ORIGINAL ARTICLE

Synaptic circuitry in the retinorecipient layers of the optic tectumof the lamprey (Lampetra fluviatilis). A combined hodological,GABA and glutamate immunocytochemical study

Jacques Reperant Æ Roger Ward Æ Monique Medina ÆNatalia B. Kenigfest Æ Jean-Paul Rio Æ Dom Miceli ÆBruno Jay

Received: 5 September 2008 / Accepted: 2 February 2009

� Springer-Verlag 2009

Abstract The ultrastructure of the retinorecipient layers

of the lamprey optic tectum was analysed using tract

tracing techniques combined with GABA and glutamate

immunocytochemistry. Two types of neurons were identi-

fied; a population of large GABA-immunonegative cells,

and a population of smaller, highly GABA-immunoreac-

tive interneurons, some of whose dendrites contain

synaptic vesicles (DCSV). Five types of axon terminals

were identified and divided into two major categories. The

first of these are GABA-immunonegative, highly gluta-

mate-immunoreactive, contain round synaptic vesicles,

make asymmetrical synaptic contacts, and can in turn be

divided into AT1 and AT2 terminals. The AT1 terminals

are those of the retinotectal projection. The origin of the

nonretinal AT2 terminals could not be determined. AT1

and AT2 terminals establish synaptic contacts with DCSV,

with dendrites of the retinopetal neurons (DRN), and with

conventional dendritic (D) profiles. The terminals of the

second category are GABA-immunoreactive and can sim-

ilarly be divided into AT3 and AT4 terminals. The AT3

terminals contain pleiomorphic synaptic vesicles and make

symmetrical synaptic contacts for the most part with glu-

tamate-immunoreactive D profiles. The AT4 terminals

contain rounded synaptic vesicles and make asymmetrical

synaptic contacts with DRN, with DCSV, and with D

profiles. A fifth, rarely observed category of terminals

(AT5) contain both clear synaptic vesicles and a large

number of dense-core vesicles. Synaptic triads involving

AT1, AT2 or AT4 terminals are rare. Our findings are

compared to these of previous studies of the fine structure

and immunochemical properties of the retinorecipient

layers of the optic tectum or superior colliculus of

Gnathostomes.

Keywords Lamprey � Optic tectum �Synaptic connections � Immunocytochemistry

Abbreviations

AOA Area optica accessoria

AT1 Type 1 axon terminals

AT2 Type 2 axon terminals

AT3 Type 3 axon terminals

AT4 Type 4 axon terminals

AT5 Type 5 axon terminals

AX2 Axon of type 2 terminal

AXg GABA-immunoreactive axon

BA Background immunolabelling

CGL Corpus geniculatum laterale

J. Reperant � R. Ward � M. Medina � N. B. Kenigfest �D. Miceli � B. Jay

Departement Regulations, Developpement et Diversite

Moleculaire, USM 501, CNRS UMR 7221,

Museum National d’Histoire Naturelle, Paris, France

J. Reperant � R. Ward � M. Medina � D. Miceli

Laboratoire de Neuropsychologie Experimentale et Comparee,

Universite du Quebec, Trois-Rivieres, Canada

N. B. Kenigfest

Laboratory of Evolution of Neuronal Interactions, Sechenov

Institute, Russian Academy of Sciences, St. Petersburg, Russia

J.-P. Rio

INSERM U-616, Hopital de la Salpetriere, Paris, France

M. Medina (&)

Departement RDDM, USM 501, CNRS UMR 7221,

Museum National d’Histoire Naturelle,

CP 55, Batiment d’Anatomie Comparee,

57 rue Cuvier, 75231 Paris Cedex 05, France

e-mail: monique.medina@neuf.fr; medina@mnhn.fr

123

Brain Struct Funct

DOI 10.1007/s00429-009-0205-9

CH Cerebral hemisphere

CV Centrifugal visual pathway

D Conventional dendrite

DCSV Dendrite of interneuron containing synaptic

vesicles

DCSV1 Dendrite of interneuron containing synaptic

vesicles of the first type

DCSV2 Dendrite of interneuron containing synaptic

vesicles of the second type

Dg? Conventional GABA-immunoreactive dendrite

of interneuron

Dg- Conventional GABA-immunonegative dendrite

of efferent neuron

DG? Conventional glutamate-immunoreactive

dendrite of efferent neuron

DG- Conventional glutamate-immunonegative

dendrite of interneuron

DRN HRP-labelled dendrite of the centrifugal

neurons of RMA

DRNg? HRP-labelled GABA-immunoreactive dendrite

of the centrifugal neurons of RMA

GABA c-Aminobutyric acid

HI Horizontal interneurons

HRP Horseradish peroxidase

HY Hypothalamus

INS Soma of interneuron

ir Immunoreactive

M5 Centrifugal visual neurons of the nucleus M5 of

Schober

MT Mesencephalic tegmentum

NP Nucleus preopticus

NPO Nucleus of the postoptic commissure

NPR Nucleus pretectalis

OB Olfactory bulb

OC Optic chiasma

OF Optic fibre

ON Optic nerve

PCSV Profiles containing synaptic vesicles

PNS Soma of projecting neuron

RI Radial interneurons

RITC Rhodamine b-isothiocyanate

RMA Centrifugal visual neurons of the reticular

mesencephalic area

RP Retinal projections

TO Tectum opticum

SCE Stratum cellulare ependimale

SCFC Stratum cellulare et fibrosum centrale

SCFE Stratum cellulare et fibrosum externum

SCFI Stratum cellulare et fibrosum internum

SCP Stratum cellulare periventriculare

SFP Stratum fibrosum periventriculare

SM Stratum marginale

Introduction

The Agnatha, or jawless vertebrates, belong to the most

ancient extant vertebrate group, which appeared over

500 million years ago (Forey and Janvier 1993; Janvier

1996). The living representatives of these ancestral verte-

brates fall into one of two groups, the Myxinidae (hagfish)

and Petromyzontidae (lampreys). In spite of their superfi-

cial resemblances, the hagfish and lampreys diverged early

in vertebrate phylogeny, and lampreys are now considered

to be more closely related to the Gnathostomes, or jawed

vertebrates, than to the hagfish (Forey and Janvier 1993;

Janvier 1996). In this light, authors are in general agree-

ment that the central nervous system of the lamprey can be

considered as the prototype of that of the Gnathostomes

(Heier 1948; Nieuwenhuys 1977; Karamian et al. 1984;

Nieuwenhuys and Nicholson 1998; Robertson et al. 2006,

2007). The numerous studies of the functional anatomical

organisation of the primary visual system of the lamprey,

carried out over several decades (Northcutt and Przybylski

1973; Kennedy and Rubinson 1977; Kosareva et al. 1977;

Rubinson and Kennedy 1979; Reperant et al. 1980, 1990;

Kosareva 1980; Vesselkin et al. 1980, 1996; Karamian

et al. 1984; de Miguel et al. 1990; Rubinson, 1990; Pierre

et al. 1992; Rio et al. 1993) are in agreement with this

hypothesis. In the lamprey, as in all Gnathostomes (cf.

Ebbesson 1970, 1972; Reperant et al. 1990), the retina

projects contralaterally to five distinct regions of the brain

(Fig. 1): the postoptic region (nucleus of the postoptic

commissure); the thalamus (corpus geniculatum laterale);

the pretectum (nucleus pretectalis); the tegmentum mes-

encephali (accessory optic area); and the superficial layers

of the tectum opticum. Of these, the retinotectal projection

is by far the most important, as it is in all nonmammalian

vertebrates, in particular the anamniotes (cf. Ebbesson

1970, 1972; Reperant et al. 1990). The organisation of this

projection in the lamprey has been studied by electro-

physiological methods (Karamian et al. 1984) and by a

variety of hodological techniques at the level of resolution

of the light microscope (Northcutt and Przybylski 1973;

Kennedy and Rubinson 1977; Kosareva et al. 1977;

Kosareva 1980; Reperant et al. 1980, 1990; Vesselkin et al.

1980, 1984, 1996; de Miguel et al. 1990; Rubinson 1990).

While the synaptic circuitry of the retinorecipient layers

of the optic tectum, or its mammalian equivalent, the

superior colliculus, has been extensively studied in all

major groups of Gnathostomes (for reviews: teleosts,

Vanegas et al. 1984; Peyrichoux et al. 1986; Kageyama and

Meyer 1989; amphibians, Lazar 1984; Antal 1991; Gabriel

and Straznicky 1995; reptiles, Reperant et al. 1997; Rio

et al. 1995; birds, Morino et al. 1991; Tombol 1998;

Tombol and Nemeth 1999; Tombol et al. 2003; mammals,

Brain Struct Funct

123

Mize 1992; Mize and Butler 1995; Mize et al. 1994; Ortega

et al. 1995) no comparable ultrastructural studies have been

carried out in the lamprey, with the exception of a pre-

liminary study by Rio et al. (1996).

The immediate aim of the present investigation was to

carry out a detailed analysis of the synaptic circuitry of the

retinorecipient layers of the optic tectum of the lamprey,

using a variety of morphological techniques: tract tracing,

immunocytochemistry, double and triple labelling, mor-

phometry, and stereology. These were intended to specify

the cytological, neurochemical and synaptological charac-

teristics of the profiles containing synaptic vesicles (PCSV)

and other neuronal profiles. We pay particular attention to

the following points: (1) the characterisation of retinal

terminals and their postsynaptic targets; (2) the same

properties of nonretinal terminals; (3) the possible exis-

tence of a projection of the centrifugal visual neurons onto

this structure, a hypothesis recently proposed by Robertson

et al. (2006); and (4) the identification of interneurons and

their dendrites containing synaptic vesicles (DCSV), of

their afferent supply and the targets of these profiles, and a

concomitant analysis of the c-aminobutyric acid (GABA)-

immunoreactive system. In an attempt to determine the

extent to which the details of the organisation of the reti-

norecipient tectal layers of this ancestral vertebrate form

are conserved in more recent Gnathostomes, we compare

our findings to those reported in the literature.

Materials and methods

A total of 64 adult specimens of Lampetra fluviatilis were

used. The animals were caught in October in the Neva

River (St. Petersburg, Russia) during their migration

upstream from the Baltic Sea. The animals, approximately

20 cm in length, were kept in aerated aquaria at a tem-

perature of 5–7�C; the experimental manipulation of these

animals followed European, Canadian and Russian guide-

lines for the treatment of experimental animals.

Degeneration experiments (n = 12)

Animals were anaesthetised in a 0.4% solution of tricaine

methane sulfonate (MS 222, Sandoz), and subsequently

received an injection of 20 ll of a 40% aqueous solution of

calcium chloride into the posterior chamber of the eye.

They were allowed to survive for 12–30 days and then,

under deep anaesthesia (MS 222), were perfused transcar-

dially with 20–30 ml of 0.7% saline followed by 100–

150 ml of a fixative composed of 1% paraformaldehyde

and 2.5% glutaraldehyde in 0.12 M phosphate buffer at

pH 7.4. The brains were dissected out and stored for 3.5 h

at room temperature in fresh fixative. Small blocks of tectal

tissue, cut transversely, were then post-fixed for 3–4 h in

2% buffered osmium tetroxide, stained en bloc in 2.5%

buffered uranyl acetate, dehydrated in a graded series of

ethanol and embedded in Araldite. Semithin (1 lm) sec-

tions were cut and stained with toluidine blue. Ultrathin

(70–80 nm) sections were cut on a Reichert OM U2

ultramicrotome, mounted on copper grids, double-stained

with ethanolic uranyl acetate and lead citrate, and exam-

ined under a Philips EM 400 electron microscope. The

same procedure was used in preparing tissue for electron

microscopy in other experiments.

