The vertebrate olfactory system has long been an attractive model for studying neuronal regeneration and adaptive plasticity due to the continuous neurogenesis and synaptic remodelling throughout adult life in primary and secondary olfactory centres, its precisely ordered synaptic network and accessibility for manipulation. After homotopic transplantation of fetal olfactory bulbs in bulbectomized neonatal rodents, newly regenerated olfactory neurons form glomeruli within the graft, and the efferent mitral/tufted cells of the transplant innervate the host brain, terminating in higher olfactory centres. However, the synaptic connections of the transplanted relay neurons within the graft and/or host's olfactory centres could not be characterized mainly because of lack of suitable cell-specific markers for these neurons. In this study, we have used olfactory bulbs from transgenic fetuses, in which the majority of the mitral/tufted cells express the bacterial enzyme beta-galactosidase, for homotopic olfactory bulb transplantation following complete unilateral bulbectomy. In the transplants, the cell bodies and terminals of the donor mitral/tufted cells were identified by beta-galactosidase histochemistry and immunocytochemistry at both light and electron microscope levels. We demonstrate that transplanted relay neurons re-establish specific synaptic connections with host neurons of the periphery, source of the primary signal and central nervous system, thereby providing the basis for a functional recovery in the lesioned olfactory system.

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Letter to Neuroscience

BETA-GALACTOSIDASE-LABELLED RELAY NEURONS OF

HOMOTOPIC OLFACTORY BULB TRANSPLANTS

ESTABLISH PROPER AFFERENT AND EFFERENT

SYNAPTIC CONNECTIONS WITH HOST NEURONS*

G. SEKERKOVA u,†‡ Z. KATAROVA,† E. MUGNAINI,‡ F. JOO u,

≥

§ J. R. WOLFF,¶

S. PRODAN,† and G. SZABO u†Q

Institutes of †Biochemistry and §Biophysics, BRC, Hungarian Academy of Sciences, Temesva´ri krt 62,

6701 Szeged, Hungary

‡Northwestern University, Institute of Neuroscience, 320 E. Superior Street, Chicago, IL 60611, U.S.A.

¶Department of Anatomy, University of Go¨ttingen, 37075 Go¨ttingen, Germany

Key words: homotopic graft, lacZ, mitral/tufted cells, neuronal regeneration, synaptogenesis, transgenic

mice.

The vertebrate olfactory system has long been an

attractive model for studying neuronal regeneration

and adaptive plasticity due to the continuous neurogen-

esis and synaptic remodelling throughout adult life in

primary and secondary olfactory centres,

10,11,15,16,18

its precisely ordered synaptic network

6,13,24

and ac-

cessibility for manipulation. After homotopic trans-

plantation of fetal olfactory bulbs in bulbectomized

neonatal rodents, newly regenerated olfactory neurons

form glomeruli within the graft,

5,8,20,29,30

and the

eff erent mitral/tufted cells of the transplant innervate

the host brain, terminating in higher olfactory cen-

tres.

14,28,30

However, the synaptic connections of the

transplanted relay neurons within the graft and/or

host's olfactory centres could not be characterized

mainly because of lack of suitable cell-specific markers

for these neurons. In this study, we have used olfactory

bulbs from transgenic fetuses, in which the majority of

the mitral/tufted cells express the bacterial enzyme

â-galactosidase, for homotopic olfactory bulb trans-

plantation following complete unilateral bulbectomy.

In the transplants, the cell bodies and terminals of

the donor mitral/tufted cells were identified by

â-galactosidase histochemistry and immunocyto-

chemistry at both light and electron microscope

levels.

We demonstrate that transplanted relay neurons

re-establish specific synaptic connections with host

neurons of the periphery, source of the primary signal

and central nervous system, thereby providing the

basis for a functional recovery in the lesioned olfac-

tory system. ? 1997 IBRO. Published by Elsevier

Science Ltd.

The cellular and subcellular distribution of

â-galactosidase in the olfactory bulb (OB) of

transgenic mice of line Tg(GAD67lacZ7.5)1 has

been characterized in detail by â-galactosidase

histochemistry using 5-bromo-4-chloro-3-indolyl-

â- -galactopyranoside (X-gal) as substrate and

immunohistochemistry with anti-â-galactosidase

antibody.

