Exp Brain Res (1990) 81:649-653
Experimental BrainResearch 9
Springer-Verlag1990
Topography of the facial musculature within the facial (VII) motor nucleus of the neonatal rat B.G. Klein 1, R.W. Rhoades 2, and M.F. Jacquin 3 1 Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA 2 Department of Anatomy, Medical College of Ohio at Toledo, C.S. 10008, Toledo, OH 43699, USA 3 Department of Anatomy and Neurobiology, St. Louis University School of Medicine, St. Louis, MO 63104, USA Received November 21, 1989 / Accepted April 17, 1990
Summary. W G A - H R P , H R P and fluorescent tracers were used to determine the representation of the facial muscles in the facial m o t o r nuclear complex ( F M N C ) of the newborn rat. Tracer injections of the superficial cervical and anterior mandibular portions of platysma, the orbicularis oculi muscle, the nasolabial musculature and the posterior auricular musculature revealed an adultlike topographic organization across F M N C subnuclei. Tracer delivery to individual vibrissa follicle loci o f the whiskerpad also demonstrated an adult-like musculotopic organization within the lateral subnucleus.
Key words: M o t o n e u r o n s - Musculotopic organization - Whisker follicle - Brainstem - Rat
Introduction It is well known that the adult rodent facial nucleus can be architectonically partitioned into discrete subnuclei. Several anatomical and electrophysiological studies have demonstrated an orderly representation of the facial nerves and musculature both across and within the subdivisions of this facial m o t o r nuclear complex ( F M N C ) (Martin and Lodge 1977; Ashwell 1982; Watson et al. 1982; Klein and Rhoades 1985; Semba and Egger 1986). In rat, the cytoarchitectonic subdivisions c o m m o n l y noted in the adult F M N C (Martin and Lodge 1977; Erzurumlu and Killackey 1979; Klein and Rhoades 1985; Semba and Egger 1986) have been shown to be present at birth (Hogg ~928). However, to our knowledge, the representation of the facial musculature within the F M N C has not been described in neonatal rats. This question is significant in light o f the fact that the highly ordered facial m o t o r system of the rat has been used to examine the extent o f musculotopic organization in the adult following neonatal facial nerve transection (AidOffprint requests to. B.G. Klein (address see above)
skogius and Thomander 1986). We have therefore utilized the retrograde transport of single or multiple tracers to determine the representation of the facial musculature, both across and within the subnuclei of the F M N C in newborn rats.
Material and methods The locations of muscles or muscle groups targeted for tracer injection in neonates were based upon studies of FMNC musculotopic organization in adult rodents (Ashwell 1982; Hinrichsen and Watson 1984; Klein and Rhoades 1985). Newborn Wistar rats were anesthetized by ice water hypothermia, less than 12 h after birth. All tracers were delivered with the aid of a dissecting microscope. Injections of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP, Sigma, 5% in water, 10-30 nl) into the anterior mandibular region of platysma, superficial cervical region of platysma and posterior auricular musculature were made through a small cutaneous incision, using a glass micropipette (tip diameter 20-40 pro). The orbicularis oculi muscle and the nasolabial musculature were targeted transcutaneously by respective WGA-HRP injections into the upper eyelid and whiskerpad. For a more detailed assessment of nasolabial musculotopic organization in the neonate, tracers were delivered subcutaneously, at the base of specific mystacial vibrissae. Using fine microdissecting forceps, HRP crystals (Sigma, Type VI) were pushed just below the skin at the base of a single vibrissa on each whiskerpad. With the aid of a glass micropipette (tip diameter 30-60 pm), multiple vibrissae on a single whiskerpad were respectivelyinjected with aqueous solutions (2.5 %, 10--50 nl) of the fluorescent tracers true blue (TB, Sigma) and diamidino yellow (DY, Dr. Illing GmbH & Co.). Survival times ranged from 10-20 h for pups receiving WGA-HRP or HRP alone and from 12-48 h for rats injected with fluorescent tracers. Topography was similar for shortest and longest survival times. However, label intensity was greater at longer survival times. Following deep ether anesthesia, pups were perfused and brains and whiskerpads were sectioned and stained according to previously described methods (Klein and Rhoades 1985).