Axonal tracing experiments (n = 20)

Animals (n = 16) were anaesthetised by flooding the gills

with a 0.3% solution of urethane. After ablation of one or

two eyes, each severed optic nerve was gently exposed and

NPR

SM +SCFE

TO

NP

CGL

AOA

ON

M5RMA

OC

RP

CV

MT

OBCH

HY

DRN

DRN

DRN

NPO

Fig. 1 A schematic drawing of

a longitudinal section of the

brain of Lampetra fluviatilisindicating the retinal projection

(RP) and the centrifugal visual

pathway (CV) visualised after

either iontophoretic deposit of

HRP into the contralateral optic

nerve or intraocular injection of

RITC into the contralateral eye

Brain Struct Funct

123

isolated from the oculomotor nerves which were covered

with paraffin oil. A glass micropipette with a tip diameter

of 20–50 lm, filled with a 4% solution of horseradish

peroxidase (HRP; either Sigma Type VI or Boehringer

Grade I) in 0.1 M PB at pH 8.0, was placed in contact with

the sectioned right optic nerve and a positive current of

10 lA was passed for 20–30 min at a rate of 7 s on/7 s off.

In some cases the same procedure was then carried out on

the left optic nerve. The animals were reanimated in oxy-

gen-saturated water, and allowed to survive for 4–16 days.

They were then anaesthetised with MS 222 and perfused

transcardially, using the procedure described above. Thick

(50–80 lm) transverse sections of the optic tectum were

cut on an Oxford Vibratome and collected in 0.1 M Tris

buffer at pH 7.6. HRP was visualised either by the method

of Adams (1981), using cobalt-nickel intensified 3,30

diaminobenzidine (DAB) or by Mesulam’s (1982) tetra-

methylbenzidine (TMB) procedure. After the reaction,

some sections were mounted directly on glass slides for

examination by light microscopy, while others were pre-

pared for electron microscopy by the methods described

above. Under anaesthesia four other animals received a

unilateral intraocular injection, into either the left or right

eye, of 10 ll (w/v) of rhodamine b-isothiocyanate (RITC,

Sigma-Aldrich) containing 2% dimethyl sulfoxide. The

injections were performed with the lamprey maintained in

a stereotaxic apparatus, using a Hamilton syringe with a

maximum capacity of 20 ll. The solution was injected

slowly into the posterior chamber of the eye, as close to the

retina as possible but avoiding direct contact of the needle

with this structure. Survival time ranged from 10 to

18 days. Following perfusion the brains were cryostat

sectioned in the transverse plane at a thickness of 20 lm.

The sections were immediately mounted in slides,

coverslipped and viewed using a Leitz Ploemopack fluo-

rescence microscope fitted with an N2 filter (barrier filter

580 nm).

Combined degeneration and HRP tracing experiments

(n = 12)

Six to 12 days after the injection of calcium chloride into

the posterior chambers of each eye, the eyeballs were

removed under urethane anaesthesia and HRP was applied

to each optic nerve by the iontophoretic procedure descri-

bed above. The lampreys were allowed to survive for 6–

25 days and then perfused; the methods described above

were used both for fixation and revelation of HRP activity

in sections of the optic tectum, and also for the preparation

of sections for electron microscopy.

In addition, four intact lampreys were prepared for

examination by light microscopy, and a further six for

electron microscopy. The brains prepared for light

microscopy were embedded in paraffin, serially sectioned

at 10 lm and stained with cresyl violet.

Immunocytochemical methods

GABA immunofluorescence procedures

(normal material, n = 6)

The animals were perfused transcardially with 20–30 ml of

0.7% saline followed by 150 ml of a fixative composed of

4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M

phosphate buffer at pH 7.4. The brains were subsequently

removed and stored in fresh fixative for 2 h at 4�C, then

transferred to a 30% solution of sucrose in phosphate

buffer, in which they remained overnight at the same

temperature. Transverse sections of the mesencephalon

were cut at 40–50 lm in a freezing microtome. They were

incubated for 48 h at 4�C in a primary rabbit anti-GABA

antibody (Immunotech, Marseille, France) diluted 1:3,000

in phosphate buffer saline (PBS). The sections were sub-

sequently incubated for 2 h in a fluorescein-conjugated

(FITC) goat anti-rabbit IgG diluted 1:50 in PBS, rinsed in

PB, mounted on slides and coverslipped with Vectashield

(Vector Laboratories, Temecula CA, USA). Sections were

observed under a Leitz Ploemopack fluorescence micro-

scope fitted with I3 (448 nm) filter mirror systems to detect

the FITC fluorescence of GABAergic neurons. Labelled

profiles in serial sections were counted either directly under

the microscope or on a monitor screen driven by a Pana-

sonic WV1900 video camera.

GABA or glutamate pre-embedding procedure

(normal material, n = 4)

After perfusion under urethane anaesthesia with the same

dialdehyde fixative as that used in the HRP experiments,

slabs of tectal tissue from normal animals were serially

sectioned at 40–50 lm in the transverse plane in a

Vibratome and processed for GABA or glutamate-immu-

nocytochemistry. Sections were collected in cold 0.1 M

PBS, and preincubated for 30 min in 0.1 M PBS con-

taining 0.2% gelatine, 0.1% Triton-X 100 and 0.1 M

lysine. After several rinses in PBS the sections were

incubated overnight in a cold chamber with a humid

atmosphere in a 1:2,000 dilution of an rabbit anti-GABA

(Immunotech, France) or a 1:300 dilution of rabbit anti-

glutamate (Chemicon Temecula CA, USA) antibodies

containing 0.2% gelatin and 0.1% Triton-X 100; sections

were then rinsed in PBS and incubated for 1 h in a 1:100

dilution of a goat anti-rabbit immunoglobulin (Chemicon,

USA), rinsed again in PBS and incubated for 1 h in a

1:200 dilution of a rabbit avidin–biotin–HRP complex

Brain Struct Funct

123

(Vectastain Elite, Vector Laboratories, USA). Peroxidase

activity was visualised with 0.03% DAB (Sigma, USA)

and 0.005% hydrogen peroxide in 0.01 M Tris–HCl buffer

at pH 7.6. Sections were then embedded in Araldite.

The GABA and glutamate-immunoreactive elements of

the tissue were then examined by optical microscopy in

semithin (B1 lm) sections counterstained with toluidine

blue, or under the electron microscope in thin (70–80 nm)

sections.

GABA or glutamate post-embedding procedure

(normal, n = 6 and experimental material:

HRP, n = 20; HRP ? degeneration, n = 12)

Light microscopy

Semithin sections throughout the optic tectum were

mounted on gelatin-coated slides, etched in saturated

sodium ethoxide, treated with 10% hydrogen peroxide, and

preincubated for 30 min in 10% bovine serum albumin

(BSA) in PBS containing 0.2% gelatin, 0.25% Triton-X

100, and 0.1 M lysine prior to being incubated overnight at

4�C, in a humid atmosphere, in a 1:5,000 dilution of rabbit

anti-GABA antiserum (Immunotech, Marseille, France)

diluted in PBS containing 0.2% gelatin, 0.25% Triton-X

100, 0.1% lysine, and 1% BSA. Sections were subse-

quently rinsed in PBS and incubated for 1 h in a 1:100

dilution of a goat anti-rabbit antiserum, rinsed again in

PBS, and then incubated for 1 h in a 1:200 dilution of a

rabbit peroxidase–antiperoxidase complex, following the

technique of Sternberger et al. (1970). After several rinses

in PBS the peroxidase activity was visualized with 0.03%

DAB and 0.005% hydrogen peroxide in 0.1 M Tris–HCl

buffer. Sections were coverslipped, and examined under

the microscope.

Electron microscopy

Thin sections were gathered onto nickel grids and immersed

in the following solutions: (1) 1% aqueous periodic acid,

7 min; (2) rinse in distilled water; (3) 1% sodium metape-

riodate, 7 min; (4) rinse in distilled water; (5) 5% BSA in

0.05 M Tris-buffered saline (TBS) at pH 7.6, 30 min; and

(6) rinse in TBS. Sections were then incubated overnight at

4�C in one of two primary antibodies. These were either

rabbit anti-glutamate (Chemicon, Temecula CA, USA)

diluted 1/300 in TBS at pH 7.6, or rabbit anti-GABA

(Immunotech, France) diluted 1/2,000 in TBS at pH 7.6.

After several rinses in TBS, the sections were treated with a

goat anti-rabbit immunoglobulin (Chemicon) conjugated

with 10, 15 or 30 nm colloidal gold particles (Janssen,

Belgium), diluted 1/75 in TBS at pH 8.2, for 1 h at room

temperature. The sections were then rinsed in TBS and

distilled water, and counterstained with uranyl acetate and

lead citrate prior to examination under the electron micro-

scope. Some sections were also processed for double

immunolabelling; after incubation in the rabbit anti-gluta-

mate antibody and the second antibody conjugated with 30

or 10 nm gold particles, the sections were exposed to the

vapour of 4% paraformaldehyde at 80�C for 1 h (Wang and

Larsson 1985) thoroughly rinsed in PBS and then incubated

in the anti-GABA antibody followed by the secondary

antibody conjugated with 10 or 20 nm particles.

Specificity of antibodies and control procedures

The two polyclonal antibodies used in this study have

already been characterised in previous investigations;

the anti-glutamate antibody (Chemicon) by Sherry and

Ulshafer (1992) and Kalloniatis and Fletcher (1993), and

the anti-GABA antibody (Immunotech) by Seguela et al.

(1984) and Miceli et al. (2008); they were generated in

rabbit against glutamate or GABA coupled to BSA with

glutaraldehyde. The cross-reactivities were determined

using ELISA or RIA techniques. It has been shown that

each antibody shows a high specificity for the particular

haptene (glutaraldehyde-fixed glutamate, or glutaralde-

hyde-fixed GABA) and negligible cross-reactivity with

the other haptene of the pair, or with other amino acids

(L-aspartate, L-glutamate, taurine and glycine for the anti-

GABA antibody, L-aspartate, taurine, glycine and GABA

for the anti-glutamate antibody).

In the present study, in each immunolabelling session,

control grids were tested for any cross-reactivity with

glutamate and GABA. Lamprey brain macromolecules

were treated with glutaraldehyde either alone or in the

presence of one of these amino acids. The conjugates thus

formed were embedded and processed according to tech-

nique developed by Ottersen (1987, 1989), Ottersen and

Storm-Mathisen (1984) and Ottersen (1990); sections of

conjugates then underwent the same immunogold proce-

dure as sections of tectal tissue. Electron micrographs were

printed at the same magnification as that used for tissue

sections, and the density of gold particles determined (see

below).

The results obtained with the conjugated material

revealed a high degree of selectivity of each antiserum.