23

We have found, that in the adult OB,

â-galactosidase is expressed in the majority of mitral/

tufted (M/T) neurons and a subpopulation of peri-

glomerular and granule granule cells (Fig. 1a and

Ref. 23). At embryonic day 17 (E17), staining is

confined exclusively to the M/T neurons and their

projections (Fig. 1b, c). Fetal OBs were removed

from E17 transgenic embryos (Fig. 1b) and subse-

quently inserted into the cavity vacated after ablation

of the right OBs of three- to five-days-old (P3–P5)

pups. The age of donor/host was chosen so as opti-

mal conditions for survival and integration of the

graft are provided.

2,14,28–30

Two to four months after

surgery, most of the transplants were stably inte-

grated and had developed into an OB-like structure

(Fig. 1d, e) occupying the space between lamina

cribrosa and forebrain. Unlike the intact OB, which

*The results presented here have appeared in an abstract

form.

22

≥

Deceased 25 February, 1996.

QTo whom correspondence should be addressed.

Abbreviations: E, embryonic day; LOT, lateral olfactory

tract; M/T, mitral/tufted cells; OB, olfactory bulb; OC,

olfactory cortex; OMP, olfactory marker protein; ORN,

olfactory receptor neurons; P, postnatal day; PBS,

phosphate-buff ered saline; PIPES, piperazine- N-N* -

bis(ethanesulphonic acid); TH, tyrosine hydroxylase;

X-gal, 5-bromo-4-chloro-3-indolyl-â --galactopyranoside

Pergamon

Neuroscience Vol. 80, No. 4, pp. 973–979, 1997

Copyright ?1997 IBRO. Published by Elsevier Science Ltd

Printed in Great Britain. All rights reserved

0306–4522/97 $17.00+0.00

PII: S0306-4522(97)00250-9

973

Fig. 1.

974 G. Sekerkova´ et al.

has an ordered laminar structure (Fig. 1a, c, Refs 6,

13, 24), the homotopic grafts appeared fairly disor-

ganized. Axons of newly regenerated host olfactory

receptor neurons (ORN), which were revealed by

staining with antibody to the olfactory marker

protein (OMP), a unique ORN marker,

19

formed

glomeruli randomly within the transplant (Fig. 1g).

Instead of segregating in layers, donor relay neurons,

clearly identified by the prominent X-gal staining

were scattered throughout the graft (Fig. 1d–h).

Their dendrites invaded OMP-positive glomeruli

(Fig. 1g), suggesting they may contact with host

ORN fibres. Some glomeruli were free from relay

cell dendrites (Fig. 1g) and might be structurally

similar to previously reported ectopic glomerular

structures.

12,21

The outgrowth of labelled axons of donor M/T

cells could be followed through the olfactory ped-

uncle towards the host olfactory cortex (OC) in

a position corresponding to the degenerated host

lateral olfactory tract (LOT) (Fig. 1d). Their

stained axons terminated in lamina 1a of piriform

cortex (not shown). Similar to regenerated fibres

reinnervating the OC after LOT transections in

neonatals,

25

these axon fibres ran in parallel, but

separately from each other instead of forming a

compact bundle.

Numerous â-galactosidase-negative small neurons

in the grafts may represent local circuit neurons

granule and periglomerular cells (Fig. 1e, f). The

latter were identified by immunostaining with anti-

tyrosine hydroxylase (TH) antibody (Fig. 1d, h).

Since the TH immunoreactivity in the periglomerular

neurons depends strictly on the sensory input, this

finding indicates that proper synaptic connections

mediating the signal from the host ORN to the

periglomerular cells in the graft have been restored.

1,9

In the mouse, the migration of periglomerular and

granule cells into the bulb is most intense during the

first week post partum and continues at a slow rate

throughout adult life.

15,16,18

Consequently, the small-

size neuronal population in the grafts is probably

predominantly of host origin.

At the ultrastructural level, labelled cell bodies,

dendritic shafts and dendritic tips of relay neurons

were recognized by the presence of a strong immuno-

reaction for â -galactosidase (Fig. 2 and Fig. 3).

Within the glomeruli, we identified numerous

Fig. 1. Light microscope visualization of normal and transplanted olfactory bulbs (OB) of transgenic

mouse Tg(GAD67lacZ7.5)1

23

: (a) In adult OB, X-gal staining is predominantly localized to mitral

(arrowhead 1) and tufted (arrowhead 2) neurons, confined to distinct layers.