Results Our results demonstrate that in newborn rats, the facial musculature is represented in an adult-like fashion, both
650
across and within FMNC subnuclei. Figure 1 illustrates the location of WGA-HRP delivery sites and the corresponding locations of retrogradely labeled cells. Since FMNC cytoarchitectonic subdivisions are similar in neonatal and adult rats, the terminology of Hogg (1928) and Martin and Lodge (1977) is used. In all cases, labeled neurons within a given subnucleus were present throughout its rostrocaudal extent. Injection of tracer into the
nasolabial musculature produced heavy labeling within the lateral and dorsal subnuclei (Fig. 1A). WGA-HRP injections targeting the orbicularis oculi muscle resulted in retrograde labeling within the most dorsal portions of the dorsal, intermediate and medial subnuclei (Fig. 1B). Injections of the anterior mandibular portion of platysma resulted in retrograde labeling primarily within the medial portion of the intermediate subnucleus (Fig. 1C),
Fig. 1A-F, Brightfield photomicrographs showing the location of W G A - H R P labeled cells in thionin stained transverse sections of neonatal F M N C . Figurines denote injection site. Three cases were analyzed for each site. All sections were taken from the approximate
midpoint of the rostrocaudal axis of the F M N C . F Depicts the F M N C snbnuclei in an unlabeled section. D = dorsal subnucleus, L = l a t e r a l subnucleus, I = i n t e r m e d i a t e subnucleus, V M = v e n tromedial subnucleus, M = medial subnucleus
651 while injections of the superficial cervical region of platysma labeled neurons in the ventromedial nucleus and at the border between the intermediate and medial subnuclei (Fig. 1D). When W G A - H R P was delivered to the posterior auricular musculature, labeled neurons were observed throughout the medial subnucleus, with heaviest labeling in the ventral two-thirds (Fig. 1E). A topographic organization of the dorsoventral axis of the whiskerpad was revealed within the lateral subnucleus. Figures 2 and 3 present the pattern o f retrograde labeling produced by unilateral implantation o f H R P crystals into single whisker follicle loci or unilateral injection of fluorescent tracers into loci of different rows or columns of vibrissae. Delivery o f H R P crystals to the A2 (nomenclature of Arvidsson 1982) follicle locus labeled cells primarily in the lateral portion of the lateral subnucleus and in the dorsal subnucleus (Fig. 2A). Neurons in the medial portion of the lateral subnucleus were most heavily labeled by tracer implantation of the E2 follicle locus (Fig. 2B). When TB and D Y were respectively delivered to A2 and E2 follicle loci of the same whiskerpad, D Y labeled cells were located medially to TB labeled neurons (Fig. 2C, C').
N o such topography was apparent for the rostrocaudal axis of the whiskerpad. Delivery of H R P crystals to the CI or C5 follicle loci of individual whiskerpads labeled cells throughout the dorsoventral extent of the central portion of the lateral subnucleus (Figs. 3A, B, respectively). In concordance, delivery of DY and TB to these respective loci of the same whiskerpad produced an overlapping pattern of labeled cells along the dorsoventral and rostrocaudal axes of the lateral subnucleus (Figs. 3C, C'). Inspection of injection sites suggested that this pattern of labeling was not a result of overlapping tracer deposits.
Discussion The data presented above describe a topographic representation of the facial musculature both across and within subnuclei of the F M N C of the newborn rat. The musculotopic organization across F M N C subnuclei was virtually identical to that observed in adult rats when direct intramuscular or percutaneous injections of H R P
A
Fig. 2. A, B Are brightfield photomicrographs of retrogradely lebeled cells in the FMNC following respective implantation of HRP crystals into the A2 or E2 vibrissa follicle loci of different whiskerpads. C ls an episcopic fluorescence photomicrograph (355 nm excitation wavelength) depicting labeled neurons resulting from respective delivery of TB and DY to the A2 and E2 whisker
follicle loci of the same whiskerpad. C' Is same section at 410 nm excitation wavelength, which accentuates the fluorescence of DY labeled neurons and virtually eliminates fluorescence of TB labeled cells. Figurines illustrate tracer delivery sites and number of cases. D = dorsal, V = ventral, M = medial, L = lateral. Note topographic separation of cells along mediolateral axis of FMNC
652
Fig. 3 A-C'. Data presented in same fashion as Fig. 2, for tracer delivery to C 1 and/or C 5 vibrissa follicle loci. Note lack of topographic separation of labeled neurons
were employed (Watson et al. 1982; Hinrichsen and W a t s o n 1984). An adult-like topographic organization has been reported in mouse, prior to the onset o f cell death, following in utero (17 days post
Experimental BrainResearch 9
Springer-Verlag1990
Topography of the facial musculature within the facial (VII) motor nucleus of the neonatal rat B.G. Klein 1, R.W. Rhoades 2, and M.F. Jacquin 3 1 Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA 2 Department of Anatomy, Medical College of Ohio at Toledo, C.S. 10008, Toledo, OH 43699, USA 3 Department of Anatomy and Neurobiology, St. Louis University School of Medicine, St. Louis, MO 63104, USA Received November 21, 1989 / Accepted April 17, 1990
Summary. W G A - H R P , H R P and fluorescent tracers were used to determine the representation of the facial muscles in the facial m o t o r nuclear complex ( F M N C ) of the newborn rat. Tracer injections of the superficial cervical and anterior mandibular portions of platysma, the orbicularis oculi muscle, the nasolabial musculature and the posterior auricular musculature revealed an adultlike topographic organization across F M N C subnuclei. Tracer delivery to individual vibrissa follicle loci o f the whiskerpad also demonstrated an adult-like musculotopic organization within the lateral subnucleus.