Thus, for the sections of glutamate conjugate, a mean

density of 3,172 ± 283 (n = 12) particles/lm2 was

observed when they were treated with the anti-glutamate

antibody, and only 108 ± 10 (n = 14) particles/lm2 when

treated with the anti-GABA antibody. Conversely, the

immunoprocessing of sections of the GABA conjugate led

to a mean density of 2,892 ± 304 (n = 12) particles/lm2

for the anti-GABA antibody, a value to be compared to the

mean density of 2.9 ± 1.2 particles/lm2 obtained with the

Brain Struct Funct

123

anti-glutamate antibody. Additional controls were made by

either omitting the primary antibody or replacing it with

non-immune normal serum, or by immunoadsorbtion of

free glutamate (for the anti-glutamate antibody) or of free

GABA (for the anti-GABA antibody) on polyacrylamide

beads with glutaraldehyde as a linking agent. Under none

of these conditions was any labelling ever observed. The

specificity of the second antibody was demonstrated by the

complete absence of labelling observed after omission of

the primary antibody.

Quantitative methods

Light microscopy

Video images of GABA-immunoreactive cells in sections

treated either for immunofluorescence or by pre and post-

embedding immunocytochemistry were observed and

mapped directly on the monitor screen; cell counts were

performed using NIH Image software. The GABA-reac-

tive cells of the dorsolateral region of the retinorecipient

layers of the optic tectum were counted within a

263 9 263 lm2 (i.e. an area of 69,169 lm2); cells tran-

sected by either the bottom or the right-hand side of this

square were not included in the count (Gundersen 1978;

Williams and Rakic 1988). The sizes of the somata were

estimated by measuring the longest and shortest axes of

the cell body and converting these to mean circular

diameters. The relative proportions of GABA-immunore-

active and GABA-immunonegative cells were estimated

in semithin sections of pre and post-embedded material by

counting the numbers in each category. Simple counting

methods yield biased estimates (cf. Williams and Rakic

1988; Guillery 2002); in our material the larger size of

GABA-negative cells led to an overestimate of their

number in comparison to that of the smaller GABA-

reactive neurons. However, since the mean cell body

diameters were known, as well as the thickness of the

tectal sections, we applied Abercrombie’s (1946) correc-

tion to the raw data in order to obtain unbiased estimates

of cell densities, expressed as cells/mm3, and the relative

proportions of each cell type.

Electron microscopy

The quantitative analysis was carried out on a total area of

6,500 lm2 of sections derived from the dorsolateral

superficial layers (SM and SCFE) of the central portion of

the optic tectum (Fig. 2c).

For each category of the profiles (axons, terminals, con-

ventional dendrites, DCSV), a sample of 40 randomly chosen

profiles was analysed to estimate various morphometric

parameters; the area of the profile and that of its synaptic

vesicles, the length of the synaptic differentiation, and the

density of immunogold particles lying over the profile.

The cross-sectional areas of dendritic and axon terminal

profiles, and the lengths of the synaptic zones, were

measured in electron micrographs printed at a final magni-

fication of 28,0009 or 46,0009, by means of the NIH Image

software package. Areas (a) and perimeters (p) of synaptic

vesicles were measured in micrographs printed at a final

magnification of 160,0009 by means of the Optilab image

analysis system. From these a dimensionless shape factor

was calculated as:

S ¼ 4pa=p2

A value of 1.0 for this factor indicates a perfect circle,

lower values indicating progressively elongated ellipsoids.

The density of immunogold particles (either GABA-ir or

Glu-ir) over different profiles was measured in micrographs

printed at a final magnification of 46,0009; the area of

each profile (axon terminals, dendrites containing synaptic

vesicles or DCSV, conventional dendrites, cell bodies) was

measured as above, and the density expressed as particles/

lm2 uncorrected for background. The same technique was

used to estimate the background intensity of labelling,

counts in this case being made over 100 lm2 areas of

empty resin or the lumina of blood vessels. Estimates of the

background intensity were made in randomly selected

micrographs of 36 sections of tissue.

All the numerical results are presented as mean val-

ues ± standard deviation; statistical analyses were made

by Student’s test or analyses of variance.

In order to estimate the relative proportions of each of

the different types of profile observed in the retinorecipient

layers of the dorsolateral tectum, we carried out a stere-

ological analysis using the point counting method

(Mayhew 1979; Underwood 1970; Weibel 1979). A 6 cm2

grid was randomly positioned on each of 30 electron

micrographs printed at a final magnification of 28,0009;

the profile underlying each of the 36 points of the grid was

noted as a conventional dendrite (noting the presence or

absence of HRP-labelling, GABA- or glutamate-immuno-

reactivity, or a combination of these), an axon (similarly

annotated) a PCSV (AT1, AT2, AT3, AT4, AT5 or DCSV),

these being defined by cytological, hodological and

immunocytochemical criteria; see below), a neuronal cell

body or a glial cell; a total of 1,080 points was counted.

Results

Light microscopy: general morphology

The optic tectum is a prominent external feature of the

adult lamprey brain. The two halves of the tectum bulge

Brain Struct Funct

123

laterally, forming a deep lateral tectal recess. The resulting

tectal ventricle is roofed over by the choroidal lamina. The

structure of the tectum has been extensively described

(Johnston 1902; Ariens-Kappers et al. 1936; Heier 1948;

Leghissa 1962; Schober 1964; Pfister 1971; Nieuwenhuys

1977; Kennedy and Rubinson 1977, 1984; Rubinson and

Kennedy 1979; Nieuwenhuys and Nicholson 1998; Iwahori

et al. 1999). These authors all agree in recognising a well-

developed tectal lamination, but differences of nomencla-

ture and the use of different histological techniques have

led them to disagree over the number, from six to nine, of

tectal laminae. In this investigation we have chosen the

nomenclature proposed by Kennedy and Rubinson (1984),

since it has the advantage of being based on the study of

the development of the tectal lamination. In this termi-

nology, the following seven layers are described from the

Fig. 2 a A low-power micrograph of a transverse section of the

central mesencephalon, 6 lm thick, stained with cresyl violet,

illustrating the general cytoarchitecture of the optic tectum. b A

micrograph at a higher magnification of a semi-thin (1 lm) transverse

section stained with toluidine blue illustrating the architecture of the

dorsolateral central region of the optic tectum. c A micrograph of a

thick (50 lm) transverse section of the dorsolateral central tectum

showing the dense anterograde HRP labelling in the superficial layers

(SM, SCFE) and the retrograde labelling of some centrifugal visual

neurons in the RMA (arrows). Note that the labelled dendrites of

some of these cells (arrowhead) run to the superficial layers. The

rectangle illustrates the region within which electron microscopic

observations were made. d–h Aspects of GABA-immunoreactive

cells in the superficial tectal layers. d Immunofluorescence in a 50 lm

vibratome section. e Immunocytochemical labelling by the pre-

embedding technique in a 50 lm frozen section. f Immunolabelling

by the post-embedding procedure in a 1 lm section. g, h Immunola-

belling by the pre-embedding method in 1 lm sections. Scale bars100 lm in (a–c), 25 lm in (d–f), 12.5 lm in (g, h)

Brain Struct Funct

123

external surface to the ventricle: (1) the stratum marginale

(SM); (2) the stratum cellulare et fibrosum externum

(SCFE); (3) the stratum cellulare et fibrosum centrale

(SCFC); (4) the stratum cellulare et fibrosum internum

(SCFI); (5) the stratum fibrosum periventriculare (SFP); (6)

the stratum cellulare periventriculare (SCP); and (7) the

stratum cellulare ependimale (SCE) (Fig. 2a, b).

Simple inspection of 10 lm paraffin sections stained

with cresyl violet (Fig. 2a) or of semithin (1 lm) sections

counterstained with toluidine blue (Fig. 2b), of the normal

optic tectum shows that the majority of tectal cell bodies is

concentrated in the deeper tectal layers (SCFI and SCP in

particular), whereas these cells are considerably rarer in the

superficial layers wherein they are widely dispersed.

HRP and RITC experiments

After application of HRP to the central stump of the optic

nerve, examination of vibratome sections revealed an

extremely dense anterograde labelling of the entire extent

of the primary visual system, in particular the superficial

layers (SM, SCFE) of the optic tectum (Fig. 2c), covering

approximately half of the tectal thickness. Optimal results

were obtained after a survival time of 4–6 days following

the iontophoretic application of the tracer and 10 days after

intraocular injection of RITC. The retinotectal fibres are

organised as the lateral optic tract, arising from the mar-

ginal optic tract; these densely packed fibres penetrate the

lateral aspect of the medial part of the SCFE in a fan-like

fashion. Collateral branches of these axons arborise within

the SM and throughout the entire thickness of the SCFE;

the retinotectal projection is predominantly contralateral,

but with a modest ipsilateral component which principally

concerns the lateroventral and mediodorsal regions of the

SM and SCFE. Comparable results were obtained after

intraocular injection of RITC.

The iontophoretic application of HRP to the stump or

intraocular injection of RITC of the optic nerve also led to

a bilateral, but predominantly contralateral, retrograde

labelling of the centrifugal visual neurons situated in the

nucleus M5 of Schober and in the reticular mesencephalic

area (RMA). The retinopetal neurons in the RMA were

mainly located in the dorsal tegmental region of the mid-

brain, but some were located in the SCFI of the

lateroventral region of the optic tectum (Fig. 2c). The

labelled cell bodies in M5 were pyramidal in shape, and

larger than those observed in RMA, which generally

appeared fusiform, or rarely multipolar. The dendrites of

the RMA retinopetal neurons are mainly oriented towards

the optic tectum; these labelled dendrites follow a latero-

medial path, reaching the deepest layers of the tectum

through its ventrolateral flank. They then cross the inter-

mediate layers (SCFI, SFC) extending finally into the

dorsolateral zone of optic terminal labelling (SCFE, SM;

Fig. 2c). These observations are entirely comparable to

those made after intraocular injection of RITC (Fig. 3). In

those specimens which received HRP after an intraocular

injection of calcium chloride, no orthograde labelling of

the primary visual system, including the retinotectal path-

way, was observed. However, the retrograde labelling of

the neurons of the centrifugal visual system in M5 and

RMA corresponded to that described above.

GABA immunofluorescence and immunocytochemistry

The general distribution of GABA-immunoreactive somata

in the SM and SCFE of the optic tectum was clearly visible

in frozen immunofluorescence and semithin pre-embedded

or post-embedded sections (Fig. 2d–h). The two layers

contained small GABA-ir cell bodies with a mean equiva-

lent diameter of 8.9 ± 2.32 lm (n = 66); a few of these

had fusiform somata oriented parallel to the tectal surface,

while the majority had vertically oriented pear-shaped

somata. The GABA-immunonegative cell bodies, observed

in semi-thin sections, were larger in size with a mean

diameter of 11.8 ± 3.44 lm (n = 57). In the neuropil

numerous GABA-ir puncta were also observed, represent-

ing axon terminals and dendritic shafts. Cell counts of

GABA-ir somata in pre- and post-embedded semithin sec-

tions of the dorsolateral part of the superficial layers of the

central region of the optic tectum, corrected by Aber-

crombie’s (1946) method, indicated their mean packing

density to be 684 ± 203 cells/mm3, and that they accounted

for 50.3% of the total population of cells in this region.

