Visual corticopontine input to the paraflocculus: a combined autoradiographic and horseradish peroxidase study

Brain Reseaich, 143 (1978) 139-146 ~', El~ewer/North-Hol[and B~omed~ca[Press

139

Short Communications

Visual corticopontine input to the paraflocculus: a combined autoradiographic and horseradish peroxidase study

R.A

B U R N E , G A M I H A | L O F F and D J W O O D W A R D

Department of Cell Biology, The UmvetstO' of Te.~a~ Health Sctence Center at Dalla,s, Dallas, TeA 75235 ( U S A )

(Accepted September 29th, 1977)

The projection of visual information to the cerebellum has been known since the early studies of Snider and Stowell e4` in which visual-evoked potentials were recorded in the mldvermal region of the cat cerebellum. Anatomical Investigations have since demonstrated in more detail pathways from the wsual cortex 4,5, superior colhculus 9.1:~ and ventral lateral geniculate nucleus 10 to the basilar pons, all of which could relay visual information to the cerebellum. However, Hoddevik et al 11 have emphasized in a recent report that visual input to the mldvermal region of the cat cerebellum originates from pontme nuclei projected upon primarily by the superior colhculus. The outstanding question considered in this investigation was the deterruination of the cerebellar target of pontlne neurons projected upon by the visual cortex In the cat, Brodal 4,~ has demonstrated that the visual projection to the pons terminates upon ~transverse bands "4 of rostral pontme cells. In our previous study in the rat 7, a comparable rostral pontme region was shown to project to the lateral lobules of the cerebellum. Therefore, ~t appeared to us that reformation from visual cortex may reach major cerebellar areas other than the m~dvermal region. Results of work revolving the processing of lndlwdual sections through both autoradlographlc and horseradish peroxldase techniques as described in this report demonstrate that the visual cortex, through pontme neurons, strongly projects to the paraflocculus. This ~s a surprising result since the paraflocculus has not been previously suspected to be a major target for wsual information. Conventional hodologlcal techniques which take advantage of the orthograde and retrogradetransport of trltlated amino acids 18 and horseradish peroxldase (HRP) 19, respectively, were systematlcally employed to explore the pattern of the wsualcortlco-ponto-cerebellar projection. A total of 14 female Long-Evans rats weighing between 180 and 220 g were used in this study. Of the 14 ammals used, the wsual cortex of 5 rats received hydrauhc reJections of 0.2-0.3 t~1 (65-75 /~C1//~1) [4,5-3H]kleuclne ([3H]leuclne) (sp. act. 139 CI/mmole, Amersham) in water through a glass

140 microplpette (10-30/zm t~p) attached to a 2 #1 Hamilton syringe. At the time of m jection, the visual cortical site was defined electrophysiologically under pentobarblla~ anesthesm by recording a photlc flash-reduced evoked surface potential through the injection micropipette (Fig. 2E). The paraflocculus of 7 ammals received injections of 0.1-0.25/zl 40'~,~ HRP (Sigma, type VI) water solution through a 50-75 #m tipped glass mlcropipette. In two additional rats, the right visual cortex and the left paraflocculus received simultaneous injections of [3H]leucine and HRP, respectively. A survival period of 24 h was found to be satisfactory for both the retrograde and orthograde transport of HRP and [3H]leucine, respectively to the basilar pons. The general methodology for fixation, tissue processing and visualization of the HRP labehng has been described previously 6. For [3H]leucine-injected brains, 40 #m frozen sections were mounted from distdled water onto gelatin-coated slides, defatted, and coated w~th Kodak NTB-2 emulsion. The shdes were dried slowly in a humid atmosphere before being sealed m hght-t~ght containers and refrigerated at 4 °C. After 4 or 5 weeks of exposure, the coated sections were developed in Kodak D-19, treated with Ektaflo fixer and stained w,th cresyl wolet. For the two brains with dual [ZH]leucine and H R P injections, two series of alternating 40 #m coronal sections were collected. In one series the t~ssue was reacted with DAB and hydrogen peroxide to visualize the H R P reaction product. These same sections, as well as the second untreated series, were then coated with NTB-2 emulsion and processed as described above for autoradiography. With this methodology one series of sections was available for observation of the [aH]leucine labehng alone, while the second series of coronal sections revealed both the H R P and [3H]leucine labeling (Fig. 1C, E). This allowed visualization of autoradiographic grains over HRP-tabeted cells in the same section. In all H R P injections, the spread of H R P by diffusion wa~ confined to the paraflocculus or parafloccular peduncle. Typically, the accessory, ventral, and ventral dorsal lobules of the paraflocculus were intensely filled with H R P granule~ (Fig. I A). In 7 animals with parafloccular injecttons two separate groups: (1) a lateral aggregate consisting of dorsolateral, lateral and ventrolateral regions and (2) a medial aggregate of HRP-labeled cells, were observed bdaterally (contralateral predominance) to extend through several transverse sections of the rostral pons (representative case m Fig. 2C). In extreme rostral sections (Fig. 2C, section 1), a few labeled neurons were scattered diffusely between the medial and lateral aggregates. Also, in two other ammals, a third group of labeled neurons was observed along the ventral border of the pons (Fig. 2C, section 9). Following one or four week exposures, the labeling resulting from the diffusion of [3H]leucine injections appeared to be restricted to area 1714 in three and extended into area t 814 in four additional animals (Figs. 1B, 2B and 3) as determined by comparing the injection site location from the occipital pole and midline in histological sections with a cortical map defined electrophysiological by Montero et al. 2°. In all cases labeling was restricted to the cortical gray matter with additional labeling of axons originating from the injection site and entering the internal capsule.

