Role of central oxytocin in the control of the milk ejection reflex

The neuropeptide oxytocin, synthetized by magnocellular neurons in the hypothalamus, is well known for its peripheral action after it is released into the bloodstream from axons in the neurohypophysis. Less familiar is the notion that it is also released centrally to control the activity of oxytocinergic neurons themselves. When injected into the third ventricle of lactating rats during suckling, oxytocin increases the basal firing rate of oxytocinergic neurons as well as their activity at the time of each reflex milk ejection. On the other hand, centrally administered oxytocin engenders the neuronal-glial and synaptic plasticity characteristic of the oxytocin system when it is physiologically activated. From numerous in vivo and in vitro observations, it appears that central oxytocin is released in the hypothalamic nuclei themselves. For example, the use of push-pull cannulae inserted into one supraoptic nucleus of suckled rats shows that oxytocin is released inside the nucleus specifically during milk ejection. Moreover, ultrastructural immunocytochemistry reveals synaptic terminals in the supraoptic nucleus where both the pre- and postsynaptic elements are oxytocinergic. Nevertheless, the mechanism of the central release of the neuropeptide has still to be determined, especially in view of electrophysiological observations indicating that the release process in the hypothalamus is different from that within the neurohypophysis.     

The cells of origin of the hypoglossal afferent nerves and central projections in the cat

The cells of origin for the hypoglossal afferent nerves of the cat and their central projections were examined using the transganglionic and somatopetal transport of horseradish peroxidase (HRP). Primary afferent neurons from the hypoglossal nerve were located in the trigeminal ganglion, the superior ganglion of glossopharyngeal and vagal nerves, and the first 3 cervical ganglia. The central projections of hypoglossal afferents were organized in a selective manner according to their cells of origin. The primary afferent nerves originating from the trigeminal ganglion terminated in the subnucleus dorsalis (Vpd) of the principal nucleus (Vp), lateral margin of the caudal pars interpolaris (Vi), interstitial nucleus and laminae I and V of the pars caudalis (Vc). The projection of the afferent nerves for glossopharyngeal and vagal origins are similarly organized in the Vi and Vc to those of trigeminal origin, but differed in that they terminated ipsilaterally in the caudal half of the solitary nucleus and bilaterally in the commissural nucleus. The primary afferents arising from the first 3 cervical ganglia terminated in laminae I and V of the corresponding cervical cord segments.     

Modulation of the spinal excitability by muscle metabosensitive afferent fibers

The aim of this study was to identify the effect of chemical activation of muscle metabosensitive afferent fibers from groups III and IV on Hoffmann (H‐) reflex modulation in the vastus medialis muscle. The experiment was conducted in rats and was divided into two experiments. The first experiment consisted of recording the metabosensitive afferent activity from femoral nerve in rats in response to KCl intraarterial injections in nontreated adults and adults treated neonatally with capsaicin. Thus, the dose‐response curve was determined. The second experiment consisted of eliciting the H‐ and M‐waves before and after KCl injection in nontreated adult animals and those treated neonatally with capsaicin. Thus, the H_max/M_max ratio was measured. Results indicated that, 1) in nontreated animals, afferent fibers peak discharge was found after 10 mM KCl injection; 2) no significant increase in afferent discharge rate was found in capsaicin‐treated animal after KCl injections, confirming that capsaicin is an excitotoxic agent that had destroyed the thin metabosensitive nerve fibers; 3) in nontreated animals, H_max/M_max ratio was significantly attenuated after a 10 mM KCl injection activating metabosensitive afferent fibers; and 4) in capsaicin‐treated animals, no significant change in H_max/M_max ratio was observed after the KCl injection. These results reinforce the hypothesis that the spinal reflex response was influenced by metabosensitive muscle fibers and provide direct evidence that activation of these fibers could partially explain the reported decrease in H‐reflex when metabolites are released in muscle. © 2010 Wiley‐Liss, Inc.     