Electron microscopy

Examination of the ultrathin sections revealed that the SM

and SCFE of the dorsolateral part of the central region of

Fig. 3 Micrograph of a transverse frozen section, 25 lm thick, of

the midbrain of the lamprey showing the retrograde labelling of

centrifugal visual neurons in M5 and RMA after contralateral

intraocular injection of RITC. The double arrow indicates the

mediolateral axis and the single arrow the dorsoventral axis. Scalebar 200 lm

Brain Struct Funct

123

the tectum contained several types of profile containing

synaptic vesicles (PCSV), dendritic profiles, unmyelinated

axons, and occasional profiles of glial cells and neurons

(Fig. 4a).

General criteria used in the identification of profiles

Several criteria were used to classify the profiles observed in

the superficial layers of the central optic tectum: the presence

or absence of tracer labelling or signs of degenerative

change, and the presence or absence of immunoreactivity to

glutamate or GABA. In addition, other criteria were used to

classify the axonal profiles and DCSV; the size, shape, ori-

entation and position (presynaptic or postsynaptic) of the

profiles; the relative density of the hyaloplasm; the presence

of microtubules, microfilaments and ribosomes; the number

of mitochondria; and the shape of the vesicles (rounded or

flattened), together with their size, distribution and density.

The final criterion was the type of synaptic junction: on

the one hand, those described as Type I (Gray 1959) or

asymmetrical (Colonnier 1968), with a synaptic cleft

approximately 30 nm wide, with a well-developed synaptic

grid and a slight degree of postsynaptic differentiation; and

on the other hand those described as Type II (Gray 1959) or

Fig. 4 a A low-power

electromicrograph showing

HRP-labelled (DAB method)

optic fibres (OF) and their AT1terminals in the central region of

the SCFE. b An optic fibre (OF)

making synaptic contact enpassant (arrow) with a dendrite

profile (D). c An unmyelinated

HRP-labelled (TMB method)

optic fibre (OF) showing a high

degree of glutamate-

immunoreactivity (post-

embedding technique, 10 nm

gold particles). d A triple-

labelled preparation showing an

HRP-labelled (TMB method),

glutamate-immunoreactivity,

OF (post-embedding technique,

10 nm gold particles) and

GABA-immunoreactive AT3

terminals (30 nm gold

particles). Note the presence of

many 10 nm gold particles,

indicating glutamate-

immunoreactivity, over the

mitochondria of the AT3

terminals. Scale bars 2 lm in

(a), 1 lm in (c), 0.5 lm in

(b, d)

Brain Struct Funct

123

symmetrical (Colonnier 1968), in which the synaptic cleft is

approximately 18 nm wide, and with a considerably poorer

degree of postsynaptic differentiation.

General considerations of GABA- and glutamate-

immunoreactivity

The levels of background immunogold labelling were

0.94 ± 0.20 particles/lm2 for the anti-GABA antibody

(Fig. 13), and 1.38 ± 0.62 particles/lm2 for the anti-gluta-

mate antibody (Fig. 12). We do not subtract these values

from those presented below concerning the immunoreac-

tivity of the PCSV, conventional dendrites and neuronal

somata.

The density of immunolabelling of the two categories

of dendritic profiles, not containing synaptic vesicles,

(conventional dendrites: D), postsynaptic to the various

categories of terminal that we describe below was, for the

first, 23.2 ± 4.5 particles/lm2 for the anti-glutamate anti-

body (Fig. 12) and for the second 17.4 ± 2.2 particles/lm2

for the anti-GABA antibody (Fig. 13) (n = 40 for each).

The analysis of double immuno-labelled preparations

(Figs. 4d, 7a) treated with anti-glutamate and anti-GABA,

showed that the GABA-ir terminals (AT3, AT4, see below)

and GABA-ir (DCSV, see below) were also moderately

glutamate-immunoreactive (8.03 ± 0.7 particles/lm2 for

AT3 and AT4, n = 37, and 8.43 ± 1.2 particles/lm2 for

DCSV, n = 21) (Fig. 12). A more detailed analysis

revealed that these particles were generally situated over

mitochondria (Fig. 7a), while in GABA-negative, strongly

glutamate-ir profiles (AT1, AT2, see below) the gold par-

ticles were more concentrated over the synaptic vesicles

(Figs. 7, 8b, e).

Profiles containing synaptic vesicles (PCSV)

Six categories of PCSV could be distinguished on the

basis of several cytological; and immunochemical criteria.

Five of these correspond to axon terminals (AT) and the

sixth to DCSV. Of a total of 430 PCSV seen to make

synaptic contact, we were able to estimate that 91% of

these were axo-dendritic contacts with spines or den-

dritic shafts which sometimes contained synaptic vesicles,

8.9% were dendro-dendritic contacts, and 1.1% were axo-

somatic. We also point out the total absence of myelin-

ated axons.

Axon terminals

AT1. These correspond to retinal terminals, since they are

consistently labelled with tracer after iontophoretic

deposit of HRP into the optic nerve, or show degenerative

changes after intraocular injection of calcium chloride.

All the other types of PCSV, which we describe below,

were unlabelled and showed no signs of degenerative

change.

Many HRP-labelled (Figs. 4, 5d–g, 6, 7) or degenerating

profiles (Fig. 5a–c) were observed in the superficial reti-

norecipient layers of the tectum. These were unmyelinated

visual fibres and their terminals. The visual fibres were

irregularly shaped (Fig. 4a), displaying numerous neuro-

filaments, few microtubules, and occasional agranular

synaptic vesicles (Fig. 4b). These highly glutamate-ir fibres

(42.6 ± 3.9 particles/lm2, Fig. 4d) occasionally made

synaptic contact en passant with small, glutamate-ir or

GABA-ir dendrites. The diameter of these fibres varied

from 0.54 to 4.2 lm, about 60% of them having diameters

between 1 and 3 lm. In lampreys having received intra-

ocular calcium chloride, the retinal terminals showed a

variety of degenerative changes (Fig. 5a–c). The first sign

of degeneration was a swelling of the synaptic vesicles,

followed by a progressive darkening of the axoplasm in

which the organelles were more and more disrupted. In

those cases involving both degeneration and HRP tracing

techniques, we never observed HRP-labelled terminals in

the superficial layers of the optic tectum, but exclusively

labelled dendrites arising from the centrifugal visual neu-

rons of the RMA.

The AT1 terminals, with a mean cross-sectional area of

0.96 ± 0.09 lm2 and an irregular and sometimes scalloped

outline, were densely packed with round agranular synaptic

vesicles with a mean size of 1,340 ± 74 nm2 and a shape

index of 0.90 ± 0.10 (n = 420) (Fig. 11). The terminals

were often gathered into clusters (Fig. 4a), and were

GABA-immunonegative (Fig. 7a) their density of labelling

(0.98 ± 0.15 particles/lm2) (Fig. 12) being close to that of

the background; on the other hand, they showed strong

glutamate-immunoreactivity (43.8 ± 2.10 particles/lm2,

compared to a background of 1.38 ± 2.3 particles/lm2,

n = 33) (Figs. 7, 13). Their active zone is small (0.23 ±

0.04 lm). These terminals make asymmetrical synaptic

contact, their postsynaptic targets being either DCSV,

(19% of cases) (Figs. 5b–d, f, g, 6a–c, 7b), the distal

dendritic segments of HRP-labelled retinopetal neurons

of the RMA (7% of cases) (Fig. 5e), or conventional

dendrites unlabelled by HRP (74% of cases) (Fig. 5d).

About one-third of these latter profiles are GABA-immu-

noreactive (17.4 ± 2.2 particles/lm2), the remainder being

glutamate-positive (23.2 ± 4.5 particles/lm2, Fig. 5d).

The HRP-labelled dendrites of the centrifugal visual neurons,

which never contain synaptic vesicles, are either glutamate-

immunoreactive or GABA-immunoreactive (Fig. 5e). The

DCSV, which are of two types, are consistently GABA-ir

(see below). The AT1 retinal terminals represented 31.3% of

the total population of PCSV in the dorsolateral retinore-

cipient layers of the contralateral optic tectum.

Brain Struct Funct

123

AT2. These terminals, which are never labelled after

iontophoretic administration of HRP to the optic nerve, and

which do not degenerate after destruction of the retina, are,

like the AT1 terminals, GABA-immunonegative (Fig. 8c)

and strongly glutamate-ir (46.2 ± 3.1 particles/lm2, n = 28)

(Figs. 8b, e, 12). They are large in size (mean cross-sec-

tional area 0.99 ± 0.15 lm2) and generally rounded or

ovoid in shape. Their axoplasm is light, and within it some

neurofilaments can be observed. They contain rounded

synaptic vesicles with a mean size of 965 ± 51 nm2 and a

shape index of 0.88 ± 0.03 (n = 152) (Fig. 11), values

which differ (P \ 0.001) from those of AT1 terminals.

Their large active zone (0.29 ± 0.04 nm) makes asym-

metrical synaptic contact, rarely with HRP-labelled

dendrites of the centrifugal visual neurons of the RMA

(Fig. 8e) or with GABA-ir DCSV (Fig. 8b, c), but more

commonly (75% of cases) with dendrites unlabelled by

HRP, (Fig. 8c) which are either glutamate-ir or GABA-ir.

In the retinorecipient layers of the tectum, alongside the

glutamate-ir labelled visual fibres, there also exist unmy-

elinated fibres, unlabelled by HRP, showing glutamate

immunoreactivity (AX2) which may establish synapses en

Fig. 5 Double-labelled

preparations combining GABA

immunocytochemistry (post-

embedding method) and either

degeneration (a–c) or HRP

labelling (d–g, DAB method).

a A degenerating AT1 terminal

making synaptic contact with a

conventional GABA-

immunonegative dendrite

(Dg-). b A similar terminal

making synaptic contact with

a GABA-ir DCSV of type 2.

c A GABA-ir DCSV poor in

synaptic vesicles, probably of

type 1, contacted by a

degenerating visual terminal in

which the synaptic

differentiation is still visible

(arrows). d An HRP-labelled

optic terminal making contact

with a GABA-ir DCSV (topof image) and a conventional

GABA-negative dendrite

(Dg-, bottom of image),

e An HRP-labelled GABA-

immunoreactive dendrite of an

RMA centrifugal visual neuron

contacted by a AT1 optic

terminal, f, g Examples of HRP-

labelled optic terminals making

synaptic contact with GABA-ir

DCSV. Scale bars 0.25 lm in

(f), 0.5 lm in other images

Brain Struct Funct

123

passant with DCSV (Fig. 8a) or with HRP-labelled den-

drite of the centrifugal visual neurons (Fig. 8d). While we

have no tangible proof, it is quite possible that the AT2

terminals, which account for 18.8% of the total population

of PCSV, arise from these unlabelled fibres.

AT3. These GABA-ir (20.8 ± 2.4 particles/mm2,

n = 26) (Fig. 13) terminals are medium-sized (0.95 ±

0.204 lm2) and display a medium to dense matrix with

densely packed flattened vesicles (535 ± 65 nm2 in size

with a shape index of 0.42 ± 0.06, n = 132) (Figs. 4d, 9a,

11). They make symmetrical synaptic contact, for the most

part with small glutamate-ir profiles. They account for

22.6% of the total population of PCSV.

