Brain Research 925 (2002) 100–106www.elsevier.com/ locate /bres

Short communication

Evolutionary significance of different neurochemical organisation ofthe internal and external regions of auditory centres in the reptilian

brain: an immunocytochemical and reduced NADPH-diaphorasehistochemical study in turtles

a a,b a c , b,c* ´M.G. Belekhova , N.B. Kenigfest-Rio , N.P. Vesselkin , J.-P. Rio , J. Reperant ,b,dR. Ward

aLaboratory of Evolution of Neuronal Interactions, Sechenov Institute, St. Petersburg, Russiab ´ ´Laboratoire d’Anatomie comparee, Museum National d’Histoire Naturelle, Paris, France

c ´ ˆ ˆ `INSERM U 106, Neuromorphologie: Developpement, Evolution, Hopital de la Salpetriere, Paris, Franced ´ ´ ´ `Laboratoire de Neuropsychologie experimentale, Universite du Quebec, Trois-Rivieres, Canada

Accepted 5 October 2001

Abstract

An immunocytochemical and histochemical study was undertaken of the torus semicircularis and nucleus reuniens, the mesencephalicand diencephalic auditory centres, in two chelonian species, Testudo horsfieldi and Emys orbicularis. The nucleus centralis of the torussemicircularis receives few 5-HT-, TH-, substance P-, and menkephalin-immunoreactive fibres and terminals, in marked contrast to theexternal nucleus laminaris of the torus semicircularis, in which 5-HT-, TH-, substance P-, and menkephalin-immunoreactive elements andcell bodies show a laminar distribution. Dense NPY-positive terminal-like profiles and cell bodies were observed in both the nucleicentralis and laminaris, and many NADPH-d-positive cell bodies were observed in the cell layers of the latter. In the nucleus reuniens, thedistribution of 5-HT-, TH-, substance P-, and menkephalin-immunolabelling resembles that seen in the torus semicircularis, but at a lowerdensity. The dorsorostral regions of the nucleus reuniens, as in the nucleus centralis, is insignificantly labelled, in contrast to theventrocaudal regions in which labelled elements abound. NPY-positive elements are uniformly distributed throughout the nucleus, but nolabelled cell bodies were observed. NADPH-d-positive fibres and terminals were observed in both dorsal and ventral regions of thenucleus reuniens, but the few labelled cell bodies to be observed were located in the peripheral regions of the nucleus. These findings arediscussed in terms of the evolution of the core-and-belt organisation of sensory nuclei observed in other vertebrate species. 2002Elsevier Science B.V. All rights reserved.

Theme: Sensory systems

Topic: Auditory systems: central anatomy

Keywords: Auditory system; Core-belt organisation; Neuroactive substances; Turtle

Several investigators [1–3,6,12,25,26,31,43] have pro- these two regions show morphological and neuro-posed that the sensory centres of the mammalian brain, physiological differences and correspond to separate, par-particularly the auditory and somatosensory nuclei, are allel, sensory channels. Although the lemniscal and ex-each composed of an internal ‘core’, receiving a lemniscal tralemniscal channels appear to overlap to a varying degreeinput, and an external ‘belt’, receiving an extralemniscal in different mammalian species [26,44], the distinctioninput. In addition to their different hodological properties, between the core and the belt of auditory and somato-

sensory nuclei continues to be supported by immuno-cytochemical data showing clear differences in the dis-*Corresponding author. Tel.: 133-142-162-678; fax: 133-145-709-tribution of some neuroactive substances in the internal990.

E-mail address: rio@infobiogen.fr (J.-P. Rio). and external regions of these nuclei [7,18,21,24,30].

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )03255-3

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In nonmammalian amniotes, parallel channels within zolium (Sigma, USA) and 0.2% Triton X-100 in 0.05 Meach of several sensory systems have been demonstrated Tris buffered saline (TBS, Sigma, USA), pH 7.6–7.8 at 37(reptiles: [4,5,22,23,33]; birds: [8,13,15,34]). It has also 8C for no more than 1 h. The reaction was stopped bybeen shown that the mesencephalic and thalamic auditory rinsing sections in cold TBS. Free-floating sections werecentres of the avian and reptilian auditory system are gathered onto gelatinised slides, some sections were coun-neurochemically nonhomogeneous [7,10,16,33,35,36,40, terstained with cresyl violet, and all were dehydrated,42,46]. Given these latter findings, and in view of the cleared, and mounted either in Canada balsam or Entellan.previous claim [37,38] that turtles may provide infor- Observations were made under a light microscope.mation concerning the ‘premammalian’ configuration of Omitting the primary antibody never produced immuno-the central nervous system, we present below the results of labelling. The distribution of immunoreactivity to 5-HT,an immunocytochemical study of subcortical auditory TH, SP, mENK, or NPY and of NADPH-d activity did notcentres: the mesencephalic (torus semicircularis, Ts) and differ conspicuously between the two chelonian species,thalamic (nucleus reuniens, nRe) nuclei of two species of nor did we detect any differences between material workedturtle, Testudo horsfieldi and Emys orbicularis, in an up as free-floating or previously mounted sections. Ourattempt to demonstrate their core-and-belt organisation. observations are summarized in the schematic Fig. 1. In

