush bristles with plastic brush backs provides for heating the tips of brush bristle strands until they are soft and heating zones of the plastic brush backs to which the bristles will be mounted until they are soft. Thereafter, the soft tips of the bristles are brought into contact with the soft zones of the brush backs to fuse the bristles to the brush backs. In the particular structure and method of the machine of this invention, two relatively movable plates alternately clamp to and unclamp from indeterminant-length bristle strands to jog the strand ends into contact with the brush backs. One of the plates carries a cutting blade which cuts the strands into bristle lengths once their tips are fastened to the brush backs. For the heating step, expandable heating plates are moved between the brush backs and the bristle strand tips and expanded to contact the bristle strand tips and the brush backs and are then contracted and retracted from between the bristles and the brush backs.

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

Pregnancy-induced uterine neuronal degeneration in the rat

Abstract.We studied the location, distribution, and density of uterine nerve bundles in virgin, full-term pregnant, and early postpartum female rats. In the virgin, a low-magnification analysis of uterine whole-mounts stained for acetylcholinesterase revealed fibers both in close association with blood vessels and coursing freely within muscle or connective tissue in the mesometrium and the uterine body. Higher magnification analysis of sections treated immunohistochemically with antibodies to the ubiquitous neuronal protein gene product 9.5, tyrosine hydroxylase, and calcitonin gene-related peptide indicated that the greatest density of nerve bundles was in the mesometrial triangle and intramyometrial region. Although the majority of bundles were associated with blood vessels, many coursed freely, particularly in the myometrial and endometrial layers. The density of innervation within the body of the uterus was heterogeneous. For instance, both innervated and noninnervated spiral arteries branched from the same innervated circumferential artery. In the term pregnant rat, we observed a profound denervation within all layers of the body of the uterus. The few remaining nerve bundles tended to be associated with blood vessels, and they were confined to the intramyometrial region. Within 48 h postpartum, both the number and density of nerve bundles increased. Long tortuous bundles, not seen in the full-term rat, were observed. The present study is the first to describe and illustrate the pattern of uterine innervation in virgin, full-term pregnant, and early postpartum rats over a wide range of magnifications.

Sensitivity of Sympathetically Correlated Spinal Interneurons, Renal Sympathetic Nerve Activity, and Arterial Pressure to Somatic and Visceral Stimuli after Chronic Spinal Injury

In the chronic stage of spinal cord injury in humans, both innocuous and noxious somatic and visceral stimuli can elicit severe autonomic dysreflexia characterized by potentially dangerous, sympathetically mediated, increases in arterial pressure. We hypothesized that a similar sympathetic hyperexcitability would be manifested in spinal sympathetic networks of chronically spinally transected rats. To test this hypothesis, we compared the responses of sympathetically correlated spinal interneurons and arterial pressure to both innocuous and noxious stimuli in acutely and chronically spinally transected rats. Experiments were conducted in anesthetized female rats, either within hours of T(3) spinal transection (rats with acute spinal transection) or one month after T(3) spinal transection (rats with chronic spinal transection). Sympathetically correlated spinal interneurons were identified by cross correlating their ongoing activity with simultaneously recorded renal sympathetic nerve activity. Cutaneous stimuli (either light brushing or noxious pinch) were delivered to a wide area of the ipsilateral side of the rat. Colorectal distension was used as a noxious visceral stimulus. The activity of sympathetically correlated interneurons was increased by stimulation of more of the body surface and decreased by stimulation of less of the body surface in rats with chronic spinal transection than in rats with acute spinal transection. Colorectal distension elicited greater increases in arterial pressure in chronically than acutely spinally-transected rats without exciting significantly more interneurons in those rats. These results suggest that spinal circuits undergo significant plastic changes in the chronic stage of spinal cord injury, and they provide a mechanism for the observation, in some human patients, that many stimuli, both noxious and non-noxious, applied caudal to the site of spinal injury increase sympathetic activity and arterial pressure.

Behavior of chronically decerebrated kittens.

Abstract Kittens were decerebrated within 1 wk after birth and the subsequent development of functional capacity of these mesencephalic animals was observed for up to 2 mo. The only detectable immediate deficit was that sucking behavior was abolished. Ingestion of solid foods and lapping of milk, however, developed at weaning age. These were reflex in nature and and required contact of the lips with the food. Development of temperature regulation was impaired only slightly. Auditory reflexes, placing reactions, defense reactions, and many other types of behavior except visual recognition and socialization developed in nearly normal chronological order. In most animals some aspects of behavior were exaggerated. Hyperkinesis, hypermetria, compulsive climbing, and exaggerated prey behavior were frequently observed. The findings suggest that many aspects of behavior and regulatory processes generally thought to be organized primarily in the prosencephalon originate instead in the mesencephalon and more caudal structures and, during ontogeny, become increasingly dependent upon subsequently developing descending synaptic inputs.