AT4. These terminals are also GABA-ir (Figs. 5a, 9c–e),

but more so (P \ 0.001) than the AT3 terminals

(24.2 ± 3.1 particles/mm2, n = 28) (Fig. 13), with a mean

cross-sectional area of 0.94 ± 0.27 lm2, their light to

moderately dense axoplasm contains numerous small,

rounded to oval, synaptic vesicles (802 ± 70 nm2 in size,

with a mean shape index of 0.82 ± 0.16, n = 143), these

Fig. 6 Examples of the two

types of DCSV observed in the

superficial tectal layers of the

lamprey. a A GABA-

immunoreactive dendrite,

oriented vertically with respect

to the tectal surface, gives off a

collateral branch containing

synaptic vesicles (arrows)

which ends in a swelling packed

with synaptic vesicles (DCSV2)

which is contacted by a HRP-

labelled AT1 terminal (DAB

method). b and c The type 1

DCSV are less frequently

observed than the former; they

generally appear as large

GABA-immunoreactive profiles

oriented parallel to the tectal

surface and contain

pleiomorphic vesicles at a lower

density than the DCSV2. The

DCSV1 are contacted by HRP-

labelled AT1 terminals (DAB

method). In c, note the synaptic

triad (arrows) involving an

HRP-labelled AT1 terminal,

a GABA-immunoreactive

DCSV1 and a small GABA

immunonegative dendrite

profile (Dg-). Scale bar 0.5 lm

in all images

Brain Struct Funct

123

values differing (P \ 0.001) from those of AT3 terminals

(Fig. 11). They make asymmetrical synaptic contact with

HRP-labelled dendrites of RMA neurons (Fig. 9c), with

GABA-ir DCSV (Fig. 9e), and small dendritic profiles

(Fig. 9d) which may be either GABA- or glutamate-ir.

They account for 14.8% of the total population of PCSV.

We point out the existence within the retinorecipient layers

of the tectum of large (C2 lm) strongly GABA-ir (23 ±

2.6 particles/mm2) unmyelinated axons (Fig. 9b), from

which these terminals may possibly arise.

AT5. These terminals (Fig. 9f, g), which are rarely

observed, accounting for 1.7% of the total population of

PCSV, are characterised by the fact that they contain both

clear synaptic vesicles and a large number of dense core

vesicles. Some of them show an accumulation of GABA-

immunoreactive particles over their synaptic vesicles.

Dendrites containing synaptic vesicles (DCSV)

These profiles are GABA-ir (23.04 ± 3.9 particles/mm2,

n = 38) (Fig. 13); their sizes vary considerably as a

function of the plane of section. Within their clear axo-

plasm microtubules, ribosomes and mitochondria may be

observed; finger-like blobs of smooth endoplasmic reticu-

lum can also be observed. Their synaptic vesicles are large

[1,270 ± 76 nm2) and ovoid to flattened in shape (shape

index 0.67 ± 0.11, n = 143) (Fig. 11)]. In general, two

types of DCSV can be distinguished by morphological

Fig. 7 a An example of triple

labelling, showing an HRP-

labelled AT1 terminal (TMB

method) expressing glutamate

immunoreactivity (10 nm gold

particles) and two GABA-ir

AT3 (30 nm gold particles);

note that in these, the smaller

particles indicating glutamate

reactivity are generally

concentrated over mitochondria.

b An example of a synaptic

triad, in which a glutamate-

immunoreactive HRP-labelled

AT1 terminal (DAB method)

makes synaptic contact with a

glutamate-immunonegative

DCSV2, which in turn contacts

a conventional glutamate-

immunoreactive dendrite

(DG?). Scale bars 0.25 lm in

(a), 0.5 lm in (b)

Brain Struct Funct

123

criteria. Those of the first type (DCSV1) are relatively rare;

large in size and polygonal in shape, their longest axes are

oriented parallel to the tectal surface (Fig. 6b, c). Their

synaptic vesicles are dispersed throughout the profile, but

tend to be concentrated in the region of the synaptic dif-

ferentiation; these DCSV are most commonly observed

in the SM. The DCSV of the second, commoner, type

(DCVS2) have a generally ovoid profile, smaller than those

of the first type, (Figs. 5f, g, 6a, 7b, 8c, 9e). The density of

their synaptic vesicles is visibly greater than that of the

DCSV of the first type; these profiles are more common in

the SCFE. In the majority of cases they appear in isolation

among other profiles of the neuropil. In particularly

favorable cases they appear as collateral, vesicle-filled

processes united to a dendritic shaft by a short pedicle

(Fig. 6a); the dendritic profiles which give rise to these

DCSV are generally oriented vertically to the tectal sur-

face. We point out, however, that in some cases (Figs. 5c,

d, f, 8b) it was not possible to clearly determine, on the

basis of these morphological criteria, to which category

some DCSV belonged.

The two type of DCSV may be postsynaptic to AT1 and

more rarely to AT2 and AT4 terminals, and may them-

selves make symmetrical synaptic contact with dendritic

profiles; these synaptic triads are, however, relatively rare

(Figs. 6c, 7b, 9e). The DCSV account for 10.8% of the

Fig. 8 Various aspects of AT2

terminals and their axons

(AX2). a A GABA-

immunoreactive DCSV,

oriented parallel to the tectal

surface and poorly endowed

with synaptic vesicles, is

contacted by an HRP labelled

(DAB method) AT1 terminal

(arrow) and en passant by an

HRP unlabelled AX2 axon

(arrow). b Two AT2 terminals

showing strong glutamate-

immunoreactivity, one of which

makes asymmetrical synaptic

contact with a DCSV. c An

example of an HRP unlabelled

AT2 terminal GABA

immunonegative making

asymmetrical synaptic contact

with a GABA-immunoreactive

DCSV2 and a conventional

GABA immunonegative

dendrite. d A glutamate-

immunoreactive (10 nm gold

particles) AX2 axon making

contact en passant with a

dendrite HRP-labelled (DAB

method) centrifugal visual

neurons of the RMA.

e A glutamate-immunoreactive

AT2 terminal making

asymmetrical synaptic contact

with a glutamate-

immunoreactive HRP labelled

(TMB method) dendrite of a

centrifugal visual neuron. Scalebar 0.5 lm in all images

Brain Struct Funct

123

PCSV: this figure may, however, be an underestimate. In

our stereological analyses of the neuropil, a small group

(1.8%) of the PCSV could not be unambiguously classified

on account of their small size, paucity of synaptic vesicles

and occasionally the absence of a synaptic differentiation.

It is thus conceivable that a small proportion of the profiles

we classify as AT3 may in fact be DCSV.

We finally point out that the different GABA-positive

elements (conventional dendrites, DCSV and terminals)

can also be recognized in sections of tissue treated for pre-

embedding. However, the immunolabelling is frequently so

intense that it partially obscures the synaptic vesicles,

which generally appear rounded in shape.

Neuronal somata

Glutamate-ir somata. These neurons can be identified in

sections treated by pre-embedding (Fig. 10a) or post-

embedding (Fig. 10b) procedures. In the later case these

are labelled at an intensity of 22.6 ± 3.8 particles/mm2,

n = 18) (Fig. 12). The shapes of these neurons are vari-

able; rounded, fusiform, triangular or multipolar. Their

Fig. 9 a Example of GABA-

immunoreactive AT3 terminals

with pleiomorphic synaptic

vesicles making symmetrical

synapse. b An unmyelinated

GABA-immunoreactive axon of

large diameter. c–e Examples of

GABA-immunoreactive AT4

terminals with rounded synaptic

vesicles making asymmetrical

synaptic contact with an HRP-

labelled dendrite (DRN) of a

centrifugal visual neuron of the

RMA (c), with a small GABA

immunonegative dendrite

(Dg-) (d), and with a GABA-

immunoreactive DCSV (10 nm

gold particles) which is

presynaptic to a GABA-

immunonegative conventional

dendritic profile (e). f, gExamples of two AT5 terminals

containing numerous dense-core

vesicles and small agranular

vesicles. Scale bar 0.5 lm

in all images

Brain Struct Funct

123

greatest diameter varies between 11 and 18 lm. The thick

layer of cytoplasm surrounding the nucleus is rich in dif-

ferent types of organelle. The somata of these neurons

receive few synaptic contacts from AT2, AT3 and AT4

terminals.

GABA-ir somata. These neurons are easily recognized in

pre-embedded material (Fig. 10c), the cytoplasm of the cell

body and dendritic trunks being densely immunolabelled. In

post-embedded sections (Fig. 10d), the somata are densely

labelled (19.8 ± 3.3 particles/mm2, n = 20 (Fig. 13).

These neurons are round to fusiform in shape. In the latter

case, some of the somata, particularly those in the SM, have

their long axes, which vary from 7 to 13 lm, oriented

parallel (horizontal interneurons, HI) to the tectal surface, or

vertically (radial interneurons, RI). All these neurons are

characterised by a voluminous nucleus enveloped by a thin

ring of cytoplasm in which organelles are infrequent. On

Fig. 10 a, b Examples of large

diameter, glutamate-

immunoreactive projection

neurons. a A low power view of

such a cell treated by the pre-

embedding procedure. b Detail,

at a higher magnification, of a

cell treated by the post-

embedding technique. c, dExamples of small GABA-

immunoreactive interneurons

(c, d), treated by the pre-

embedding (c) or post-

embedding (d) techniques. Note

their relatively large nuclei,

each surrounded by a thin fringe

of cytoplasm. Scale bars 1 lm

in (a, c, d), 0.5 lm in (b, e, f)

0

Shap

e fa

cto

r

200 400 600 800 1000 1200 1400 1600

AT3

AT4

AT2 AT1

DCSV

Surface Area (nm²)

0,2

0,4

0,6

0,8

1,0

1,2

Fig. 11 The relationship between the mean shape factor and surface

area (±SD) in synaptic vesicles from (1) glutamate-immunoreactive

AT1 and AT2 termninals, (2) GABA-immunoreactive AT3 and AT4

terminals, and (3) GABA-immunoreactive DCSV

GLUTAMATE

0

10

20

30

40

50

60

Mea

nn

um

ber

ofg

old

par

ticu

les

/µm

²

AT1 AT2 OF DG+ PNS AT3 AT4 DCSV BA

Fig. 12 The intensity of glutamate immunoreactivity expressed as

the mean (±SD) number of gold particles/lm2 over GABA-immu-

nonegative AT1 and AT2 terminals, in GABA-immunoreactive AT3

and AT4 terminals, GABA-immunoreactive DCSV, the somata of

GABA- immunonegative projection neurons (PNS) and their den-

drites (DG?), and over empty resin (BA)

Brain Struct Funct

123

occasion, cilia issuing from the somatic cytoplasm can be

observed. The somata of these neurons receive some syn-

aptic contact from AT2 and AT4 terminals.

The synaptic circuitry that we propose is summarised in

a schematic diagram (Fig. 14).

Discussion

Comparison with previous findings in the lamprey

Light microscopy

Cyto/chemoarchitecture of the superficial layers of the

optic tectum. The fact that the superficial layers of the optic

tectum of the lamprey are generally poorly furnished with

neurons has been mentioned in many studies of Lampetra

and other lamprey species (Ariens-Kappers et al. 1936;

Heier 1948; Leghissa 1962; Schober 1964; Pfister 1971;

Nieuwenhuys 1977; Kennedy and Rubinson 1984; Rio

et al. 1996; Nieuwenhuys and Nicholson 1998; Iwahori

et al. 1999). We are able to support this finding with some

quantitative data, concerning in particular the GABA-ir

neurons of this region. These cells, which account for half

the neurons of the superficial layers of the dorsolateral

region of the central portion of the tectum, are sparsely

distributed at a density of 684 ± 203 cells/mm3. In a

recent study of the distribution of GABA in the central

nervous system of L. fluviatilis, Robertson et al. (2007)

note that GABA-ir neurons are particularly numerous in

the deeper tectal layers, the stratum griseum periventricu-

lare in particular, whereas in the superficial layers they are

widely dispersed and weakly labelled. They note, as we do,

that in the latter region the neurons are for the most part

small or medium sized. They also describe several large

GABA-containing cells, which we did not observe in our

material. The fact that the small cells are GABAergic

indicates that they are most likely local circuit neurons or

interneurons (see below). The nature of the larger, GABA-

immunonegative neurons, which are highly glutamate-

immunoreactive, is more difficult to determine, but it is

quite likely that they are intra- or extratectal projection

neurons.