Physiological evidence for branching of peripheral unmyelinated sensory afferent fibers in the rat

Single unmyelinated sensory afferent nerve fibers were recorded in dorsal root filaments in urethane-anesthetized or in decerebrate-spinal rats. The peripheral branch of these axons ran in the sural nerve where they were stimulated by tungsten microelectrodes. All action potentials showed the characteristics of single fiber responses with a fixed all or none shape and a fixed latency at a given stimulus strength. In all units, the action potential evoked from a proximal stimulus site collided with the action potential evoked from a distal stimulus site. Of the 44 single units isolated, 17 showed the expected small progressive decrease of latency of the recorded impulse as the stimulus strength at a fixed point on the sural nerve was progressively raised above threshold. However, in 27 units there was an abrupt jump decrease of latency as the stimulus rose above the threshold. The average size of this latency shortening was 2.2 msec, which occurred as the stimulus strength rose a mean 21% above threshold. As the stimulus rose above threshold, 7 fibers showed 3 different fixed latencies and 2 fibers showed 4 fixed latencies. In order to test the possibility that the peripheral nerve contained 2 branches of the same axon with one conducting slower than the other, the peripheral nerve was stimulated at progressively longer conduction distances. As predicted, the difference between the 2 fixed latencies became larger as the conduction distance increased. We discuss 6 possible explanations for the results and conclude they are consistent with the proposals that some fibers branch distal to the dorsal root ganglion and some branches do not establish a functional sensory ending in the periphery.     

Fine afferent fibers from viscera do not terminate in the substantia gelatinosa of the thoracic spinal cord

Transganglionic transport of horseradish peroxidase (HRP) has been used to study the anatomy of the central projection of somatic and visceral afferent fibers to the thoracic spinal cord of the cat. A dense concentration of somatic afferent fibers and terminals was found in laminae I and II of the dorsal horn and more scattered terminals were present in laminae III, IV and V and in Clarke's column. In contrast, visceral afferent fibers and terminals were found only in lamina I or reaching lamina V via a small bundle of fibers located in the lateral border of the dorsal horn. These results indicate that fine afferent fibers from viscera, unlike those of cutaneous origin, do not project to the substantia gelatinosa (lamina II) of the dorsal horn.     

Direct connections of primary afferent fibers with central cervical nucleus neurons projecting to the cerebellum in the cat

The synaptic organization of the central cervical nucleus (CCN) in the cat was examined by means of anterograde and retrograde axonal transport of horseradish peroxidase and the anterograde degeneration tracing technique. Light microscopic studies confirmed that cervical primary afferents terminate heavily in the region of CCN cerebellar projection neurons and electron microscopic findings demonstrated that primary afferent fibers of cervical dorsal roots form synapses mainly with distal dendrites of CCN neurons projecting to the cerebellum.     

Origin and function of spiral fibers projecting to the goldfish mauthner cell

Two neuron types contact the Mauthner cell (M cell) in the axon cap, a specialized region of high electrical resistance surrounding the initial segment of the M cell axon. One type produces a mixed electrical and chemical inhibition of the M cell. The second sends axons into the central core of the axon cap, where they spiral around the initial segment making both conventional synapses and gap junction contacts. The origin and synaptic effects of these spiral fibers have not been studied previously. When goldfish M cells were filled with Lucifer yellow, presynaptic spiral fibers were seen in the axon cap. These fibers could be traced back through the medial longitudinal fasciculus to their somata, near the contralateral fifth nerve motor nucleus. The same somata were labeled by horseradish peroxidase injected extracellularly into the axon cap. Recordings were made in the axon cap and the M cell after stimulation of hindbrain areas near the spiral fiber somata and axons. Extracellularly, a negative potential was observed close to the termination of the spiral fibers and termed the spiral fiber potential (SFP). Intracellularly, a graded, short latency depolarization of the M cell corresponded to the SFP and could cause the M cell to spike. This depolarization did not shunt the membrane, indicating that it may be produced through gap junctions. Intracellular responses to hindbrain stimulation also had a chloride-dependent, second component that shunted the membrane during paired-pulse testing. This inhibitory second component was probably evoked by cells other than the spiral fiber cells themselves. © 1994 Wiley-Liss, Inc.     

The afferent innervation of the thymus gland in the rat

The afferent nervous supply to the thymus gland has been investigated by means of the retrograde transport of horseradish peroxidase. It has been shown that the thymus receives an afferent supply from the nodose ganglia of the vagus and from the dorsal root ganglia C₁–C₇. The afferent innervation of the right and left thymic lobes is bilaterally organized; the fibers of a small celled population of nodose ganglion neurons cross outside the thymus and those of a larger celled population cross within the thymus gland. The functional implications of these findings are discussed in the context of central nervous system-immune system interactions.     

