Reorganization of the Peripheral Projections of the Trigeminal Ganglion Following Neonatal Transection of the Infraorbital Nerve

Two different anatomical techniques were used to obtain evidence that transection of the infraorbital (IO) nerve on the day of birth would result in reorganization of the peripheral projections of the trigeminal nerve. In 14 of 19 neonatally nerve-damaged adult rats, injection of horseradish peroxidase (HRP) directly into the IO nerve, proximal to the point of the neonatal transection, resulted in labeled cells in the ophthalmic-maxillary portion of the ganglion and labeled fibers in mandibular sensory nerves. In an additional 28 neonatally nerve-damaged adult rats, double-labeling techniques were employed to document the reorganization suggested by the HRP tracing experiments. In these experiments, one fluorescent tracer, diamidino yellow (DY), was injected directly into the regenerate IO nerve, proximal to the point of the neonatal transection; a second tracer, true blue (TB), was deposited into peripheral ophthalmic and/ or mandibular fields. These combinations of injections invariably resulted in the demonstration of a small number (46-401) of double-labeled cells that were located in the ophthalmic-maxillary part of the ganglion. Identical combinations of injections in normal adult rats and the intact sides of nerve-damaged animals never produced more than 6 double-labeled cells per ganglion.

In two additional series of experiments, sequential double-labeling techniques were employed to demonstrate that the multiply projecting ganglion cells probably arose in at least two ways: (1) development of non-IO projections by ganglion cells that contributed axons to the IO nerve at the time of the lesion; (2) elaboration of IO axon branches by primary afferent neurons that had non-IO projections at the time of the lesion. A final two-stage double-labeling experiment demonstrated that approximately 75% of the ganglion cells that projected to the whisker pad at birth, and survived transection of the IO nerve on the first postnatal day, regenerated axons into this trigeminal branch.     

Trigeminal projections to contralateral dorsal horn: central extent, peripheral origins, and plasticity

Prior studies have documented a trigeminal (V) mandibular primary afferent projection to the dorsomedial portion of the contralateral medullary and cervical dorsal horns in cat, hamster, and rat. We now report the existence of a much more substantial V ophthalmic primary afferent projection to the ventrolateral portion of contralateral medullary and cervical dorsal horns in rat. Horseradish peroxidase (HRP) injections into the V ganglion or V brainstem complex anterogradely labeled a fascicle of primary afferent axons that exited the caudal ventrolateral V spinal tract to form a rostrocaudally continuous, transversely oriented, V primary afferent decussation. These fibers terminated most heavily in laminae III-V of the ventrolateral dorsal horn in contralateral caudal medulla and the first and second cervical segments. Retrograde tracing with diamidino yellow (DY) or fluorogold and anterograde tracing with Phaseolus vulgaris leucoagglutinin also demonstrated a substantial commissural projection of central origin in medullary dorsal horn laminae I-VII. The latter projection had a more diffuse trajectory and termination pattern than that of the V primary afferent decussation. Unilateral HRP injections into medullary and cervical dorsal horns also retrogradely labeled V primary afferent collaterals contralateral to the injection site in corresponding regions of dorsal horn, and also in ventromedial interpolaris, oralis, and principalis, rostral to their decussation. Axons (1.5 +/- 0.8 microns mean diameter; 0.4-3.9 microns range) therefore terminated both ipsi- and contralateral to their cells of origin. These HRP injections also labeled an average of 40.4 +/- 13.0 V ganglion cells (mean +/- SD, corrected for split somata) in dorsomedial, ophthalmic regions of the contralateral ganglion. Their mean diameter was slightly larger than that of cells labeled ipsilaterally (29.9 vs. 26.3 microns). Double-labeling studies assessed possible ophthalmic receptor surfaces innervated by centrally crossing primary afferents. DY was injected into right medullary and cervical dorsal horns, and HRP was applied to either the left cornea, the ethmoid nerve, or the dura overlying cerebral cortex. Though DY labeled from 75 to 125 left ganglion cells per animal, no cells were double-labeled. All of these findings suggest that nociceptive-specific ganglion cells are not a source of the crossed ophthalmic primary afferent projection. Unilateral transection of the infraorbital nerve on the day of birth did not alter the crossed primary afferent projection to the partially deafferented side of the brainstem. This is further evidence of an absence of central sprouting in spared V primary afferents following neonatal V deafferentation.     