Fifteen adult specimens of Testudo horsfieldi and seven general, 5-HT- (Fig. 2A, F) and TH-immunolabelled (Fig.adult specimens of Emys orbicularis were used. Ex- 2B) elements appeared as fragments of thin fibres withperimental procedures conformed to the guidelines for varicosities or as punctae (terminal-like profiles), andanimal care of the Sechenov Institute. Under deep (40–50 NADPH-d-positive varicose fibres and terminals beingmg/kg, i.p.) Nembutal anaesthesia, animals were perfused more slender (Fig. 2E), while SP- (Fig. 2C, G), mENK-through the heart with an excess of 0.7% heparinised saline (Fig. 2D) and NPY-immunopositive (Fig. 2H) elementsand subsequently with 300 ml of 4% paraformaldehyde in appeared as fragments of thicker, smooth fibres or as0.1-M phosphate buffer. Brains were dissected out and granular elements of larger size.postfixed for 4–15 h in fresh fixative, then cryoprotected The mesencephalic torus semicircularis (Ts) of eachby storage for 24 h in 20–30% sucrose at room tempera- species contains a well-outlined and clearly distinct centralture. Frozen sections (25–40 mm) were cut and divided nucleus (nCe), whose neurophysiological and hodologicalinto two parallel series, one being immediately mounted on properties [5,23] correspond to those of a ‘core’ region. Itgelatinised slides, the other being worked up as free- is characterized by a very low density of 5-HT-, TH-, SP-floating sections. For immunocytochemistry, after pretreat- and mENK-immunoreactive (ir) fibres and terminals,ment with 0.3% H O , sections were treated for 30 min at which are mainly located at the periphery of the nucleus2 2

room temperature in 3% normal goat serum (NGS) (Figs. 1A–D and 2A–D). In contrast, a very dense(Vector, USA) diluted in phosphate buffered saline con- immunolabelling of these substances was observed in thetaining 0.3% Triton X-100 (PBS-Tr), and then transferred nucleus laminaris (nL) of the Ts (Figs. 1A–D and 2A–D),to one of the following polyclonal antibodies, diluted in whose characteristics correspond more to those of a ‘belt’PBS-Tr containing 1% NGS (as a control procedure, some region. Immunolabelling of NPY, seen mostly as thatsections were not exposed to the primary antibody): anti- typical of terminal-like structures, was observed through-serotonin (5-HT, Immunotech, France, 1:500), anti- out the Ts, most densely in the nCe and particularly in itstyrosine hydroxylase (TH, Chemicon, USA, 1:1000), anti- peripheral regions (Fig. 1E). Somata immunolabelled bysubstance P (SP, Peninsula Laboratories, USA, 1:500), anti-SP and anti-mENK were occasionally observed in theanti-menkephalin (mENK, generous gift of Dr. G. Tramu, nL (Figs. 1C and 2D), and NPY-ir neurons were present inFrance, 1:2500), or anti-neuropeptide Y (NPY, Peninsula both nL and nCe (Fig. 1E). The laminar distribution ofLaboratories, USA, 1:1000). Sections remained for 15–20 immunoreactive elements varied; terminal-like TH-ir ele-h in the primary antibody, under continuous agitation. ments were most dense in the cell layers of the nL (Fig.After several rinses in PBS, sections were incubated for 1 2B), whereas fragments of labelled fibres were mainlyh under the same conditions in a biotinylated goat anti- located between them, as well as in the periventricularrabbit IgG (Vector, USA) diluted 1:100 in PBS-Tr con- layer (Pe; Figs. 1B and 2B). Elements immunoreactive totaining 1% NGS, washed and were treated, at room 5-HT, mENK, SP or NPY were more homogeneouslytemperature, for 1 h, in an avidin–biotin–peroxidase distributed throughout the layers of the nL (Figs. 1A, C–Ecomplex (Vector Elite ABC, Vector, USA) diluted 1:100 in and 2A, C, D), and labelled punctae surrounding immuno-PBS-Tr. Peroxidase was visualized by treating the sections negative cell bodies outlined their contours; this wasfor 5–15 min in a 0.03% solution of diaminobenzidine especially clear in SP and mENK preparations (Figs. 1C, D(DAB, Sigma, USA) in Tris buffer (0.05 M, pH 7.6) and 2C, D). The distribution of NADPH-d activity wascontaining 0.03% H O . For reduced nicotinamide adenine different from the distribution of immunoreactivity to other2 2

dinucleotide-diaphorase (NADPH-d) histochemistry, sec- substances. First of all, a great number of positive neuronstions were incubated in a reaction mixture containing 1 (up to 50 per section) were observed throughout the cellmM b-NADPH (Sigma, USA), 0.2 mM Nitro Blue Tetra- layers of the nL (Fig. 1F). These cells had an ovoid or