Hodological data. The distribution of retinal projec-

tions to the optic tectum, and that of the retinopetal

neurons, that we describe above after intraocular injection

AT1AT2AT4

AT1AT2AT4

DCVS1

HI

Dg+

AT2AT4

DG+

AT1AT2AT4

DCVS1

AXg(?)

Dg+

RI

AT1AT2AT4

AT1AT2AT4

DCSV2DG+

AT1AT2AT4

AT2AT4

DCSV2

AXg(?)

DCF, G+, g+

AT1AT2AT4

DG+

AT1AT2

AT4AT3

Fig. 14 A schematic

representation of the proposed

synaptic circuitry in the

retinorecipient layers of the

optic tectum of the lamprey

GABA

0

5

10

15

20

25

30

Mea

nn

um

ber

ofg

old

par

ticu

les

/µm

²

AT3 AT4 INS BADg+DCSV

Fig. 13 The intensity of GABA-immunoreactivity expressed as the

mean (±SD) number of gold particles/lm2 over AT3 and AT4

terminals, DCSV, the somata of interneurons (INS) and their

dendrites (Dg?), and over empty resin (BA)

Brain Struct Funct

123

of RITC or iontophoretic deposit of HRP into the optic

nerve of L. fluviatilis are entirely comparable to those

obtained in previous studies of this and other species

of lamprey using differents tracers: [3H] proline, [3H]

adenosine, [3H] glycine, HRP (Kosareva et al. 1977;

Kosareva 1980; Reperant et al. 1980, 1982, 1985; Ves-

selkin et al. 1980, 1984, 1996; de Miguel et al. 1990;

Pierre et al. 1992; Rio et al. 1992, 1996; Rodicio et al.

1995). We nevertheless point out that in our experiments

involving the degeneration of optic fibres by intraocular

injection of calcium chloride, followed several days later

by the iontophoretic deposit of HRP into the optic nerve,

anterograde labelling was totally absent from the primary

visual centres and in particular from the superficial tectal

layers. In these latter regions the only labelled structures

were a few dendrites of the centrifugal visual cells of the

RMA, labelled by retrograde transport of the enzyme. No

labelled prolongations, possibly corresponding to axons of

the centrifugal neurons of M5, were observed to pass in

their direction of the tectum. These observations indicate

(see also below) that none of the centrifugal visual neu-

rons project by way of collaterals in the superficial tectal

layers, a hypothesis recently formulated by Robertson

et al. (2006).

Electron microscopy

A single study of the fine structure of the optic tectum of

the lamprey (Rio et al. 1996) has been carried out; it was

primarily concerned with the synaptic relationships of the

intratectal dendrites of the centrifugal visual neurons of the

RMA with retinal terminals and other categories of ter-

minal. Our results are, on the whole, in general agreement

with several of those reported by Rio et al. (1996), par-

ticularly those concerning the cytological, morphometric

and synaptological properties of the retinal terminals. Rio

et al. (1996) also describe seven other types of PCSV,

including DCSV, of which the majority were situated in the

central and deep layers of the optic tectum. In the present

study devoted to the retinorecipient layers, in addition to

the retinal terminals (AT1) and the DCSV, we were able to

identify only four categories of terminal, of which one

(AT5) was not described by Rio et al. (1996). The

remaining three (AT2, AT3 and AT4) are difficult to

compare to the nonvisual terminals contacting the RMA

dendrites described by Rio et al. (1996), since these latter

PCSV were mainly observed in the nonretinorecipient

tectal layers.

Comparison with Gnathostomes

We propose to compare our ultrastructural data concerning

the retinorecipient layers of the optic tectum of the lamprey

with those of the literature concerning the same layers of

the optic tectum or superior colliculus of jawed vertebrates.

Retinal terminals (AT1)

The cytological features of these terminals (scalloped

outline, electron-lucent axoplasm, round synaptic vesicles

and asymmetrical synaptic contacts) in the lamprey are

totally comparable to those of retinotectal or retinocollic-

ular terminals described in Gnathostomes (for reviews:

teleosts: Peyrichoux et al. 1986; Vanegas et al. 1984;

Kageyama and Meyer 1989; amphibians, Lazar 1984;

Antal 1991; Gabriel and Straznicky 1995; reptiles, Repe-

rant et al. 1981, 1997; birds, Reperant and Angaut 1977;

Acheson et al. 1980; Morino et al. 1991; Tombol 1998;

Tombol and Nemeth 1999; Tombol et al. 2003. mammals:

Mize 1983, 1988; Huerta and Harting 1984; Schonitzer and

Hollander 1984; Ortega et al. 1995; Mize and Butler 1996).

The proportion of the total population of PCSV represented

by the retinal terminals of the lamprey (31.3%, present

results) lies within the range of values of this proportion

described in Gnathostomes (Teleosts: Carassius, 37% for

Murray and Edwards (1982) but 27% for Airhart and

Kriebel (1984); Rutilus, 23.5% (Peyrichoux et al. 1986);

Holocentrus, 16% (Ito et al. 1980); Reptiles: Vipera aspis,

25% (Reperant et al. 1981); Birds: Columba livia, 23%

(Angaut and Reperant 1976); Mammals: rabbit, 20%

(Vrensen and de Groot 1977); cat, 24% (Sterling 1971); the

primate Galago, 45% (Tigges et al. 1973). This variation

may reflect interspecific differences as well as different

methods of estimation.

The retinal terminals (AT1) of the lamprey tectum are

GABA-immunonegative and strongly glutamate-immuno-

reactive. It has been shown that the same is true not only of

retinotectal or retinocollicular terminals in all major groups

of Gnathostomes (fish, Kageyama and Meyer 1989;

amphibians, Gabriel and Straznicky 1995; reptiles, Repe-

rant et al. 1997; birds, Morino et al. 1996; mammals,

Ortega et al. 1995; Mize and Butler 1996) but also of all

retinal terminals in general (Reperant et al. 1997; Kenigfest

et al. 1998 for review), with the exception of a small

population of GABAergic visual terminals (Kenigfest et al.

1998; Miceli et al. 2008 for review) of which we found not

the slightest trace in the lamprey.

The fact that the retinal terminals are highly glutamate-

reactive, and that this immunoreactivity is preferentially

expressed over synaptic vesicles, strongly suggests that

these terminals use glutamate as a neurotransmitter (cf.

Montero 1990, 1994; Mize and Butler 1996; Reperant et al.

1997; Kenigfest et al. 1998). Other data support this

hypothesis. In the optic tectum and other primary visual

centres (dorsal lateral geniculate nucleus, nucleus of the

optic tract, suprachiasmatic nucleus) of a wide variety of

Brain Struct Funct

123

species (goldfish, frog, pigeon, chicken, mouse, rat, rabbit,

cat) extra- and intracellular recordings in vivo or in vitro

have shown that antagonists of excitatory amino acids such

as glutamate block the excitatory postsynaptic potentials

induced by stimulation of the retina. In addition, the

iontophoretic administration of glutamate produces an

excitatory response of visually sensitive neurons (Felix and

Frangi 1977; Langsdon and Freeman 1986; Shibata et al.

1986; Debski et al. 1987; van Deusen and Meyer 1988;

Cahill and Menaker 1989; Murphy et al. 1989; Hartveit and

Heggelund 1990; Heggelund and Hartveit 1990; Sillito

et al. 1990a, b; Funke et al. 1991; Kim and Dudek 1991;

Kwon et al. 1991; Roberts et al. 1991; Schmidt 1991;

Hestrin 1992; Binns and Salt 1994; Dye and Karten 1996).

In the pigeon optic tectum (Canzek et al. 1981) and the

rabbit superior colliculus (Sandberg et al. 1982), electrical

stimulation of the optic nerve leads to a release of endog-

enous glutamate and aspartate in vivo. In vitro, a calcium-

dependent potassium-induced release of glutamate has

been demonstrated in pigeon brain slices (Reubi 1980;

Toggenburger et al. 1982). The high-affinity uptake of

glutamate is greatly reduced in synaptosomes prepared

from the retinorecipient layers of enucleated chickens

(Henke et al. 1976) or pigeons (Bondy and Purdy 1977);

retinal ablation also leads to a significant reduction of

glutamate and aspartate levels in the optic nerve and reti-

norecipient tectal layers in the pigeon (Fonnum and Henke

1982; Morino et al. 1991) and the corresponding collicular

layers in the rat (Sakurai and Okada 1992; Sakurai et al.

1990). Mize and Butler (1995) have shown that, in the

superficial layers of the cat superior colliculus, antibody

labelling of NMDA R1 receptor subtypes is present at the

postsynaptic densities opposite to retinal terminals. Finally,

NMDA and nonNMDA agonists have been shown to excite

the retinorecipient layers of the feline superior colliculus in

vitro (Grantyn et al. 1987; Perouansky and Grantyn 1990).

Our data show that the retinotectal terminals in Lam-

petra make synaptic contact essentially with dendritic

profiles, of which the majority are devoid of synaptic

vesicles (conventional dendritic profiles, D), and with a

minority of DCSV. The D profiles are either GABA-ir

(Dg?) or glutamate-ir (DG?). It has been shown that

the same applies to all major groups of Gnathostomes

(fish, Kageyama and Meyer 1989; amphibians, Antal 1991,

Gabriel and Straznicky 1995; reptiles, Rio et al. 1995,

Reperant et al. 1997; birds, Morino et al. 1991; mammals,

Houser et al. 1983; Mize 1988; Mize and Butler 1996).

However, the relative proportions of the two types of

postsynaptic target vary somewhat. In Lampetra, 19% of

the retinal terminals contact DCSV (present results), a

value to be compared with 4% in the amphibian Ambys-

toma mexicanum (Ingham and Guldner 1981), 10% in the

teleost Rutilus (Peyrichoux et al. 1986), 11% in the viper

(Reperant et al. 1981), 33–50% in the cat (Sterling 1971;

but 20–34% in the same species for Behan 1981) and 57%

in the cane toad Bufo marinus (Gabriel and Straznicky

1995). The precise cellular origins of these dendritic targets

are for the most part unknown, but it is highly probable that

in all taxonomic groups the GABA-ir DCSV arise from

interneurons (see below), as do a proportion of GABA-ir D

profiles. However, in the lamprey, we have seen that some

of these latter profiles arise from the retinopetal neurons of

the RMA, and a small proportion of GABA-negative,

glutamate-positive dendritic profiles have the same origin

(present results).