Trigeminal Projections to Contralateral Dorsal Horn Originate in Midline Hairy Skin

The present study tested the hypothesis that the trigeminal (V) primary afferent projection to the contralateral dorsal horn originates in midline hairy skin. A prior study (Jacquin et al., 1990) showed that this crossed projection is heaviest to ophthalmic regions of medullary and cervical dorsal horns, and that it does not arise from V ganglion cells that innervate cornea, nasal mucosa, or cerebral dura mater. Here, retrograde double-labeling methods were used to show that many ophthalmic ganglion cells that innervate midline hairy skin via the supraorbital nerve project to the contralateral medullary and upper cervical dorsal horns. Diamidino yellow injections into the right dorsal horn labeled an average of 104 cells in the left V ganglion. Of these contralaterally projecting ganglion cells, an average of 45% were also labeled by horseradish peroxidase (HRP) injections into the left supraorbital nerve, and 25% were also labeled by HRP injections into the midline opthalmic hairy skin. However, only 2% were labeled by HRP injections restricted to left supraorbital vibrissae follicle nerves. Almost all of the double-labeled cells were located in the dorsal one-half of the V ganglion, and they did not differ in size from single-labeled cells.

On the basis of these and prior data, we conclude that a high percentage of contralaterally projecting V ganglion cells originate in midline hairy skin. It is also likely that the contralaterally projecting V ganglion cells serve a low-threshold mechanoreceptive function, given the relatively large ganglion cells and axons giving rise to this pathway and their central terminations in dorsal horn laminae III-V.     

Intersubnuclear connections within the rat trigeminal brainstem complex

Prior intracellular recording and labeling experiments have documented local-circuit and projection neurons in the spinal trigeminal (V) nucleus with axons that arborize in more rostral and caudal spinal trigeminal subnuclei and nucleus principalis. Anterograde tracing studies were therefore carried out to assess the origin, extent, distribution, and morphology of such intersubnuclear axons in the rat trigeminal brainstem nuclear complex (TBNC). Phaseolus vulgaris leucoagglutinin (PHA-L) was used as the anterograde marker because of its high sensitivity and the morphological detail provided. Injections restricted to TBNC subnucleus caudalis resulted in dense terminal labeling in each of the more rostral ipsilateral subnuclei. Subnucleus interpolaris projected ipsilaterally and heavily to magnocellular portions of subnucleus caudalis, as well as subnucleus oralis and nucleus principalis. Nucleus principalis, on the other hand, had only a sparse projection to each of the caudal ipsilateral subnuclei. Intersubnuclear axons most frequently traveled in the deep bundles within the TBNC, the V spinal tract, and the reticular formation. They gave rise to a number of circumscribed, highly branched arbors with many boutons of the terminal and en passant types. Retrograde single- or multiple-labeling experiments assessed the cells giving rise to TBNC intersubnuclear collaterals. Horseradish peroxidase (HRP) and/or fluorescent tracer injections into the thalamus, colliculus, cerebellum, nucleus principalis, and/or subnucleus caudalis revealed large numbers of neurons in subnuclei caudalis, interpolaris, and oralis projecting to the region of nucleus principalis. Cells projecting to more caudal spinal trigeminal regions were most numerous in subnuclei interpolaris and oralis. Some cells in lamina V of subnucleus caudalis and in subnuclei interpolaris and oralis projected to thalamus and/or colliculus, as well as other TBNC subnuclei. Such collateral projections were rare in nucleus principalis and more superficial laminae of subnucleus caudalis. TBNC cells labeled by cerebellar injections were not double-labeled by tracer injections into the thalamus, colliculus, or TBNC. These findings lend generality to currently available data obtained with intracellular recording and HRP labeling methods, and suggest that most intersubnuclear axons originate in TBNC local-circuit neurons, though some originate in cells that project to midbrain and/or diencephalon.     