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F~g l The mjectmn sites and resulting labeling of pontlne neurons following combined HRP and [,JH]leucme injections in rat 46 A a coronal sectmn of the cerebellum and brain stem through the HRP reject,on site m the left paraflocculus B: a coronal section through the [aH]leuclne injection site in the right visual cortex (1 week exposure) C and E. dark-field photomicrographs of the right rostral basllar pons showing the dlstnbutmn of both HRP-labeled neurons and silver grain labehng m the same section at low magnification (C) and high magnification (E). D and F. dark-field photom,crographs of the brain stern section adjacent to the one in C showing only the dlstrlbutmn of [JH][eucme labehng at low magmficatlon (D) and high magmficatlon (F). Arrows m C - F mdlcate a bundle of axons forming a convenient landmark Abbreviations : B P., brachlum pontis ; FI., flocculus ; PED, cerebral peduncle, Pfl a , accessory paraflocculus; Pfl d , dorsal paraflocculus; Pfl v , ventral paraflocculus.

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Fig. 2. D~agrams of the m lectlon sites and resulting HRP and [aH]leucine labehng m rat 46 dlustratmg the congruence of the wsual cort~co-pontme and ponto-parafloccular projections. A: a lateral view of the rat brain showing the H R P rejection site in the paraYlocculus. B: a dorsal vlew of the rat brain showing the [aH]leucine rejection site in areas 17 and 18, defined here according to the map of Kriegl~ C and D: drawings (1-15) represent consecutwe coronal pontine sections. Odd numbered sections (C) show the bilateral distrnbution of HRP-labeled neurons in medial and lateral aggregates Even numbered sectnons (D)show the lpsdateral silver gram labehng (4-week exposure) over the lateral pons. E ' vtsual-evoked potential recorded through the [~H]leucme rejection ptpette (3 traces superimposed). F a ventral wew of the rat brain stem mdvcatmg the rostrat-caudal level of the coronal sections shown m C and D For abbreviations see Fig I

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Fig 3 Vzsual c o r t l c o - p o n t m e proJectxons following a [aH]leuclne mJectzon into area 17 (rat 44) and an rejection extending into areas 17 a n d 18 (rat 18) A : the [3H]leucme rejection s~te a n d resulting sliver grain l a b e h n g m rat 44. B. the [3H]leucme reJection site and resulting silver g r a m l a b e h n g m rat 18. Sectlon n u m b e r s are the same as m F~g. 2 D