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Fig. 1. Schematic drawings of the distribution of 5-HT (A, G), TH (B, H), SP (C, I), mENK (D, J) and NPY (E, K) immunoreactivity and activity ofNADPH-d (F, L) in mesencephalic and thalamic auditory centres of the turtle. A–F: Torus semicircularis, H–L: Nucleus reuniens at their mid-level (frontalsections). For further explanation see the text. Abbreviations: Ce, central (core) region of the nRe; D, dorsal; Dma, nucleus dorsomedialis anterior; IGL,intergeniculate leaflet; L, lateral; nCe, nucleus centralis (core) of the Ts; nL, nucleus laminaris (belt) of the Ts; nRe, nucleus reuniens; nV, nucleus ventralis;Pe, periventricular region of the Ts; pe, periventricular region of the nRe; pedd, dorsal pedunculus of the lateral forebrain bundle; Rot, nucleus rotundus;Ts, torus semicircularis; Ve, ventral (belt) region of the nRe.

M.G. Belekhova et al. / Brain Research 925 (2002) 100 –106 103

Fig. 2. Examples of frontal sections at the levels of torus semicircularis and nucleus reuniens, demonstrating the difference in the labelling of their central(core) and peripheral (belt) regions. A–E: distribution of 5-HT (A), TH (B), SP (C), mENK (D) and NADPH-d (E) in the Ts. Note that nCe in all cases isalmost free from labelled fibres and terminals, whereas the nL has a dense labelling. Also notice the laminar distribution of SP-ir elements in the nL (C).Arrows show mENK-ir cells in the nL (D). In E, NADPH-d-positive neurons in the nL with dendrites entering nCe are seen together with a single neuronin nCe. The strip of denser innervation of the peripheral nCe is also seen. F–H: distribution of 5-HT-ir (F), SP-ir (G) and NPY-ir (H) fibres and terminalsin the nRe. Notice that only NPY is relatively homogeneously distributed in this nucleus. Other substances are mostly present in its ventral (Ve) andperiventricular (pe) regions (belt structures). Abbreviations and orientation of sections are the same as for Fig. 1. Scale bar: 100 mm.

104 M.G. Belekhova et al. / Brain Research 925 (2002) 100 –106

fusiform shape, and they were oriented perpendicularly to auditory centres in turtles are generally in agreement withthe curvature of the layers of the nucleus. As a rule, they previous results obtained in turtles and other reptileshad two dendrites originating from the opposite poles of [33,35,36,40–42,46]. However, with a few exceptionsthe cell body. One of these was directed towards the [10,33], these authors have not interpreted their results inventricular surface of the tectum, and the second one ran to terms of the ‘core-and-belt’ organisation of sensory cen-the nCe, branching in its peripheral parts. NADPH-d- tres. In birds and mammals, little attention appears to havepositive neurons in the nCe were much less abundant (up been paid to the distribution of the aforementioned mono-to 10 per section), variable in their shape and size, with amines and neuropeptides in projection sensory centres,different numbers of dendrites having no definitive orienta- with the exception of those of the visual system. This lacktion and never passing through the border of the nucleus of interest can be likely explained by the generally lower(Figs. 1F and 2E). The pattern of NADPH-d innervation of level of immunoreactivity in sensory centres in comparisonthe Ts was the same as that observed for the distribution of to nonsensory centres. It has, however, been shown in5-HT, TH, SP and mENK immunoreactivity. However, in some mammalian species that SP and mENK are morethe peripheral parts of the nCe, a wide band of dense concentrated in fibres and terminals of the peripheralNADPH-d-positive fibres and terminals, similar to the regions of the inferior colliculus and medial geniculatedistribution of NPY-ir elements, was observed. It is not nuclei together with adjacent satellite nuclei [14,16,47].impossible that part of these labelled elements is formed of More recently, studies of the distribution of CGRP-,branching dendrites of NADPH-d-positive neurons in the parvalbumin- and calbindin-immunoreactivity [7,21,24,30]nL. have shown that these are differentially distributed in the