The distribution with the spinal cord of visceral primary afferent axons carried by the lumbar colonic nerve of the cat

Horseradish peroxidase taken up by the sensory axons in the lumbar colonic nerves in 5 cats was observed in the dorsal root ganglia and in the spinal cord in segments L1 through L5. Reaction product was observed in Lissauer's tract, the dorsal columns and laminae I, V, VII and X in a pattern typical of visceral primary afferents from other nerves. A small number of preganglionic neurons were also labeled.     

Ascending and descending effects of joint afferent discharge on forelimb and hindlimb flexion reflex excitability in decerebrate cats

In decerebrate cats with intact innervation of the fore- and hindlimbs, flexion reflexes are most easily elicited in forelimb muscles when the hindlimb is extended, and hindlimb flexion reflexes are most easily elicited when the forelimbs are extended. After intra-articular injection of local anaesthetic, this modulation of reflex excitability is abolished. Thus, in addition to their known segmental effects, joint afferents also exert significant ascending and descending effects on motoneurone excitability.     

Central projections of coarse and fine vagal axons of the cat

We used the wallerian degeneration of vagal afferents and the retrograde transport of WGA-HRP microinjected in the nucleus of the tractus solitarius (NTS) to study the central projections of myelinated and unmyelinated vagal axons. We concluded that the set of largest nodose cells projected to the dorsolateral, interstitial, ventral, ventrolateral and intermediate NTS subnuclei, while the smaller nodose cells terminated in the medial, dorsal, gelatinosus and commissural NTS subnuclei.     

Deficits in the function of small and large afferent fibers in confirmed cases of carpal tunnel syndrome

Nerve conduction studies and measurements of thermal thresholds (hot and cold) were performed in 25 healthy control subjects and 24 patients with carpal tunnel syndrome: first, to compare the extent of axons; second to correlate these abnormalities with clinical severity;and third to validate a modified from of palmar stimulation as an additional neurophysiological test in suspected carpal tunnel syndrome. The routine nerve conuction studies compared the amplitudes and conduction velocities from digit II and digit V to wrist and for the digit-II potential between wrist and elbow. With palmar stimulation, the palm-to-wrist segment was compared to the simultaneously evoked antidromic digital potential (palm-to-index). Thermal threshold testing involved determination of hot and cold thresholds for digit II and digit V using an automated forced-choice procedure. In the patients there was selective slowing of conduction across the palm-to-wrist segment with normal velocities distally and proximally, and there was clear evidence of abnormal small-fiber function, with higher thresholds for both hot and cold sensation. However, the thermal abnormalities also involved the ulnar territory, equally so for cold threshold but significantly less so for hot threshold. Thermal threshold testing confirmed the clinical impression that small-fiber dysfunction is important in carpal tunnel syndrome, but the abnormal findings for the ulnar-innervated digit V suggest that this test will not be routinely useful for the early detection of compression or entrapment neuropathies. © 1994 John Wiley & Sons, Inc.     

Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase

Retrograde and transganglionic transport of horseradish peroxidase (HRP) was used to trace afferent and efferent pathways in the left inferior cardiac nerve of the cat. Cardiac efferent and afferent neurons were located, respectively, in the stellate ganglion (average cell count per experiment: 2679) and in the ipsilateral dorsal root ganglia (DRG) from C8 to T9 (average cell count per experiment: 213). Labeled cardiac afferent projections to the spinal cord were most dense in segments T2–T6 where they were located in Lissauer's tract and in lamina 1 on the lateral border of the dorsal horn. Labeled affrent axons extended ventrally through lamina 1 into lamina 5 and the dorsolateral region of lamina 7 in proximity to the intermediolateral nucleus. A weak projection was noted on the medial side of the dorsal horn. These sites of termination are similar to projections by other sympathetic afferent pathways (i.e. renal, hypogastric and splanchnic nerves) to the lower thoracic and lumbar spinal cord, indicating that visceral afferents may have a uniform pattern of termination at various segmental levels. This pattern of termination in regions of the gray matter containing spinothalamic tract neurons and neurons involved in autonomic mechanisms is consistent with the known functions of sympathetic afferent pathways in nociception and in the initiation of autonomic reflexes.     