Key words: M o t o n e u r o n s - Musculotopic organization - Whisker follicle - Brainstem - Rat
Introduction It is well known that the adult rodent facial nucleus can be architectonically partitioned into discrete subnuclei. Several anatomical and electrophysiological studies have demonstrated an orderly representation of the facial nerves and musculature both across and within the subdivisions of this facial m o t o r nuclear complex ( F M N C ) (Martin and Lodge 1977; Ashwell 1982; Watson et al. 1982; Klein and Rhoades 1985; Semba and Egger 1986). In rat, the cytoarchitectonic subdivisions c o m m o n l y noted in the adult F M N C (Martin and Lodge 1977; Erzurumlu and Killackey 1979; Klein and Rhoades 1985; Semba and Egger 1986) have been shown to be present at birth (Hogg ~928). However, to our knowledge, the representation of the facial musculature within the F M N C has not been described in neonatal rats. This question is significant in light o f the fact that the highly ordered facial m o t o r system of the rat has been used to examine the extent o f musculotopic organization in the adult following neonatal facial nerve transection (AidOffprint requests to. B.G. Klein (address see above)
skogius and Thomander 1986). We have therefore utilized the retrograde transport of single or multiple tracers to determine the representation of the facial musculature, both across and within the subnuclei of the F M N C in newborn rats.
Material and methods The locations of muscles or muscle groups targeted for tracer injection in neonates were based upon studies of FMNC musculotopic organization in adult rodents (Ashwell 1982; Hinrichsen and Watson 1984; Klein and Rhoades 1985). Newborn Wistar rats were anesthetized by ice water hypothermia, less than 12 h after birth. All tracers were delivered with the aid of a dissecting microscope. Injections of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP, Sigma, 5% in water, 10-30 nl) into the anterior mandibular region of platysma, superficial cervical region of platysma and posterior auricular musculature were made through a small cutaneous incision, using a glass micropipette (tip diameter 20-40 pro). The orbicularis oculi muscle and the nasolabial musculature were targeted transcutaneously by respective WGA-HRP injections into the upper eyelid and whiskerpad. For a more detailed assessment of nasolabial musculotopic organization in the neonate, tracers were delivered subcutaneously, at the base of specific mystacial vibrissae. Using fine microdissecting forceps, HRP crystals (Sigma, Type VI) were pushed just below the skin at the base of a single vibrissa on each whiskerpad. With the aid of a glass micropipette (tip diameter 30-60 pm), multiple vibrissae on a single whiskerpad were respectivelyinjected with aqueous solutions (2.5 %, 10--50 nl) of the fluorescent tracers true blue (TB, Sigma) and diamidino yellow (DY, Dr. Illing GmbH & Co.). Survival times ranged from 10-20 h for pups receiving WGA-HRP or HRP alone and from 12-48 h for rats injected with fluorescent tracers. Topography was similar for shortest and longest survival times. However, label intensity was greater at longer survival times. Following deep ether anesthesia, pups were perfused and brains and whiskerpads were sectioned and stained according to previously described methods (Klein and Rhoades 1985).