In the thalamic nucleus reuniens (nRe), the distribution central and peripheral regions of sensory centres in theof immunoreactivity was generally similar to that observed avian and mammalian brain, and it has been suggested thatin the Ts, although the density of labelled elements was their complementary distribution in the ‘core’ and ‘belt’lower. Fragments of 5-HT-ir, TH-ir, SP-ir and mENK-ir zones of auditory thalamic centres is a feature common tofibres and terminal-like punctae were most dense in the all amniotes [7]. Our findings concerning the monoaminer-pars ventralis (Ve) of the nucleus (Figs. 1G–J and 2F, G), gic, neuropeptidergic and NO innervation of the mesence-particularly at its caudal levels, whose neurophysiological phalic and thalamic auditory centres of turtles support theand hodological characteristics [5,23] together with the notion that the core-and-belt organisation of sensorydistribution of calcitonin-gene related peptide (CGRP) [7] centres arose in the common ancestor of all amnioteidentify it as a peripheral or ‘belt’ region. In rostral regions species.of the pars dorsalis of the nucleus, which may be identified The morphological and neurochemical distinction be-as a central ‘core’ region by the same criteria (Ce), the tween the ‘core’ and ‘belt’ of a sensory nucleus reflectsdensity of these immunoreactive elements was insignificant their functional difference: purely sensory perceptual func-(Figs. 1G–J and 2F, G). The distribution of NPY-ir tions are more characteristic for the core zone, whereaselements was fairly homogeneous throughout the nRe with less specific functions related to autonomic and motora slightly greater density in its lateral part (Figs. 1K and activity such as orientation to the source of sound and an2H). No immunolabelled somata were found in this appropriate acoustico-motor behavior are mostly served bynucleus. NADPH-d activity in the nRe did not show a the belt zone of the nucleus. Thus, in mammals, [20] forgreat difference in the distribution of positive fibres and review, the central nucleus of the inferior colliculus, theterminals in the dorsal and ventral subdivisions of the ventral nucleus of the medial geniculate body and thenucleus, being thus similar to the distribution of NPY primary auditory cortex form a highly specialized coch-immunoreactivity (Fig. 1L). However, the density of leotopic system devoted to fine frequency discrimination oflabelled fibres and terminals in the nRe is much lower for auditory signals. On the other hand, the peripheral nucleiNADPH than for NPY. Some NADPH-d-positive neurons of the inferior colliculus receive multimodal auditory andwere also seen in the peripheral part of the nucleus and in somatosensory information, and project to peripheral divi-the tectofugal tract passing to the nRe (Fig. 1L). On the sions of the medial geniculate body, which in turn relaywhole, SP- and NPY-ir elements were numerous in the signals to non-primary auditory and somatosensory fieldsnRe, 5-HT-ir, TH-ir, mENK-ir and NADPH-d-positive of the cortex. These in turn strongly project back to theelements being less abundant. At more caudal levels, the belt regions of the acoustico-motor nucleus of the inferiordifference in density of all labelled elements between the colliculus, which further projects to the superior colliculus,dorsal and ventral regions of the nucleus became pro- cerebellum and somatosensory and vocalisation systems,gressively diminished as reactive elements became com- involved into the acoustico-motor function. The beltmoner in the Ce. subdivisions of the inferior colliculus and medial genicu-

Our findings concerning the distribution of some mono- late body are also connected with limbic centres in theamines (5-HT, TH), neuropeptides (SP, NPY, mENK) and hypothalamus and amygdala [26,27] and thus related toNADPH-d in mesencephalic (Ts) and thalamic (nRe) autonomic, autonomo-motor and emotional (fear condi-

M.G. Belekhova et al. / Brain Research 925 (2002) 100 –106 105

tioning to acoustic stimuli) reactions [4,20]. According to finely-tuned sensory projections subserving more complexHuffman and Henson [20], these latter functions of the processing of sensory information.auditory system are phylogenetically ancient.

In reptiles, the organisation of parallel channels in theauditory system is less well documented. However, cyto- Acknowledgementsarchitectonic [9], hodological [4,5,11,29,45,49], neuro-physiological [22,23] and neurochemical [7,10,33, The authors wish to thank D. Le Cren for his skillful35,36,46] findings strongly suggest that a core-versus-belt photographic assistance. This work was supported bydistinction may be drawn in the reptilian auditory system. grants of Academy of Sciences of Russia 00-15-97935 andIn all reptilian species, the central nucleus of the Ts is the 99-04-49848, INSERM and MNHN (EA 2587) France,principal target of cochlear projections and contains mono- FCAR Canada.modal auditory neurons with narrow tuning curves[5,19,22,23,28,32], while the peripheral regions of the Ts(the laminar nucleus) and nRe contain wider tuned mul-

Referencestimodal neurons receiving both auditory and somatosen-sory inputs [5,17,22,23] as well as project to telencephalic

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