144 In two ammals with cortical mject~ons of [ZH]leucine restricted to aret~ 17, three aggregates of silver grains in the rostral pons were noted : medial and lateral group~ oriented transversely below the cerebral peduncle and a separate ventrolatcral aggregate (Fig. 3A). In three animals with injections extending into both areas 17 and 18, a dense accumulation of grams was observed over the dorsolateral and lateral pontine regions (Figs. 1D and 2D). In 4 additional ammals with similar rejection ~ltes, terminal labeling was observed over slightly dtfferent lateral and ventrolateral pontine regions (Fig. 3B). From the H R P results described above, these same dolsolateral, lateral and ventrolateral regions were shown to project to the parafloccutu~ To demonstrate more d~rectly the congruence of wsual afferent termlndl zone~ with the origins of efferent projections in the pons, two animals were subjected to simultaneous rejections of [3H]leucine in the visual cortex and H R P m the contralateral paraflocculus. Consecutive transverse sections of the pontine nuclei treated for H R P and autoradiography proved that lateral regions projected upt)ll by the ipsilateral visual cortex contain neurons whose axons reach the contralateral paraflocculus (Figs. 1C, I E, 2C and 2D). This congruence of lateral visual afferent termmat zones with the origin of parafloccular efferents was a consistent obserwlt~on Results from previous H R P studies 6 have mdicated that pontme neurons projecting to the cerebellar hemispheres were not located within the visual cortical terminal zones, thus suggestmg that visual cortical information is not primarily directed to ~)ther lateral cerebellar regions. Therefore, the results of the combined application of the Ietrograde H R P method and the orthograde autoradlographic techmque employed m this study demonstrates a strong visual cort~co-ponto-paraftoccular projection m the rz~t The visual cortical projection to the rostral half of the basilar pons ha~ been a common finding m all species of animals stud~ed so far. The demonstration of a visual cortical projection to the dorsolateral, lateral and ventrolateral pontine nucle~ confirms and extends the results of previous studies using the method of anterograde degeneration in the rat TM rabbit ~,s and monkey ~--','~. The medial and lateral labehng observed following [3H]leucine injections restricted to area 17 has not been prewously reported for the rat, rabbit or monkey. However, the medml and lateral labehng, which Is oriented horizontally below the cerebral peduncle, does resemble the "transverse bands '4 observed m the ventral aspect of the rostral pons m the c~tt :. ~.; and opossum 27. Based on H R P studies m the rat, Grayblel reported 1° that the midvermal lobules VI and VII of Larsel117 receive a dual projection from medial and lateral pontlne nucleL We have shown in th~s study that terminals arriving from wsual cortex overlap minimally with her described o n g m of midvermal projections, but maximally with areas projecting to paraflocculus. These data have suggested that the paraflocculus and not the midvermal posterior cerebellum is the primary target zone for visual cortex projections. The paraflocculus in rat may recewe other inputs as welt, since wc have also described electrophysmologlcal and anatomical evidence for input from the ~upenor colliculus 7. Supporting this conclusion is ewdence m cat that the paraflocculus receives an input from the superior ¢olliculus via the dorsolateral pontine region ~'',~3.

145 M o r e o v e r , H o d d e v l k et al. 11 have suggested that the m l d v e r m a l region, rather than paraflocculus, is the m a j o r target for tectal information. Previous studies 16,'3,~6 have i m p h c a ted the paraflocculus in functions ranging from the m a in te n a n c e o f muscle tone m ~psdateral extremities to the control o f auton o m l c activity: however, the exact primary f u n c t i o n remains obscure. A role in vestibular functions has been suggested by the finding 3 of a p r i m a r y vestibular projection to paraflocculus m cat, but m rat we have not observed cell projections to originate f r o m vestibular nuclei. W e have also observed that the mare parafloccular efferent projection zone was the dentate nucleus and not the brain stem nuclei (unpublished). Ou r hypothes~s is that the visual projection to paraflocculus may medmte higher VlSUO-motor integration, perhaps to m~tiate or co n t r o l m o v e m e n t guided by wsual information. We are r e m i n d e d of the fact that Cetacea have a very large paraflocculus 16. It would be o f interest to determine the visual mformat~on that is processed by such a large paraflocculus. It remains to be d e m o n s t r a t e d what visually d e p e n d e n t m o t o r patterns are mediated by paraflocculus in rat. This project was supported by N . I . H . G r a n t N S 13225 and N.S.F. G r a n t G B 43301 to D. J. W o o d w a r d .

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