Neonatal transection alters the percentage of substance-P-positive trigeminal ganglion cells that contribute axons to the regenerate infraorbital nerve

Neonatal transection results in a marked reduction of the number of trigeminal (V) ganglion cells that contribute axons to the regenerate infraorbital nerve (ION; Jacquin and Rhoades, 1985; Chiaia et al., 1987). Such lesions also produce a profound deafferentation of the V brain stem complex that appears to spare the innervation of layers I and II of subnucleus caudalis (SpC) by substance-P-positive (SP-positive) primary afferents (Jacquin and Rhoades, 1985; Rhoades et al., 1988). In the present study, we combined retrograde tracing with immunocytochemistry to determine whether neonatal transection of the ION alters the percentage of SP-positive V ganglion cells that contribute axons to this V branch upon regeneration. In V ganglia ipsilateral to the intact ION (n = 8), 11.6% +/- 3.2% of the cells labeled after application of true blue (TB) to the ION were also SP-positive. In ganglia ipsilateral to the neonatally damaged nerve (n = 8), 18.6% +/- 4.7% of the cells labeled after application of TB to the regenerate ION were also SP-positive (p less than 0.001). We also compared the SP content of intact ganglia (n = 10) with that of ganglia ipsilateral to the damaged nerve (n = 10) by means of radioimmunoassay. The normal V ganglia contained (mean +/- SD) 3496 +/- 774 pg SP/mg protein. The value for the ganglia ipsilateral to the damaged nerve was 5533 +/- 1746 pg SP/mg protein (p less than 0.01). There was no significant difference between SP levels on the control and partially deafferented sides of the brain stem in neonatally nerve-damaged adult rats. In one additional experiment, we injected TB into both vibrissa pads of seven rats on the day of birth prior to transection of the ION. After an 8-hr delay, the nerve on one side was then cut and allowed to regenerate, and both V ganglia were then processed for immunocytochemistry. On the nerve-damage side, 25.8% of the TB-labeled cells were SP-positive. The value for the intact side was 12.0% (p less than 0.000001). This result demonstrated that the lesion-induced change in the percentage of SP-positive ION cells was not the result of either late-growing axons from SP-positive ganglion cells that may have been missed by our nerve cuts or collateral sprouting into the regenerate ION by undamaged SP-positive ganglion cells.     

Intersubnuclear Connections within the Rat Trigeminal Brainstem Complex

Prior intracellular recording and labeling experiments have documented local-circuit and projection neurons in the spinal trigeminal (V) nucleus with axons that arborize in more rostral and caudal spinal trigeminal subnuclei and nucleus principalis. Anterograde tracing studies were therefore carried out to assess the origin, extent, distribution, and morphology of such intersubnuclear axons in the rat trigeminal brainstem nuclear complex (TBNC). Phaseolus vulgaris leucoagglutinin (PHA-L) was used as the anterograde marker because of its high sensitivity and the morphological detail provided. Injections restricted to TBNC subnucleus caudalis resulted in dense terminal labeling in each of the more rostral ipsilateral subnuclei. Subnucleus interpolaris projected ipsilaterally and heavily to magnocellular portions of subnucleus caudalis, as well as subnucleus oralis and nucleus principalis. Nucleus principalis, on the other hand, had only a sparse projection to each of the caudal ipsilateral subnuclei. Intersubnuclear axons most frequently traveled in the deep bundles within the TBNC, the V spinal tract, and the reticular formation. They gave rise to a number of circumscribed, highly branched arbors with many boutons of the terminal and en passant types.