Analysis of reflex activity in cardiac sympathetic nerve induced by myelinated phrenic nerve afferents

Electrical stimulation of the phrenic nerve afferents evoked excitatory responses in the right inferior cardiac sympathetic nerve in chloralose-anaesthetized cats. The reflex was recorded in intact and spinal cats. The latency and threshold of the volley recorded from the phrenic nerve as well as of the cord dorsum potentials evoked by electrical stimulation of the phrenic nerve indicated that group III afferents were responsible for this reflex. The phrenicocardiac sympathetic reflex recorded in intact cats was followed by a silent period. The maximum amplitude of the reflex discharges was 800 microV, the latency was 83 ms and the central transmission time 53 ms. Duration of the silent period lasted up to 0.83 s. In spinal cats the reflex was recorded 5.5-8 h after spinalization. The maximum amplitude of the spinal reflex discharges ranged from 22 to 91 microV and the latency from 36 to 66 ms.     

Electron microscopic observations of terminals of functionally identified afferent fibers in cat spinal cord

Using the method of intra-axonal injection of horseradish peroxidase, functionally identified afferent fibers from three slowly adapting (Type I) receptors and one Pacinian corpuscle in the glabrous skin of the hind paw of the cat were stained. Electron microscopic observation of the terminals of these fibers revealed predominantly axodendritic asymmetric synapses containing round, clear vesicles. Multiple synapses on a single dendrite were observed, separated by as little as 900 mm from one another.     

Organization of cutaneous primary afferent fibers projecting to the dorsal horn in the rat: WGA-HRP versus B-HRP

Primary afferent projections from cutaneous afferents in the forelimb and hindlimb digits to the dorsal horn (DH) were examined using 4 tracers: (1) 25% free horseradish peroxidase (HRP), (2) 2.5% wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), (3) a mixture of 25% free HRP and 2.5% WGA-HRP (WGA-HRP/HRP) or (4) 0.1% HRP conjugated to cholera toxin (B-HRP). The tracer was injected intracutaneously into the digits. Three to 4 days later, the rats were perfused transcardially, transverse sections (60-microns thick) were cut and the HRP was reacted using the tetramethyl benzidine (TMB) method. The location of the label was reconstructed by camera lucida drawings. In rats which received an injection of HRP alone, no label was detected in the DH. Rats injected with WGA-HRP had projection patterns similar to those injected with WGA-HRP/HRP. Patterns of labelling with WGA-HRP differed markedly from those with B-HRP. WGA-HRP labelled cutaneous afferents projecting to Rexed's laminae I-III, with the densest label in lamina II; in contrast, B-HRP labelled cutaneous afferents projecting to laminae II-V, with the densest label in laminae III-IV. These results indicate that, for cutaneous primary afferents projecting to the DH, WGA-HRP and B-HRP labelled different subpopulations of fibers, with the B-HRP-labelled subpopulation biased toward afferents of larger diameter. Rostrocaudally, the extent of the densest fiber projections, whether labelled by WGA-HRP or by B-HRP, was essentially the same, but the extent of the less densely labelled projections was much greater with B-HRP than with WGA-HRP. Comparisons of the projection maps from each of the five digits, using either WGA-HRP or B-HRP, indicated that, as seen in transverse sections through the DH, there was extensive overlapping among the labelled cutaneous afferent fibers from adjacent, or even non-adjacent digits.     

Surgical determination of the site of crossing of jaw-opening reflex evoked by tooth pulp stimulation in the cat

Tooth pulp stimulation in the cat evokes a jaw-opening reflex that is usually bilateral. We defined the location of fibers crossing to activate the contralateral reflex by making midline sagittal transections at peripheral and central levels in 11 anesthesized cats. A local midsagittal transection of the brain stem 2–4 mm above the obex selectively eliminated the contralateral reflex response. Midline sagittal transections of the maxilla, mandible, cervical cord, or upper brain stem did not.     