With the exception of a relatively modest proportion of

synaptic contacts between retinal terminals and the den-

drites of the glutamate-ir retinopetal neurons of the RMA

(present results), we have no data concerning the intratectal

origin of the glutamate-ir neurons whose conventional

dendrites are postsynaptic to retinal terminals. They may

possibly arise from the GABA-immunonegative neurons of

the superficial tectal layers, of which the somata are

strongly glutamate-immunoreactive. It is also possible that

these dendrites arise from efferent neurons situated in the

deep tectal layers (Heier 1948; Kennedy and Rubinson

1984; Iwahori et al. 1999), whose long vertical dendrites

penetrate the retinorecipient layers (Iwahori et al. 1999).

Such an organisation has been clearly demonstrated in the

superior colliculus of the cat (Jeon et al. 1997) and the

optic tectum of the chicken (Tombol and Nemeth 1999;

Tombol et al. 2003).

We point out that one of the synaptological peculiarities

of the retinorecipient layers of the lamprey tectum, in

comparison with those of Gnathostomes, is that the visual

fibres make direct synaptic contact with the dendrites of

retinopetal neurons, thus forming a fast visual feedback

loop (retina ? tectum ? centrifugal neurons ? retina;

cf. Rio et al. 1996; Vesselkin et al. 1996; Reperant et al.

2006). While a similar feedback loop exists in birds and

crocodiles, in these organisms the dendrites of the cen-

trifugal neurons do not penetrate the optic tectum, and the

feedback loop contains an additional mono- or polysyn-

aptic pathway (cf. Reperant et al. 1989, 2006, 2007;

Uchiyama 1989; Ward et al. 1991; Medina et al. 2004).

Nonretinal terminals

In contrast to the retinal terminals, we have no experi-

mental hodological data concerning the origins of the

nonretinal GABA-ir and glutamate-ir terminals. However,

the recent light microscopic studies by Robertson et al.

(2006, 2007) which combine axonal tracing techniques and

immunohistochemistry in an attempt to identify the origins

of tectal afferent supplies and their neurochemical prop-

erties provide us with the means to advance several

Brain Struct Funct

123

hypotheses concerning the origin of these terminals. In the

same light, the comparison of our ultrastructural findings

with those in the literature concerning the superficial tectal

or collicular layers of Gnathostomes enables us, on the

basis of cytological, neurochemical and synaptological

criteria, to establish some evident points of comparison.

GABA-negative, glutamate-positive, nonvisual AT2

terminals

A relatively important proportion (18.8% of PCSV) of this

category of terminals has been recognised in the retinore-

cipient layers of the tectum of Lampetra (present results).

Given the relatively large proportion of AT2 terminals, it is

quite likely that they represent several subgroups arising in

different structures. In this context, Robertson et al. (2006)

have identified several structures containing GABA-im-

munonegative neurons (possibly glutamate-ir) which

provide afferent supplies to the tectum, for example those

situated in the caudo-ventral telencephalic pallium, the

pretectal region, the isthmic region and the octavolateral

area.

In the same tectal or collicular regions of Gnathostomes,

profiles with the same cytological characteristics as the

AT2 terminals of the lamprey (size and shape of the

synaptic vesicles, asymmetrical synaptic contact and con-

ventional dendrites or DCSV as targets) have been

identified (teleosts, Ito et al. 1980; Meek 1981; Peyrichoux

et al. 1986; amphibians, Antal 1991; reptiles, Reperant

et al. 1981; birds, Angaut and Reperant 1976; Tombol et al.

2003; mammals, Lund 1969, 1972; Sterling 1971; Tigges

et al. 1973; Tigges and Tigges 1975; Mize and Butler

1996), and some of these studies (Antal 1991; Mize and

Butler 1996) have shown that, like the AT2 terminals of the

lamprey, they are GABA-negative and glutamate-ir. The

proportion of these terminals varies considerably between

groups: our value of 18.8% is to be compared with 1% in

the pigeon (Angaut and Reperant 1976), 4% in the viper

(Reperant et al. 1981), 11% in the teleost Rutilus (Peyric-

houx et al. 1986), and 30% in the primate Galago (Tigges

et al. 1973). Despite these cytological, synaptological

and neurochemical similarities between these terminals

observed in lampreys and Gnathostomes, it is, at present,

impossible to affirm that they are homologous between the

two groups, since their origins remain largely unknown.

Thus, for example, in mammals it has been shown that

these glutamate-ir terminals (Mize and Butler 1996) are the

endings of the cortico-collicular pathway (Lund 1969,

1972; Sterling 1971; Tigges et al. 1973; Behan 1980;

Graham and Casagrande 1980; Mathers 1977; Valverde

1973; Mize and Butler 1996). Ito et al. (1980) have shown

that in the retinorecipient layers of the tectum of the teleost

Holocentrus rufus, there exists a category of nonvisual

terminals with round synaptic vesicles and asymmetrical

synaptic contact (S4), which degenerate after ablation of

the ipsilateral telencephalon. In the lamprey, the existence

of a telencephalo-tectal pathway has been demonstrated by

a variety of tracing techniques (Polenova and Vesselkin

1993; Northcutt and Wicht 1997; Robertson et al. 2006).

This pathway arises in the primordium hippocampi (Pole-

nova and Vesselkin 1993; Northcutt and Wicht 1997), a

structure also described as the medial pallium (Robertson

et al. 2006). Using a combination of tracing and GABA-

immunocytochemical techniques, Robertson et al. (2006)

conclude that some of these cells at the origin of this

projection, situated in the caudo-ventral pallium, are

GABA-immunoreactive, whereas the majority of these

neurons are GABA-immunonegative and probably gluta-

mate-ir.

GABAergic terminals (AT3, AT4)

In the superficial layers of the lamprey tectum, 37.4% of

PCSV are GABAergic terminals, and fall into one of two

categories; those with pleiomorphic synaptic vesicles and

symmetrical synaptic contacts (22.6%), and those with

round vesicles and asymmetrical synaptic contacts (14.8%)

(present results).

In the lamprey, it is highly likely that the GABAergic

terminals of the superficial tectal layers have several

origins. Some are almost certainly those of interneurons,

while others may be part of the afferent supplies to these

layers. In this context, Robertson et al. (2006) have shown

that in L. fluviatilis many sources of the GABAergic

afferent supply to the tectum arise in the caudo-ventral

pallium, the ventral and dorsal thalamus, the torus semi-

circularis and the nucleus M5 of Schober, within which

lie some of the centrifugal visual neurons. These authors

therefore advance two hypotheses: (1) the tectum is

innervated by collateral branches of the axons of the

centrifugal visual neurons of M5, a large number of

which are GABAergic (Rio et al. 1993); (2) the tectum

receives a GABAergic projection from M5 arising in

exclusively tectopetal neurons. This is quite plausible,

since the nucleus M5 is not composed exclusively of

centrifugal visual neurons (cf. Rio et al. 1993). Our

observations lead us to support the second of these

hypotheses (see also above). As we have pointed out,

after chemical destruction of the retina by calcium chlo-

ride, and subsequent administration of HRP to the optic

nerve several days later, only the elements of the cen-

trifugal visual neurons (axons, somata and dendrites) are

labelled with tracer. Examination of the retinorecipient

tectal layers reveals only the presence of dendrites of the

neurons of the RMA, with not the slightest trace of

labelled axon terminals.

Brain Struct Funct

123

In double-labelled preparations we have noticed that

these terminals, like other GABAergic elements in the

superficial tectal layers of the lamprey (cell bodies, den-

drites, DCSV) also display glutamate-immunoreactivity.

However, the density of particles from the anti-glutamate

antibody is consistently lower than those of the anti-GABA

antibody (see above), and that the latter are seen predom-

inantly over synaptic vesicles, whereas the former are

concentrated over mitochondria. Similar observations have

been made in other contexts (cf. Montero and Wenthold

1989; Montero 1990, 1994; Reperant et al. 1997; Kenigfest

et al. 1998). In contrast to the neurochemical identification

of other substances such as GABA, for which positive

immunoreactivity appears to be a specific marker of

GABAergic neurons, immunoreactivity to glutamate

appears to be more difficult to interpret. This amino acid is

present in most cellular compartments, and is involved in a

number of metabolic pathways. Several authors (Ottersen

and Storm-Mathisen 1984; Ottersen 1987, 1989; Storm-

Mathisen and Ottersen 1990) have suggested that gluta-

mate immunoreactivity reveals both the ‘‘metabolic’’ and

‘‘transmitter’’ pools of this amino acid in neural processes.

However, a high concentration of particles indicating

glutamate-immunoreactivity over the vesicles of AT1 and

AT2 terminals suggests that this substance is used as a

neurotransmitter (see above), while in the GABAergic

PCSV these particles are concentrated over mitochondria

and very likely reveal the metabolic pool of glutamate,

which serves as a precursor of GABA by the action of

glutamate decarboxylase (see Ottersen and Storm-Mathisen

1984).

A comparison of our findings with the data presented in

the literature concerning the superficial tectal or collicular

layers of Gnathostomes shows that in the two cases, while

the proportions of the two types of GABAergic terminal

vary from one species to another, they show a number of

cytological and synaptological features in common.

For example, Antal (1991) recognises, as in the lamprey,

two types of GABA-immunoreactive terminals in the ret-

inorecipient tectal layers of Rana esculenta; those with

pleiomorphic vesicles and symmetrical synaptic contact,

and those with rounded vesicles and asymmetrical contact;

in the same way, Tombol et al. (2003) recognize two types

of GABA-ir terminals in these regions of the chicken

tectum, one with pleiomorphic synaptic vesicles and

symmetrical contact, the other with round vesicles but with

symmetric contact. On the other hand, Rio et al. (1996)

distinguish four types of GABAergic terminal in the reti-

norecipient layers of the tectum of Vipera. Three of these

contain pleiomorphic synaptic vesicles and make sym-

metrical synaptic contact, while the fourth contains

rounded vesicles and makes asymmetrical contact. In

mammals, the majority of the GABAergic terminals of the

retinorecipient layers of the superior colliculus contain

pleiomorphic vesicles and make symmetrical synaptic

contact (cf. Lund 1969, 1972; Tigges et al. 1973; Valverde

1973; Houser et al. 1983; Mize 1992). However, Houser

et al. (1983) consider that in the retinorecipient layers of

the rat superior colliculus 4% of the GABAergic terminals

make assymmetrical synaptic contacts. In the lamprey, as

in Gnathostomes, the GABAergic terminals make synaptic

contacts with the dendrites of efferent neurons, and also

with the GABAergic DCSV of interneurons which may in

turn be presynaptic to the dendrites of efferent neurons.

Despite these similarities, it is difficult to establish

homologies between different taxonomic groups for these

different GABAergic terminals, since in the majority of

cases their origins have not been determined with

precision.

AT5 terminals

These terminals, with their many dense-core vesicles, are

rare in the retinorecipient tectal layers of the lamprey. They

have been described, without comment, in the corre-

sponding tectal layers of the viper (Rio et al. 1995), in the

isthmo-optic nucleus of the pigeon (Miceli et al. 2002; Rio

et al. 2002), the nucleus dorsolateralis anterior of the same

species (Miceli et al. 2008), and in the nucleus geniculatus

lateralis dorsalis of mammals (Papadopoulos and Parnav-

elas 1990; Pasik et al. 1988). In the latter cases it has been

shown that they correspond to serotoninergic terminals. At

the level of resolution of the light microscope, serotonin-

immunoreactive fibres have been described in the superfi-

cial tectal layers of the lamprey (Pierre et al. 1992), and it

is thus possible that the AT5 terminals correspond to the

terminals of these fibres.