6,13,24

A small population of

granule and periglomerular cells are also stained. (b) E17 transgenic embryo stained for â-galactosidase in

whole-mount after removal of the right OB. The M/T neurons in the left OB (asterisk) and the LOT on

both sides (arrowheads) are strongly stained. The M/T neurons of the ablated bulb obviously undergo

axotomy at the time of transplantation. (c) Section through the OB of an E17 transgenic embryo. The

majority of M/T cells (arrows) are postmitotic at this age and stain for â-galactosidase. Arrows point to

early-formed glomeruli in the presumptive glomerular layer (II). None of the periglomerular or granule

cells are stained. I, olfactory nerve layer; II, presumptive glomerular layer; III, external plexiform layer;

IV, mitral cell layer; V, granule cell layer. (d) An OB transplant (T) derived from a transgenic mouse

formed in place of the removed non-transgenic host OB. The transplanted relay neurons are stained in

blue by X-gal. Arrows point to â-galactosidase-positive fibres in the newly-regenerated LOT. OP,

olfactory peduncule; Te, telencephalon. (e) Sagittal section through a tansplanted bulb (T) stained with

X-gal and counterstained with Neutral Red. The border between transplant and host (arrowheads) is

sharply delineated by densely packed, darkly stained small-size neurons-putative granule/periglomerular

cells. Arrow points to the rostral end of the subventricular zone (SVZ). Asterisks mark lightly-stained

neuropil in the transplant that may contain glomeruli-like structures.

12

Te, telencephalon. (f) Grafted

â-galactosidase-positive neuron with a dendrite (D) terminating (arrow) in a glomerulus (arrowheads)

(counterstained with Neutral Red). (g) â-galactosidase-positive dendrite (D) enters (arrow) an OMP-

positive glomerulus (arrowheads). (h) Sagittal section through a transplant shown in (d) stained with X-gal

and anti-TH antibody. TH-positive periglomerular cells (arrows); D, dendrites of transplanted relay

neurons (arrowheads). Scale bars: a, d and e=500 mm, b=1 mm, c, f, g and h=100 mm. METHODS: Mice

were maintained in a conventional animal facility and all experiments were conducted according to the

statement issued by the Society for Neuroscience (1995). The establishment and maintenance of

Tg(GAD67lacZ7.5)1 transgenic mouse line will be published elsewhere (Z. K. et al., unpublished

observations). E17 mouse embryos (the day of vaginal plug was considered E0) were derived from

time-pregnant B6/CBA females mated to transgenic males. The brains were taken out, meninges carefully

peeled off, OB primordia were dissected and immediately transferred to a glucose–saline medium. The rest

of the brain was stained for â-galactosidase. Meanwhile, non-transgenic neonatal B6/CBA P3–P5 mice

were anaesthetized by hypothermia in crushed ice. A small window was cut over their right OB, the

exposed bulbs were ablated by vacuum suction and immediately replaced by OBs derived from

â-galactosidase-positive brains. Two to four months after surgery the transplanted mice were perfused

transcardially with 2% formaldehyde–0.25% glutaraldehyde in 0.1 M PIPES buff er (pH 6.9).

â-galactosidase histochemistry was performed as described

3

for 3–5 h at 37)C in a medium containing

1 mg/ml X-gal as a substrate in whole-mounts, 15–30 mm cryostat or 50–60 mm Vibratome sections.

Counterstaining was with 1% Neutral Red. After rinsing with phosphate-buffered saline (PBS), the

sections were processed for immunocytochemistry with anti-OMP antibody

19

(generous gift from Dr F.

Margolis) or anti-TH antibody (Boehringer Mannheim) using the avidin–biotin–peroxidase procedure

(Vectastain ABC Elite kit, Vector Labs).

Homotopic olfactory bulb transplatation 975

axodendritic synapses between ORN axons and den-

dritic branches of labelled relay cells and unlabelled

periglomerular cells (Fig. 2a, b). Adjacent to afferent

axodendritic synapses in the glomerulus, we found

synapses between labelled dendrites of relay neurons

and unlabelled dendrites presumably belonging

to short axon and periglomerular cells (Fig. 2b).

Reciprocal synapses between labelled relay cell den-

drites and unlabelled granule cell dendrites (Fig. 3c)

were seen in the graft neuropil outside the glomeruli.