Retrograde single- or multiple-labeling experiments assessed the cells giving rise to TBNC intersubnuclear collaterals. Horseradish peroxidase (HRP) and/or fluorescent tracer injections into the thalamus, colliculus, cerebellum, nucleus principalis, and/or subnucleus caudalis revealed large numbers of neurons in subnuclei caudalis, interpolaris, and oralis projecting to the region of nucleus principalis. Cells projecting to more caudal spinal trigeminal regions were most numerous in subnuclei interpolaris and oralis. Some cells in lamina V of subnucleus caudalis and in subnuclei interpolaris and oralis projected to thalamus and/or colliculus, as well as other TBNC subnuclei. Such collateral projections were rare in nucleus principalis and more superficial laminae of subnucleus caudalis. TBNC cells labeled by cerebellar injections were not double-labeled by tracer injections into the thalamus, colliculus, or TBNC.

These findings lend generality to currently available data obtained with intracellular recording and HRP labeling methods, and suggest that most intersubnuclear axons originate in TBNC local-circuit neurons, though some originate in cells that project to midbrain and/or diencephalon.     

Trigeminal projections to contralateral dorsal horn originate in midline hairy skin

The present study tested the hypothesis that the trigeminal (V) primary afferent projection to the contralateral dorsal horn originates in midline hairy skin. A prior study (Jacquin et al., 1990) showed that this crossed projection is heaviest to ophthalmic regions of medullary and cervical dorsal horns, and that it does not arise from V ganglion cells that innervate cornea, nasal mucosa, or cerebral dura mater. Here, retrograde double-labeling methods were used to show that many ophthalmic ganglion cells that innervate midline hairy skin via the supraorbital nerve project to the contralateral medullary and upper cervical dorsal horns. Diamidino yellow injections into the right dorsal horn labeled an average of 104 cells in the left V ganglion. Of these contralaterally projecting ganglion cells, an average of 45% were also labeled by horseradish peroxidase (HRP) injections into the left supraorbital nerve, and 25% were also labeled by HRP injections into the midline opthalmic hairy skin. However, only 2% were labeled by HRP injections restricted to left supraorbital vibrissae follicle nerves. Almost all of the double-labeled cells were located in the dorsal one-half of the V ganglion, and they did not differ in size from single-labeled cells. On the basis of these and prior data, we conclude that a high percentage of contralaterally projecting V ganglion cells originate in midline hairy skin. It is also likely that the contralaterally projecting V ganglion cells serve a low-threshold mechanoreceptive function, given the relatively large ganglion cells and axons giving rise to this pathway and their central terminations in dorsal horn laminae III-V.     

In vitro formation of the Merkel cell‐neurite complex in embryonic mouse whiskers using organotypic co‐cultures

A Merkel cell‐neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch‐sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell‐neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell‐neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co‐culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell‐neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co‐cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell‐neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament‐H‐positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell‐neurite complex can be observed under a microscope using our organotypic co‐culture method.     

Neonatal Transection Alters the Percentage of Substance-P-Positive Trigeminal Ganglion Cells That Contribute Axons to the Regenerate Infraorbital Nerve

Neonatal transection results in a marked reduction of the number of trigeminal (V) ganglion cells that contribute axons to the regenerate infraorbital nerve (ION; Jacquin and Rhoades, 1985; Chiaia et al., 1987). Such lesions also produce a profound deafferentation of the V brain stem complex that appears to spare the innervation of layers I and II of subnucleus caudalis (SpC) by subtance-P-positive (SP-positive) primary afferents (Jacquin and Rhoades, 1985; Rhoades et al., 1988). In the present study, we combined retrograde tracing with immunocytochemistry to determine whether neonatal transection of the ION alters the percentage of SP-positive V ganglion cells that contribute axons to this V branch upon regeneration. In V ganglia ipsilateral to the intact ION (n = 8), 11.6% ± 3.2% of the cells labeled after application of true blue (TB) to the ION were also SP-positive. In ganglia ipsilateral to the neonatally damaged nerve (n = 8), 18.6% ± 4.7% of the cells labeled after application of TB to the regenerate ION were also SP-positive (p < 0.001). We also compared the SP content of intact ganglia (n = 10) with that of ganglia ipsilateral to the damaged nerve (n = 10) by means of radioimmunoassay. The normal V ganglia contained (mean ± SD) 3496 ± 774 pg SP/mg protein. The value for the ganglia ipsilateral to the damaged nerve was 5533 ± 1746 pg SP/mg protein (p < 0.01). There was no significant difference between SP levels on the control and partially deafferented sides of the brain stem in neonatally nerve-damaged adult rats.