Vagal afferent modulation of a nociceptive reflex in rats: involvement of spinal opioid and monoamine receptors

Modulation of the spinal nociceptive tail flick (TF) reflex by electrical stimulation of subdiaphragmatic or cervical vagal afferent fibers was characterized in rats lightly anesthetized with pentobarbital. Cervical vagal afferent stimulation (VAS) inhibited the TF reflex in a pulse width-, frequency-, and intensity-dependent fashion. The optimum parameters for inhibition of the TF reflex were determined to be 2.0 ms pulse width, 20 Hz frequency with a threshold (T) current of 60 microA. Cervical VAS at 0.2-0.6 T facilitated the TF reflex. Cervical VAS at T typically produced a depressor arterial blood pressure response, but inhibition of the TF reflex by VAS was not due to changes in blood pressure. Subdiaphragmatic VAS also inhibited the TF reflex and generally produced a pressor effect, but did not facilitate the TF reflex at intensities of stimulation less than T as did cervical VAS. The parameters of cervical VAS required for inhibition of TF reflex suggest that excitation of high-threshold, unmyelinated fibers are important in VAS-induced descending inhibition. The intrathecal administration of pharmacologic receptor antagonists into the subarachnoid space of the lumbar enlargement indicated that the opioid receptor antagonist naloxone produced a dose-dependent antagonism of cervical VAS-produced inhibition of TF reflex, but single doses of either phentolamine or methysergide (30 micrograms each) failed to affect the inhibition by VAS. Combined intrathecal injection of both phentolamine and methysergide (30 micrograms each), however, significantly attenuated inhibition of the TF reflex by cervical VAS. These results suggest that cervical VAS engages a spinal opioid system and co-activates descending serotonergic and noradrenergic systems to modulate spinal nociceptive processing.     

Electrical activity of dorsal horn neurons during the suckling-induced milk ejection reflex in the lactating rat

In lactating rats, suckling elicits the milk ejection reflex which consists of an intermittent synchronous activation of hypothalamic oxytocin neurons which releases oxytocin into the bloodstream. We here investigated the electrophysiological behaviour of spinal cord neurons linked to mammary innervation in relation to suckling and the suckling-induced milk ejection reflex. Experiments were carried out on 58 urethane-anaesthetized rats, paralysed with gallamine triethiodide and artificially ventilated. Fixation of the spinal cord and laminectomy significantly slowed down the reflex, which occurred in only 27% of the animals. In these rats, 31 dorsal horn neurons at the thoraco-lumbar level were found to be excited by nipple stimulation. During suckling by a litter of at least nine pups, they displayed an irregular pattern of brief bursts of activity (peak firing rate 22.0 ± 3.2 Hz, mean ± SD) correlated to the bouts of suckling of the pups. Seventeen out of 19 cells tested by stimulation of at least 2 adjacent nipples received convergent input from different ipsilateral nipples. Out of 11 cells tested, 8 were also activated by stimulation of a contralateral nipple. Fourteen out of 30 units were recorded through at least one reflex milk ejection. Their firing rate was significantly higher than the firing rate of cells recorded in animals which failed to milk eject (4.4 Hz ± 2.8 versus 1.5 Hz ± 0.7). At the moment of the high frequency discharge of action potentials, occurring in oxytocinergic cells 10 to 15 s before each milk ejection, spinal neurons showed no systematic change in electrical activity. In contrast, the stretch reaction of the pups, which corresponds to an intense period of suckling when milk ejection has started, induced, in 12 cells, a considerable increase in electrical activity. One unit was found to be inhibited by suckling and during the stretch reaction. Ten more units, which were not activated by stimulation of the nipples but responded to stimulation of excitatory receptive fields near the last three pairs of nipples, were recorded through reflex milk ejections: 8 remained silent during reflex milk ejections but 2 were activated when the pups stimulated their excitatory receptive field. We conclude that some dorsal horn neurons, able to respond readily to the suckling movements of pups, appear to receive an ungated input from the nipples. At the time of the activation of oxytocin neurons, they present no particular pattern of activation or inhibition which could account in a simple manner for the intermittence of the high frequency discharge in oxytocinergic cells. However, in so far as these dorsal horn neurons may be part of the milk ejection reflex pathway, their activity, showing convergence and summation of input, and being facilitated in milk ejecting animals, indicates that the reflex does undergo a certain degree of processing at the spinal cord level.