They also occurred on the somata of grafted relay

neurons (Fig. 3b).

In the olfactory peduncle and in the anterior

piriform cortex myelinated and non-myelinated

Fig. 2. Dendritic tips (arrows) of transplanted relay neurons within glomerular arrays. (a) Olfactory

receptor axons (asterisks) of host origin identified by their darker cytoplasm and high density of round

synaptic vesicles contact the dendritic profiles. N, presumed periglomerular neurons. D, â-galactosidase-

negative dendrites. bv, blood vessel. (b) Detail from a glomerulus showing electron-dense dendritic tip

(Dt) postsynaptic (arrowhead) to an olfactory receptor axon (At). The arrow points to postsynaptic

densities of a synapse between the labelled dendritic terminal (Dt) and â-galactosidase-negative den-

drite (D). Scale bars: a=2 µm, b=0.4 µm. METHODS: 40mm-thick Vibratome sections were immuno-

stained with anti-â-galactosidase monoclonal antibody (Promega) using the avidin–biotin–peroxidase

procedure and daiaminobenzidine as chromogen. After staining the sections were postfixed overnight in

a mixture of 2% formaldehyde and 2% glutaraldehyde in PBS, washed several times in PBS and treated

with 1% sodium borohydrate in PBS for 30 min. After osmification, sections were dehydrated and

embedded in Durcupan (Fluka). Ultrathin sections were cut and observed under Zeiss electron

microscope.

976 G. Sekerkova´ et al.

Fig. 3. Electron migrographs of transplanted, â-galactosidase-positive relay cells. (a) Dense immuno-

reaction product marks the perikarya (asterisk) and dendrite (D

1

) of a relay neuron. Granule cells (Gc) are

â-galactosidase-negative. D2, â-galactosidase-negative, and D3, â-galactosidase-positive dendrites of

nearby relay neurons. Nu, nucleus. (b) Dendrosomatic contacts of transplanted relay cell and gemmules

(g) of granule cell dendrites; arrowheads indicate postsynaptic densities. Nu, nucleus. (c) Dendrodendritic

contacts between a â-galactosidase-positive shaft and gemmules of granule cell dendrites (g). Arrowheads

indicate individual synapses and arrows indicate reciprocal synapses. Symbols point to postsynaptic

densities. m, mitochondria. Scale bars: a=2 µm, b and c=0.4µm.

labelled axons were found (Fig. 4a). Their terminals

made synaptic contacts with dendrites of pyramidal

neurons in lamina 1a and 1b of the piriform cortex

(Fig. 4b, c).

The synaptic arrangements observed in the graft

indicate that typical local circuits

4,6,7,13,24

are estab-

lished between transplanted M/T neurons and peri-

glomerular and granule cells, derived largely from the

host. It is known, that the M/T-to-granule and

granule-to-M/T dendrodendritic synapses in the EPL

are formed much later than ORN-to-M/T or ORN-

to-periglomerular synapses in the glomerular layer

and this takes place only after M/T axons have

reached their proper targets in the primary OC.

4,7,17

Thus, the presence of reciprocal dendrodendritic syn-

apses between labelled relay neurons and granule

cells is another indication that a number of grafted

relay neurons have made proper synaptic contacts in

the primary OC.

Our results confirm previous findings about stable

integration and long-term postoperative survival of

fetal OB grafts transplanted in neonatal mice after

complete unilateral bulbectomy. In agreement with

previous data,

14,27,30

we have found axons of trans-

planted relay neurons terminating in the primary OC.

Furthermore, making use of the cell-autonomous

expression of â -galactosidase by grafted M/T neu-

rons, we could show at the ultrastructural level that

the formation of synaptic connections between trans-

planted M/T cells and neurons of the host olfactory

system can occur with a high degree of specificity

demonstrated by the restoration of the characteristic

synaptic circuitries at both their dendritic and axonal

ends.

This exceptional capacity for specific reintegra-

tion of transplanted olfactory relay neurons into

the host olfactory system is probably influenced

by, but not solely dependent on mechanisms that

guide the formation of glomeruli, since the latter

can be induced by ORN in the absence of relay

neurons.

12,20

It may be in part related to the

special wiring of M/T cells, which transmit pri-

mary sensory signals directly to the cortex without

an obligatory relay in the thalamus.