In one additional experiment, we injected TB into both vibrissa pads of seven rats on the day of birth prior to transection of the ION. After an 8-hr delay, the nerve on one side was then cut and allowed to regenerate, and both V ganglia were then processed for immunocytochemistry. On the nerve-damaged side, 25.8% of the TB-labeled cells were SP-positive. The value for the intact side was 12.0% (p < 0.00001). This result demonstrated that the lesion-induced change in the percentage of SP-positive ION cells was not1. the result of either late-growing axons from SP-positive ganglion cells that may have been missed by our nerve cuts or collateral sprouting into the regenerate ION by undamaged SP-positive ganglion cells.     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

Formation of hair follicles from a single-cell suspension of embryonic rat skin by a two-step procedure in vitro

Summary A technique for culturing skin was devised whereby hair follicles in a normal state were generated from a single-cell suspension of embryonic rat skin. Dissociated cells obtained by trypsinization of the day-15 embryonic lip were cultured by a two-step procedure in vitro. Reorganization of hair-follicle rudiments was accompanied by reaggregation of the cells during a 24-hour initial culture with rotation, and the rudiments differentiated into hair follicles within a week during subsequent subculture of the cell aggregates by floatation. The light-microscopic features and the size of the follicles were similar to those of day-18 vibrissa follicles during normal development in vivo. Furthermore, the stratification of cells, including subcellular differentiation, and the ultrastructure of the hair follicles generated in vitro were similar to those of normal hair follicles with well-keratinized hair shafts. The present system appears to be a useful model for analytical studies in vitro on the formation of hair follicles and for studies designed to facilitate the transplantation of human hair.     

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.     

Neural correlates of vibrissa resonance; band-pass and somatotopic representation of high-frequency stimuli

The array of vibrissae on a rat's face is the first stage of a high-resolution tactile sensing system. Recently, it was discovered that vibrissae (whiskers) resonate when stimulated at specific frequencies, generating several-fold increases in motion amplitude. We investigated the neural correlates of vibrissa resonance in trigeminal ganglion and primary somatosensory cortex (SI) neurons (regular and fast spiking units) by presenting low-amplitude, high-frequency vibrissa stimulation. We found that somatosensory neurons showed band-pass tuning and enhanced sensitivity to small amplitude stimuli, reflecting the resonance amplification of vibrissa motion. Further, a putative somatotopic map of frequency selectivity was observed in SI, with isofrequency columns extending along the representations of arcs of vibrissae, in agreement with the gradient in vibrissa resonance across the vibrissa pad. These findings suggest several parallels between frequency processing in the vibrissa system and the auditory system and have important implications for detection and discrimination of tactile information.     

Localization of vtiamin A by autofluorescence during induced metaplastic changes in cultures of skin

A technique was devised for following the uptake and location of vitamin A in organ cultures. Explants of 12- and 13-day embryonic mouse upper lip skin were grown for 3, 6 or 9 days in biological medium to which was added 0, 4.1 or 6.9 micrograms per ml of retinyl acetate. This form of vitamin A caused glandular morphogenesis of vibrissa follicles, and keratinization in epidermis and follicles was completely suppressed in 12-day explants and partially suppressed in 13-day explants. Frozen sections at 16 microns showed the white, nonfading fluorescence of keratin and the green, rapidly-fading fluorescence due to vitamin A which was captured by high-speed photography. Although more concentrated within lipid droplets in the dermis, the vitamin penetrated both the epidermis and the hair follicles. The ability to obtain permanent photographic records of the fading fluorescence makes this a useful method for analyzing vitamin A distribution as well as keratin distribution.     

Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals

It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.     

Principalis- or parabrachial-projecting spinal trigeminal neurons do not stain for GABA or GAD

Retrograde transport and immunohistochemical double-labeling methods (Weinberg et al., 1985) were used to assess the distribution and projection status of spinal trigeminal (SpV) neurons that stain positively for glutamic acid decarboxylase (GAD) or gamma-aminobutyric acid (GABA). Large bilateral injections of diamidino yellow into the rostral and lateral pons, inclusive of V nucleus principalis and the parabrachial nucleus, retrogradely labeled large numbers of cells in each SpV subnucleus. Many cells in SpV subnuclei caudalis, interpolaris, and oralis also exhibited GABA immunoreactivity; the largest numbers were in caudalis and the smallest numbers were in oralis. However, none of the GABA- or GAD-immunoreactive SpV cells were double-labeled with diamidino yellow, though some reticular neurons displayed both GABA and the retrograde tracer. This negative result refutes a previously offered hypothesis that SpV local-circuit neurons with principalis collaterals are GABA-ergic (Jacquin et al., 1989b). These data also indicate that parabrachial-projecting SpV neurons are not GABA-ergic.     

Occurrence of parasympathetic vasodilator fibers in the lower lip of the guinea-pig

The present study was designed to examine whether there are parasympathetic vasodilator fibers in the lower lip of the guinea-pig. Electrical stimulation of the central cut end of the lingual nerve of guinea-pigs evoked intensity- and frequency-dependent decreases in lower lip blood flow and systemic arterial blood pressure (SABP). Pretreatment with guanethidine, a postganglionic sympathetic nerve blocker and antihypertensive drug (30 mg kg⁻¹, s.c., 24 h prior to experiments), reduced the magnitude of the decrease in SABP while the intensity- and frequency-dependent increases of the lip blood flow occurred by the lingual nerve stimulation only on the side ipsilateral to stimulation. Increases in the lip blood flow evoked by lingual nerve stimulation in guanethidine pretreated guinea-pigs were reduced by hexamethonium (an autonomic ganglion cholinergic blocker) in a dose-dependent manner. When fluoro-gold (a retrograde neural tracer) was injected into the lower lip, labeled neurons were observed in the ipsilateral otic ganglion. The present study indicates the presence of parasympathetic vasodilator fibers originating from the otic parasympathetic ganglion in the guinea-pig lower lip, similar to those reported previously in rats, cats, rabbits and humans.     

Parasympathetic control of the heart. II. A novel interganglionic intrinsic cardiac circuit mediates neural control of heart rate.

Intracardiac pathways mediating the parasympathetic control of various cardiac functions are incompletely understood. Several intracardiac ganglia have been demonstrated to potently influence cardiac rate [the sinoatrial (SA) ganglion], atrioventricular (AV) conduction (the AV ganglion), or left ventricular contractility (the cranioventricular ganglion). However, there are numerous ganglia found throughout the heart whose functions are poorly characterized. One such ganglion, the posterior atrial (PA) ganglion, is found in a fat pad on the rostral dorsal surface of the right atrium. We have investigated the potential impact of this ganglion on cardiac rate and AV conduction. We report that microinjections of a ganglionic blocker into the PA ganglion significantly attenuates the negative chronotropic effects of vagal stimulation without significantly influencing negative dromotropic effects. Because prior evidence indicates that the PA ganglion does not project to the SA node, we neuroanatomically tested the hypothesis that the PA ganglion mediates its effect on cardiac rate through an interganglionic projection to the SA ganglion. Subsequent to microinjections of the retrograde tracer fast blue into the SA ganglion, >70% of the retrogradely labeled neurons found within five intracardiac ganglia throughout the heart were observed in the PA ganglion. The neuroanatomic data further indicate that intraganglionic neuronal circuits are found within the SA ganglion. The present data support the hypothesis that two interacting cardiac centers, i.e., the SA and PA ganglia, mediate the peripheral parasympathetic control of cardiac rate. These data further support the emerging concept of an intrinsic cardiac nervous system.     