Interneurons and DCSV

It has been shown many times that in Gnathostomes, as in

the lamprey (Robertson et al. 2007), GABA-ir neurons

exist in the various layers of the optic tectum or superior

colliculus In anamniotes (Franzoni and Morino 1989; Antal

1991; Medina et al. 1994), as in the lamprey (Robertson

et al. 2007; present results) these neurons are considerably

more numerous in the deeper tectal layers, and scattered at

a lower density in the superficial layers. In amniotes, par-

ticularly mammals, on the other hand, the situation is

inversed; the GABA-ir neurons are much commoner in the

superficial collicular layers than in the deeper ones (cf.

Mugnaini and Oertel 1985; Mize 1988, 1992).

The GABA-ir neurons of the superficial tectal layers

described in the lamprey (Robertson et al. 2007; present

results) resemble those described in the corresponding

tectal or collicular regions of Gnathostomes (teleosts:

Brain Struct Funct

123

Medina et al. 1994; amphibians, Franzoni and Morino

1989; Antal 1991; reptiles, Bennis et al. 1991; Rio et al.

1995; birds, Domenici et al. 1988; Granda and Crossland

1989; Veenman and Reiner 1994; mammals, Mugnaini and

Oertel 1985; Mize 1988, 1992; Mize et al. 1991,

1994).They are generally described as small in size, of

variable shape, and oriented either vertically or horizon-

tally with respect to the tectal or collicular surface. In

Lampetra they account for 50.1% of the neurons of the

superficial tectal layers, a figure close to the value of 50.3%

obtained by Rio et al. (1995) for the corresponding tectal

layers of Vipera aspis. However, different proportions of

these cells have been described in amphibians (61%; Antal

1991), the cat (45%; Mize 1988) and the rhesus monkey

(32.5%; Mize et al. 1991).

The GABA-immunoreactive profiles containing synap-

tic vesicles which we have observed in the superficial tectal

layers of the lamprey are undoubtedly of dendritic origin.

In contrast to axonal profiles, they contain polyribosomes

and some form of granular reticulum. In addition, micro-

tubules are very common, and neurofilaments are absent.

For want of serial sections, we were unable to determine

the origin of these profiles, but their high degree of

immunoreactivity strongly suggests that they arise from the

small GABAergic neurons, which show a high degree of

immunoreactivity, while the larger ones are GABA-im-

munonegative and highly glutamate-ir. We were not able to

show whether these small neurons were devoid of axons or

possessed very short axons, but in other respects they show

the cytological features of interneurons. In contrast to

projection neurons, these show a voluminous nucleus,

surrounded by a thin fringe of cytoplasm, which is poorly

endowed with organelles (cf. Peters et al. 1970; Tigges and

Tigges 1975; Mize 1992; Mize et al. 1994), as do the small

GABAergic neurons observed in our material.

In Lampetra, the DCSV are postsynaptic to glutamate-ir

retinal AT1 terminals and AT2 terminals, to GABAergic

AT4 terminals, and presynaptic to conventional dendritic

profiles (present results). In many Gnathostomes, the

DCSV of the superficial tectal or collicular layers are also

postsynaptic in most cases to retinal terminals, but also

to terminals with pleiomorphic synaptic vesicles (fish,

Peyrichoux et al. 1986; amphibians, Szekely et al. 1973;

Ingham and Guldner 1981; Antal 1991; reptiles, Reperant

et al. 1981; Rio et al. 1995; birds, Angaut and Reperant

1976; mammals, Lund 1969, 1972; Tigges et al. 1973;

Valverde 1973; Tigges and Tigges 1975; Graham and

Casagrande 1980; Vrensen and de Groot 1977; Houser

et al. 1983; Mize et al. 1991; Pinard et al. 1991).

Two types of DCSV exist in the superficial tectal layers

of the lamprey (present results). Those of the first type are

dendritic profiles generally oriented in the horizontal plane

that contain discrete clusters of pleiomorphic synaptic

vesicles located at sites of synaptic contact; those of the

second type are more richly endowed with synaptic vesi-

cles. They appear either in isolation or as varicosities

arising from collateral dendritic branches. These two types

of DCSV have been observed in Gnathostomes, most

systematically in the superficial layers of the mammalian

superior colliculus. The DCSV of the first type have been

described in the rat (Lund 1969), the mouse (Valverde

1973), the rabbit (Mize et al. 1994), the cat (Mize et al.

1982; Mize 1988), the rhesus monkey (Mize et al. 1991)

and the chimpanzee (Tigges and Tigges 1975), and it has

been shown that these DCSV arise from the horizontal cells

(cf. Mize 1992). It is likely that the same is true in the

lamprey, given that the horizontal interneurons are present

in the superficial tectal layers. These DCSV have also been

described in the superficial tectal layers of birds (Streit

et al. 1978) and reptiles (Reperant et al. 1981; Rio et al.

1995). In mammals, these profiles receive few synaptic

contacts, particularly from retinal terminals (cf. Mize

1992); in reptiles the inverse is true. In these latter forms

the DCSV are richly innervated, particularly by visual

terminals, and are frequently involved in synaptic triads

(Reperant et al. 1981; Rio et al. 1995), which does not

appear to be the case in mammals (cf. Mize 1992). In the

lamprey, these DCSV receive different types of synaptic

contact, including retinal terminals, and may be involved in

synaptic triads.

The DCSV of the second type have been described in

the superficial tectal layers of reptiles (Reperant et al.

1981), birds (Angaut and Reperant 1976) and on several

occasions in the superficial collicular layers of mammals

(Lund 1969; Sterling 1971; Valverde 1973; Tigges and

Tigges 1975; Mize 1988, 1992; Mize et al. 1982, 1991,

1994).In the lamprey, as in Gnathostomes, these DCSV

may be involved in synaptic triads of which the presynaptic

elements are generally retinal terminals. In mammals the

DCSV have been described in a variety of terms; balloon-

like dilatations (Sterling 1971), vesicle-filled spines (Lund

1969), spine-like appendages or spine-bearing dendrites

(Mize 1988, 1992), gemmule-like dendritic excrescences

(Houser et al. 1983), or as varicose dendritic enlargements

(Mize et al. 1991). It follows from this that the profiles are

interpreted as arising from dendritic spines, dendritic var-

icosities, or both of these. In the lamprey it is highly likely

that the DCSV2 arise from radial interneurons since the

collateral dendrites which give rise to them are generally

oriented perpendicular to the lateral surface. However, it is

also possible that some arise from dendritic spines. Iwahori

et al. (1999) find, in Golgi preparations of Lampetra

japonica, that the majority of neurons of the superficial

tectal layers, particularly those of small size, bear numer-

ous dendritic spines; this in marked contrast to Kennedy

and Rubinson (1984), whose Golgi preparations of

Brain Struct Funct

123

Petromyzon marinus indicate that the superficial tectal

neurons are devoid of dendritic spines. Our own findings

under the electron microscope fall between these positions,

tending towards that of Kennedy and Rubinson (1984). The

different dendritic branches of GABA-ir neurons which

appear in many of the sections we examined are very

poorly endowed with dendritic spines, and in no case did

we observe synaptic vesicles in any of these latter

structures.

Conclusions

A comparison of our data with other observations leads us

to conclude that the dorsolateral retinorecipient layers of

the optic tectum of the lamprey show many synaptological

and neurochemical similarities with the retinorecipient

layers of the tectum or superior colliculus of Gnathosto-

mes, even though it has some idiosyncrasities such as the

direct relationship of the retinal terminals to the dendrites

of retinopetal neurons and the total absence of myelinated

fibres.

These similarities are of several types. For example,

the nonvisual glutamatergic and GABAergic terminals of

lampreys and many Gnathostome species share many

common cytological, synaptological and neurochemical

characteristics; although, as we have pointed out, while

the origins of these terminals remain in many cases

unknown it is difficult to claim that the circuitry in which

they are involved is comparable from one taxonomic

group to another. On the other hand, the functional

anatomical data concerning the retinotectal system are

sufficiently extensive to reveal many similarities between

Gnathostomes and Agnathans. Thus, with the exception of

a small number of GABAergic retinal terminals (Kenigfest

et al. 1998; Miceli et al. 2008 for reviews) which are

absent in lampreys (present results), the retinotectal ter-

minals of all vertebrates are glutamatergic, have rounded

synaptic vesicles, and make asymmetrical synaptic contact

with three types of profile. The first of these are the

dendrites of glutamatergic efferent neurons, which are

activated by the retinal terminals. In the remaining two

cases, which are less numerous, the retinal terminals make

contact either with conventional GABAergic dendrites of

interneurons, or with GABAergic DCSV which may in

turn be presynaptic to the glutamatergic dendrites of

efferent neurons. In this latter triadic configuration, the

retinal terminals activate the DCSV which in turn inhibit

the efferent neurons.

It thus appears that the retinotectal system of verte-

brates, including the Petromyzontiform Agnatha, seems to

be characterised by a fundamental consistency of synaptic

organisation. This leads us to suppose that its underlying

plan probably existed in the ancestral Petromyzontiforms,

from which the Gnathostomes diverged some 450 million

years ago, and also that the consistency of synaptic cir-

cuitry probably implies a similarity of function in visual

information processing.

Numerous correlations between anatomy and function

suggest that interneurons play a powerful inhibitory role, in

particular by way of the dendro-dendritic synapses which

appear to act as a neuron-saving device (cf. Miceli et al.

2008). In addition, the microcircuits represented by the

synaptic triads involving retinal terminals (optic termi-

nal ? DCSV ? conventional dendrite) appear to play a

fundamental role in the local treatment of visual informa-

tion. Sherman (2004), for example, has suggested that they

are involved in the gain control of the visual relay cells to

maintain their modulation transfer functions within a linear

region.

In the superficial tectal layers of the lamprey this

GABAergic interneuronal network appears poorly devel-

oped in comparison to that described in the equivalent

region of Gnathostomes and particularly of Amniotes. In

the superficial tectal layers of the lamprey, the DCSV

account for 10.8% of the total population of PCSV (present

results); this proportion is considerably greater in amniotes

(21% in the viper, Rio et al. 1995; 23.5% in the rabbit,

Vrensen and de Groot 1977; 60% in the pigeon, Angaut

and Reperant 1976). In addition, in the lamprey the pro-

portion of dendro-dendritic synapses in these layers (8.9%

of synaptic contacts, present results) rises to 20% in the

viper (Rio et al. 1995) and cat (Sterling 1971) and to 55.2%

in the pigeon (Angaut and Reperant 1976). Finally, the

axo-dendro-dendritic synaptic triads involving retinal

terminals are extremely rare in the lamprey tectum but

commoner in Amniotes (reptiles; Rio et al. 1995; birds;

Angaut and Reperant 1976; mammals; Lund 1972; Houser

et al. 1983). In the light of these findings it is entirely

reasonable to suppose that the degree of information

processing is considerably less elaborate in the tectal

retinorecipient layers of Lampetra than in those of Gnat-

hostomes and in particular amniotes.

Acknowledgments We thank Denis LeCren for his extremely

skilful photographic assistance. This work was supported by MNHN,

CNRS (France), Academy of Sciences (Russia) NSERC (Canada).

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