6,13,24

The

OC is thought to provide guiding cues during

development and maturation of the LOT,

17,26

a

process which is especially intensive during the

time of transplantation (P3–P5). Although elusive

at present, these signals might be similar in nature

to the molecular-guidance cues operating during

regeneration of the retinotectal projections.

2

Future experiments could be designed, based on

the present and/or similar model systems, that

would help their identification. In combination

with electrophysiological and behavioural studies,

this approach may also allow the functional re-

covery in the regenerated olfactory system to be

evaluated.

Acknowledgements—The authors are greatly indebted

to Mrs Annelies Wolff, Mrs Ildiko´ Harmos and Martha

Synekova´ for excellent technical assistance. This research

was supported by the Hungarian Research Fund, OTKA

T-14645, F-013104, T006373 and T016971 (to G. Sz. and

Z. K.), NIH grant DC No. 01805 (to E. M.), grants from

the Deutsche Forschungsgemeinschaft, SFB 406 and

Volkswagen Stiftung No. I-7-777 (to J. W.) and partially

from grant No. 1319 of the Slovak Academy of Sciences

(to G.S.).

Fig. 4. â -galactosidase-positive axonal profiles from the

olfactory peduncle (a) and layer 1a of the piriform cortex (b,

c). (a) Small myelinated axon (A). (b) Synaptic boutons

(At) marked by the presence of synaptic vesicles. Arrow-

head, postsynaptic density on a dendritic shaft (D). (c)

Presynaptic bouton (At) synapsing on a dendritic spine (S).

Arrowhead, postsynaptic density. Scale bars: a, b=1 µm,

c=0.2 µm.

978 G. Sekerkova´ et al.

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(Accepted 21 May 1997)

Homotopic olfactory bulb transplatation 979

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme β-galactosidase (β-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X-gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully—with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls—then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial β-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli β-gal activity.

  • Anthony J. Castro
  • Jens Zimmer Jens Zimmer

Since the advent of modern neurotransplantation research about two decades ago, considerable attention has focused on its potential use in the treatment of neurodegenerative disorders, particularly Parkinson's disease. However, although clinical application is limited to a few medical centers, other pure experimental studies have added considerable knowledge of the basic principles of development and plasticity of the central nervous system (CNS). This is evident in studies involving neural grafting into adult, as well as newborn, recipients. However, neural grafting into the developing CNS is by nature particularly suited for addressing fundamental questions of nervous system growth and development, because it relates for example to neuronal cell division, differentiation, and migration, as well as to axonal elongation and axon-target interactions, trophic mechanisms, and neural plasticity and repair. Clinical application of neuronal transplantation in the newborn is not imagined in the near future, although nervous system damage remains the most common birth defect (e.g., hypoxic-ischemic encephalopathy occurs in approx 2–4 neonates/1000 liveborn at term), but experimental neurotransplantation provides intriguing data regarding repair of the damaged neonatal brain and spinal cord.

Alternative splicing of the metabotropic glutamate receptor 1 (mGluR1) receptor gene generates two major receptor isoforms, mGluR1a and mGluR1b, differing in intracellular function and distribution. However, little is known on the expression profiles of these variants during development. We examined the mRNA expression profile of mGluR1a/b in microdissected layers and acutely isolated mitral cells in the developing mouse olfactory bulb. This analysis showed that the two mGluR1 variants are differentially regulated within each bulb layer. During the first postnatal week, the mGluR1a isoform replaces GluR1b in the microdissected mitral cell layer (MCL) and in isolated identified mitral cells, coinciding with a developmental epoch of mitral cell dendritic reorganization. Although mGluR1a mRNA is expressed at high levels in both the adult external plexiform layer (EPL) and MCL, Western blotting analysis reveals a marked reduction of the mGluR1a protein in the MCL, where mitral cell bodies are located, and strong labeling in the EPL, which contains mitral cell dendrites. This suggests that there is increased dendritic trafficking efficiency of the receptor in adult. The temporal and spatial shift in mGluR1b/a expression suggests distinct roles of the mGluR1 isoforms, with mGluR1b potentially involved in the early mitral cell maturation and mGluR1a in dendritic and synapse function.