Essence of keratin in lips and associated structures of a freshwater fish Puntius sophore in relation to its feeding ecology: Histochemistry and scanning electron microscope investigation

Morphological specializations in the lips and associated structures of Puntius sophore were examined by scanning electron microscopy and histochemically. The upper lip (UL), in P. sophore, is associated with the horny upper jaw sheath (HUJS) on its ventral side and with the rostral cap (RC) on its dorsal side through a thin and extensive fold of skin (FSUR). The lower lip (LL) is greatly enlarged, conspicuous and associated with horny lower jaw sheath (HLJS) on the dorsal side and ventrally continues with ventral head skin (VHS). On the lateral sides there is a thin and extensive fold of skin (FSLS) between the lower lip and VHS. In contrast to the mucogenic epithelia of the UL, LL, the RC and fold of skins, the horny jaw sheaths are keratinized in nature and surface epithelial cells are characteristically modified into unculi. The UL and the LL are equipped with epithelial cells (EC), mucous cells (MC) and taste buds (TB) while in addition to these cells club cells (CC) are also present in the RC. Keratin found in unculi is an extremely strong protein which is tough and insoluble, they form the hard but un-mineralized structures. Keratin in unculi could be regarded as an adaptation for browsing or scraping food materials from the substrate as the fish grubs about the bottom. The elaboration of mucus is considered to lubricate the surface and protect the epithelia from abrasions. Taste buds are associated to locate and select palatable food and to trigger a ‘pick-up’ reflex.     

Organization of the cortico-ponto-cerebellar pathway to the dorsal paraflocculus. An experimental study with anterograde and retrograde transport of WGA-HRP in the cat.

To reveal the organization and relative magnitude of connections from various parts of the cerebral cortex to the dorsal paraflocculus via the pontine nuclei, WGA-HRP was injected in the dorsal paraflocculus in conjunction with injection of the same tracer in various parts of the cerebral cortex in 17 cats. Termination areas of cortical fibres (anterogradely labelled) and pontine neurons projecting to the dorsal paraflocculus (retrogradely labelled) were carefully plotted in serial transverse sections. As an average of countings in ten cats, 90% of the labelled cells were found in the pontine nuclei contralateral to the injection, and the majority (70%) were located in the rostral half of the nuclei. The highest degree of overlap between anterograde and retrograde labelling was found after injections of the parietal association cortex (areas 5 and 7). In an experiment with double anterograde tracing, it was shown that both area 5 and 7 contribute substantially to the cerebral inputs to the dorsal paraflocculus. High degree of overlap also occurred after injections of several visual cortical areas (areas 17, 18, 19, 20 and the posteromedial lateral suprasylvian visual area, PMLS). Cases with injections restricted to individual visual areas indicate that they all contribute to the parafloccular input. Considerably less overlap occurred after injections of the primary sensorimotor region (SI, MI) and second somatosensory area (SII), while the supplementary motor area, the auditory cortex and gyrus cinguli probably have no or very restricted access to the dorsal paraflocculus. It is concluded that the dorsal paraflocculus has its major cortical input from the parietal association cortex and the visual cortical areas. Since all the various cortical regions studied project to largely different parts of the pontine nuclei, and overlap with neurons projecting to the dorsal paraflocculus takes place at numerous places, it follows that the pontine neurons projecting to the dorsal paraflocculus must consist of many subgroups differing with regard to their cortical input.