Sialidase enhances recovery from spinal cord contusion injury

Axons fail to regenerate in the injured spinal cord, limiting motor and autonomic recovery and contributing to long-term morbidity. Endogenous inhibitors, including those on residual myelin, contribute to regeneration failure. One inhibitor, myelin-associated glycoprotein (MAG), binds to sialoglycans and other receptors on axons. MAG inhibition of axon outgrowth in some neurons is reversed by treatment with sialidase, an enzyme that hydrolyzes sialic acids and eliminates MAG–sialoglycan binding. We delivered recombinant sialidase intrathecally to rats following a spinal cord contusive injury. Sialidase (or saline solution) was infused to the injury site continuously for 2 wk and then motor behavior, autonomic physiology, and anatomic outcomes were determined 3 wk later. Sialidase treatment significantly enhanced hindlimb motor function, improved bulbospinally mediated autonomic reflexes, and increased axon sprouting. These findings validate sialoglycans as therapeutic targets and sialidase as a candidate therapy for spinal cord injury.

The spinal course and medullary termination of myelinated renal afferents in the rat

Myelinated afferent fibers, recorded in the left renal nerve of rats, were antidromically activated by discrete electrical stimulation of the cervical spinal cord and the caudal medulla. The lowest thresholds for activation of these fibers were found in the most medial portion of the ipsilateral fasciculus gracilis. This region of minimum threshold continued rostrad through the nucleus commissuralis. Based on threshold vs depth contours, fibers appeared to terminate in the ipsilateral nucleus gracilis and nucleus solitarius. Myelinated fibers could be activated by punctate pressure on the renal hilus. Action potentials generated by hilar pressure collided with antidromically-conducted action potentials elicited by electrical stimulation at cervical levels. We conclude that myelinated renal afferents carry information from intrarenal receptors, via the dorsal column system, to both visceral afferent and dorsal column nuclei.

The conduction velocities and spinal projections of single renal afferent fibers in the rat

This study was designed to examine the conduction velocities and spinal projections of renal afferent fibers in the rat using electrophysiological techniques. In chloralose-anesthetized rats, we electrically stimulated the peripheral ends of cut, lower thoracic and upper lumbar dorsal roots and recorded and averaged antidromically conducted action potentials in the renal nerves. Of 284 single axons responding to stimulation of ipsilateral dorsal roots T9-L1, the majority were activated by stimulating roots T11-T13. No antidromic responses could be elicited by stimulating the contralateral dorsal roots. Afferent fibers were divisible into two groups, distinguished by their conduction velocities: a population of slowly conducting axons, presumably composed of both unmyelinated (0.3-2 m/s, 76%) and thinly myelinated (2-9 m/s, 19%) fibers, and a population of more rapidly conducting, small myelinated axons (12-32 m/s, 5%). Slowly and more rapidly conducting fibers were not differentially distributed among dorsal roots. Postexperimental histological examination of nerves revealed small myelinated axons with diameters appropriate for some, but not for all, of the axons with conduction velocities in the myelinated range. These results indicate that single myelinated and unmyelinated primary afferent axons can be identified by antidromic stimulation in autonomic nerves of rat. They provide the first electrophysiological description of afferent renal nerve fibers in the rat, and they verify the predominantly unmyelinated nature of these fibers.

Sympathetic preganglionic neurons in the isolated spinal cord of the neonatal rat

A preparation of the isolated spinal cord of the neonatal rat was developed for the study of sympathetic preganglionic neurons (PGNs). PGNs were identified for extracellular single unit recording by their location and by antidromic activation by ventral root stimulation. PGNs could be synaptically activated by stimulation of the dorsal root and spinal pathways. Spontaneous firing was observed in 18% of the PGNs. The average firing rate was 1 Hz with a range of 0.3 to 2 H z. PGNs (and motoneurons) were visualized by incubating ventral roots in horseradish peroxidase (HRP) solutions. The location and morphology of PGNs were similar to those reported in studies using adult animals. Primary afferent fibers were visualized by incubating dorsal roots in HRP solutions. Dorsal root projections appeared mature in the neonatal rat. Primary afferents did not appear to project directly to PGNs. It is concluded that PGNs are viable in this preparation and that spinal sympathetic systems are relatively mature in the neonatal rat.