Results Our results demonstrate that in newborn rats, the facial musculature is represented in an adult-like fashion, both
650
across and within FMNC subnuclei. Figure 1 illustrates the location of WGA-HRP delivery sites and the corresponding locations of retrogradely labeled cells. Since FMNC cytoarchitectonic subdivisions are similar in neonatal and adult rats, the terminology of Hogg (1928) and Martin and Lodge (1977) is used. In all cases, labeled neurons within a given subnucleus were present throughout its rostrocaudal extent. Injection of tracer into the
nasolabial musculature produced heavy labeling within the lateral and dorsal subnuclei (Fig. 1A). WGA-HRP injections targeting the orbicularis oculi muscle resulted in retrograde labeling within the most dorsal portions of the dorsal, intermediate and medial subnuclei (Fig. 1B). Injections of the anterior mandibular portion of platysma resulted in retrograde labeling primarily within the medial portion of the intermediate subnucleus (Fig. 1C),
Fig. 1A-F, Brightfield photomicrographs showing the location of W G A - H R P labeled cells in thionin stained transverse sections of neonatal F M N C . Figurines denote injection site. Three cases were analyzed for each site. All sections were taken from the approximate
midpoint of the rostrocaudal axis of the F M N C . F Depicts the F M N C snbnuclei in an unlabeled section. D = dorsal subnucleus, L = l a t e r a l subnucleus, I = i n t e r m e d i a t e subnucleus, V M = v e n tromedial subnucleus, M = medial subnucleus
651 while injections of the superficial cervical region of platysma labeled neurons in the ventromedial nucleus and at the border between the intermediate and medial subnuclei (Fig. 1D). When W G A - H R P was delivered to the posterior auricular musculature, labeled neurons were observed throughout the medial subnucleus, with heaviest labeling in the ventral two-thirds (Fig. 1E). A topographic organization of the dorsoventral axis of the whiskerpad was revealed within the lateral subnucleus. Figures 2 and 3 present the pattern o f retrograde labeling produced by unilateral implantation o f H R P crystals into single whisker follicle loci or unilateral injection of fluorescent tracers into loci of different rows or columns of vibrissae. Delivery o f H R P crystals to the A2 (nomenclature of Arvidsson 1982) follicle locus labeled cells primarily in the lateral portion of the lateral subnucleus and in the dorsal subnucleus (Fig. 2A). Neurons in the medial portion of the lateral subnucleus were most heavily labeled by tracer implantation of the E2 follicle locus (Fig. 2B). When TB and D Y were respectively delivered to A2 and E2 follicle loci of the same whiskerpad, D Y labeled cells were located medially to TB labeled neurons (Fig. 2C, C').
N o such topography was apparent for the rostrocaudal axis of the whiskerpad. Delivery of H R P crystals to the CI or C5 follicle loci of individual whiskerpads labeled cells throughout the dorsoventral extent of the central portion of the lateral subnucleus (Figs. 3A, B, respectively). In concordance, delivery of DY and TB to these respective loci of the same whiskerpad produced an overlapping pattern of labeled cells along the dorsoventral and rostrocaudal axes of the lateral subnucleus (Figs. 3C, C'). Inspection of injection sites suggested that this pattern of labeling was not a result of overlapping tracer deposits.
Discussion The data presented above describe a topographic representation of the facial musculature both across and within subnuclei of the F M N C of the newborn rat. The musculotopic organization across F M N C subnuclei was virtually identical to that observed in adult rats when direct intramuscular or percutaneous injections of H R P
A
Fig. 2. A, B Are brightfield photomicrographs of retrogradely lebeled cells in the FMNC following respective implantation of HRP crystals into the A2 or E2 vibrissa follicle loci of different whiskerpads. C ls an episcopic fluorescence photomicrograph (355 nm excitation wavelength) depicting labeled neurons resulting from respective delivery of TB and DY to the A2 and E2 whisker
follicle loci of the same whiskerpad. C' Is same section at 410 nm excitation wavelength, which accentuates the fluorescence of DY labeled neurons and virtually eliminates fluorescence of TB labeled cells. Figurines illustrate tracer delivery sites and number of cases. D = dorsal, V = ventral, M = medial, L = lateral. Note topographic separation of cells along mediolateral axis of FMNC
652
Fig. 3 A-C'. Data presented in same fashion as Fig. 2, for tracer delivery to C 1 and/or C 5 vibrissa follicle loci. Note lack of topographic separation of labeled neurons
were employed (Watson et al. 1982; Hinrichsen and W a t s o n 1984). An adult-like topographic organization has been reported in mouse, prior to the onset o f cell death, following in utero (17 days post