Olfactory bulb (OB) transplantation is a well characterized model that has been widely used for studying neuronal plasticity and regeneration [G. Sekerková, Z. Katarova, E. Mugnaini, F. Joó, J.R. Wolff, S. Prodan, G. Szabó, Intrinsically labeled relay neurons of homotopic olfactory bulb transplants establish proper afferent and afferent synaptic connections with host neurons, Neuroscience, 80 (1997) 973-979 [10]; G. Sekerková, Z. Malatová, J. Orendácová, T. Zigová, Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study, Restor. Neurol. Neurosci., 6 (1993) 1-8 [11]; E. Raceková, I. Vanický, T. Zigová, Correlation of functional alteration with lesion extent after olfactory bulbectomy in rats, Int. J. Neurosci., 79 (1994) 13-20 [12]; T. Zigová, P.P.C. Graziadei, A.G. Monti Graziadei, Olfactory bulb transplantation into the olfactory bulb of neonatal rats: an autoradiographic study, Brain Res., 539 (1991) 51-58 [13]]. In previous studies, the OB grafts have been routinely labeled by tritiated thymidine [S.M. Onifer, L.A. White, S.R. Whittemore, V.R. Holets, In vitro labelling strategies for identifying primary neural tissue and neuronal cell line after transplantation in the CNS, Cell Transplant., 2 (1993) 131-149 [7]; [13]] allowing distinction of graft from the surrounding tissue by the presence of silver grains over the cell nuclei of the transplant. However, this approach has some disadvantages, namely: partial or insufficient labeling of a defined neuronal subclasses due to the length of the period of their generation, variation in the number of labeled cells due to differences in the gestation stage between individual embryos at the time of i.p. injection of tritiated thymidine, inability to follow the dendritic arborization and axonal outgrowth of the transplanted neurons or to detect directly their actual synaptic contacts, and finally, the need to work with radioactive isotopes. In this paper, we describe an alternative approach, in which the donor OBs in a homotopic OB transplantation were derived from transgenic mice carrying the bacterial gene lacZ under control from the regulatory region of GAD67 gene. In these mice, beta-galactosidase (beta-gal), encoded by lacZ is stably, ectopically expressed in the vast majority of mitral/tufted (M/T) cells of the OB and served as their intrinsic cellular marker in the OB transplant. By using a simple histochemical reaction for beta-gal or immunocytochemistry with anti-beta-gal antibody, we could detect the cell bodies and processes of the donor M/T cells and their synaptic contacts with host neurons after long-term survival using both light and electron microscopy. Given the great number of existing transgenic mouse lines that express in the nervous system, this approach may have an even wider application in neural transplantation.

  • Eiki Takahashi
  • Norimasa Miyamoto
  • Noriko Kajiwara
  • K. Yagami

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of beta-galactosidase activity.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous peptide agonist for the opioid receptor homolog, N/OFQ receptor, and serves for the central control of autonomic functions. Morphological details including the cell types that may account for such N/OFQ functions, however, remain unclear. By using X-gal histochemistry for the detection of receptor-expressing cells at both light and electron microscopic levels, we examined the hypothalamus from the receptor-deficient mice bearing a lacZ insertional mutation in the N/OFQ receptor gene. The N/OFQ receptor reflected by lacZ expression was seen at high levels in the anterior hypothalamic area. With electron microscopy, lacZ expression was observed in a subset of neurons showing large cell size and indented nucleus.

  • G. A. Monti Graziadei
  • P.P.C. Graziadei

The vertebrate CNS acquires the morphology observed in the adult animal by selective differentiation of groups of neurons, occurring at specific times during embryogenesis, and by subsequent establishment of specific neuronal connections.1–4

  • Rochelle K. Small
  • Christiana Leonard Christiana Leonard

Details of neuronal reorganization were investigated in golden hamster pups 10 days after neonatal olfactory lesions. Pups sustained unilateral olfactory tract section (ULOT) or unilateral olfactory bulbectomy (UOB) on postnatal day 5 (P5) and were then behaviorally tested through P15. UOBs were sacrificed after P15 to assess the extent of lesions. ULOTs were processed with degeneration techniques to demonstrate the terminal distribution of bulb fibers whose path had been either deflected or severed by the early tract section.

  • P.P.C. Graziadei
  • R R Levine
  • G. A. Monti Graziadei

We removed the right olfactory bulb in neonatal mice, leaving the bulb on the left side intact as an internal control. At 5 days of survival time, we observed that the right cerebral hemisphere was displaced forward to occupy the region made vacant by removal of the bulb. The frontal cortex was, consequently, in close proximity to the lamina cribrosa. As a result of bulb ablation and severance of the fila olfactoria, the sensory perikarya underwent total retrograde degeneration, which peaked at 8 days. New neurons differentiated in the neuroepithelium from basal stem cells and, at 30 days of survival, mature sensory neurons were reconstituted. These new elements sent their axons through the lamina cribrosa to reach the protruding cerebral hemisphere, penetrating it and forming glomeruli-like structures directly in the host tissue. The "glomerulization" of the sensory fibers persisted and actually expanded between 60 and 120 days. The new glomeruli were organized intimately within the brain tissue, and large neurons of the cortex were observed to be in close proximity. Ultrastructural observations of the newly formed glomeruli demonstrated that typical sensory axon terminals profusely branched and synapsed with unidentified postsynaptic processes that penetrated the glomeruli from the surrounding cerebral tissue.

  • P.P.C. Graziadei
  • R R Levine
  • G. A. Monti Graziadei

Neonatal mice underwent unilateral bulbectomy, which included the main and accessory olfactory bulbs. From 5 days of survival onward, there was a marked anterior displacement of the frontal cortex into the cavity previously occupied by the bulb. As a result of the bulbectomy and consequent damage to olfactory axons, the olfactory perikarya underwent retrograde degeneration. New neurons were then reconstituted from stem cells within the olfactory neuroepithelium. By 20 postoperative days the new olfactory axons had reached the level of the lamina cribrosa and by 30 days the fibers had penetrated into the telencephalon and had formed typical glomerular structures within the host tissue. Fibers were directed to either the paleo- or neocortex where they were observed in close proximity to large cortical neurons. The formation of glomeruli persisted over the course of the study (180 days) and showed an expansion within the cortical tissue up to 60 days of survival. The identification of these fibers and glomerular structures as olfactory was confirmed by immunohistochemical techniques using antisera to the specific olfactory protein. Ultrastructural observations clearly indicated the typical glomerular pattern of the structures and demonstrated synaptic contacts between the sensory terminals and dendritic processes, as yet unidentified, originating from the surrounding cerebral matrix.Our observations thus demonstrate that following bulbectomy and retrograde degeneration of olfactory neurons, the cells can regenerate in the absence of their normal target. Furthermore, the newly formed axons can penetrate a 'foreign' environment, the cerebral cortex, and form typical glomerular structures and corresponding sensory synapses. The findings suggest a heretofore unsuspected degree of plasticity in the olfactory system as well as in the cerebral cortex.

  • T Zigova
  • P.P.C. Graziadei
  • Ariella G. Monti-Graziadei

After unilateral bulbectomy in neonatal (P1-P5) rats, autoradiographically prelabeled presumptive olfactory bulbs from E15 and E17 embryos were transplanted in place of the removed tissue. After 2-7 months, the animals received injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the piriform cortex. Nine of the twenty animals revealed WGA-HRP-positive neurons among neurons autoradiographically labeled, providing thus evidence that the axons of the output neurons from the homotopically transplanted olfactory bulb reconnect with the host piriform cortex.

  • Ariella G. Monti-Graziadei
  • P.P.C. Graziadei

In this study, in order to provide the anatomical basis for future behavioral and electrophysiological experiments, we describe the effects of unilateral bulbar lesion on the peripheral sensory neurons and the parameters of reinnervation of the damaged olfactory bulb. Neonatal mice and rats were subjected to removal of portions of the olfactory bulb. At survival times from 2 to 6 months, the animals were killed by transcardial perfusion and processed for light (histological, immunohistochemical, autoradiographic) and electron microscopic observations. As a result of this surgery, in the basal layer of the olfactory neuroepithelium the rate of mitotic activity increased while the number of mature olfactory neurons was greatly reduced. The regrowing olfactory axons, by forming ectopic glomerular structures in the damaged target, profoundly influenced its reorganization. The typical layered morphology of the olfactory bulb was often disrupted in the bulbar remnant; the large dendrites of the deafferented mitral cells bent toward the ectopically located glomerular structures establishing numerous synaptic contacts. The results from this study indicate that the olfactory input plays an important role in the reorganization of the damaged olfactory bulb. Behavioral experiments in partially bulbectomized animals should provide essential information about the importance of a topological map in the processing of olfactory cues.