Long distance signalling in volume transmission. Focus on clearance mechanisms

L. E Agnati, K. Fuxe, C. Nicholson and E. Sykov~i(Eds.) Progressin BrainResearch,Vol 125 © 2000 Elsevier Science BV. All fights reserved.

CHAPTER

26

Long distance signalling in volume transmission. Focus on clearance mechanisms Anders Jansson 1., Andrea Lippoldt 2, Tomas Mazel 3, Tamas Bartfai 4, Sven-Ove Ogren l, Eva Sykov~i3, Luigi F. Agnati 5 and K. Fuxe 1 1Department of Neuroscience, Karolinska lnstitutet, 171 77 Stockholm, Sweden 2Centrum fiir Molekulare Medizin, 13122 Berlin-Buch, Germany J3Department of Cellular Neurophysiology, Joint Institution of ASCR and Charles University, Institute of Experimental Medicine ASCR, 142 20 Prague 4, Czech Republic 4F. Hoffmann-La Roche Ltd, Department: PRPN, Building. 69/415, CH-4070 Basel, Switzerland 5Department of Human Physiology, University of Modena, 41100 Modena, Italy

Introduction There now exists substantial evidence that large numbers of VT signals such as transmitters and modulators travel in the microenvironment of the extracellular space (Fuxe and Agnati, 1991; Zoli et al., 1999). It then becomes important to study through which mechanisms these VT signals are cleared from the extracellular environment and if they can sometimes reach the ventricular system to act also as CSF signals. It is well-known that catabolic enzymes exist in the extracellular space and in glial and neuronal membranes facing the extracellular space for various transmitters, such as monoamines and neuropeptides (Schwartz et al., 1991; del Rio-Garcia and Smyth, 1991). These enzymes play an important role in the clearance of VT signals together with the also well-known reuptake mechanisms for various transmitters such as DA, NA and 5-HT located in the entire neuron (terminals, dendrites, axons) at the membrane level but outside the synapses. The efficient recapture of some transmitters into their source neurons when *Corresponding author: Tel: 46-8-728 7028; fax: 46-8-33 79; e-mail: [email protected]

they reach the extracellular fluid is an important regulator of volume transmission (VT) (Giros et al., 1996). Glial cells also play an important role in clearance of VT signals especially ions such as K ÷ (see SykovL Chapter 6, this volume). In this chapter we will, however, mainly focus on other clearance mechanisms of VT signals, namely clearance over the brain-blood barrier and over the leaky brain-CSF interface as well as through receptor mediated uptake into other discrete nerve cell systems through internalisation (Fig. 1). The brain-CSF interfaces Recently, Lippoldt and colleagues (Lippoldt et al., 2000) have analysed the tight and adherens junctions in the ependymal cells and in the choroid plexus epithelium, since diffusion of solutes from the brain microenvironment into the ventricles would facilitate clearance and thus contribute to avoid VT signal overflow. Furthermore, this passage of chemical messages into the CSF is an important step for the endocrine-like type of VT (see Agnati and Fuxe, Chapter 1, this volume). It is known that the ependymal cells only form a leaky

400

Clearance through the brai n-cerebrospinal leaky barrier

Clearance through the brain-blood barrier

/

Cellscource

/

Celltarget

\

Fig. 1. Illustrationof clearance mechanismsfor VT signals in the CNS that may involvenot only enzymaticbreakdownand uptake in the cells but also clearance over the brain-blood barrier and the brain-CSF interface. barrier at the brain-CSF interface (Peters and Swan, 1979; Rodriguez and Bouchaud, 1996). The epithelial cells of the choroid plexus instead form a true barrier between blood and CSF and are essential for the chemical homeostasis of the CSF (Brightman and Reese, 1969; Dermietzel, 1975; Dermietzel et al., 1977; van Deurs and Koehler, 1979; van Deurs, 1980; Mack et al., 1987; Dziegielewska et al., 1988; Dermietzel and Krause, 1991; Saunders et al., 1999). At barriers tight and adherens junction molecules are essential. For adherence of cells the cadherins and catenins are necessary, while for tightness molecules such as ZO-1, occludin and claudins appear responsible (Furuse et al., 1993, 1998a, b; Anderson et al., 1993; Anderson and van Itallie, 1995; Dejana et al., 1995; Lampugnani and Dejana, 1997; Mitic and Anderson, 1998; Tsukita and Furuse, 1998, 1999; Morita et al., 1999). In the study of Lippoldt et al. (Lippoldt et al., 2000) adherens junction molecules were demonstrated both between the tight epithelial cells of the plexus choroideus and between leaky ventricular ependymal cells. The tight junction molecules ZO-1, claudin and occludin were present in substantial amounts between the epithelial cells but less so between the ependymal cells in line with the existence of an ineffective barrier in the ependymal cell layer.

The most interesting results were obtained when the phorbolester phorbol myristate acetate (PMA) was given intraventricularly (1.5 Ixg) 24 hr earlier. PMA is known to open up cell-cell contacts (Lombardi et al., 1987; Balda et al., 1991, 1993; Wolburg et al., 1994; Citi and Denisenko, 1995; Stuart and Nigam, 1995; Kaya et al., 1996; Mullin et al., 1996, 1997) and its influence on the adherens and tight junctions were studied immunocytochemically and changes in permeability were studied by evaluating effects on dextran (mol. wt. 3000) diffusion microinjected into the striatum (Jansson et al., 1999b; Lippoldt et al., 2000). As seen in Table 1, the spread of biotinylated dextran (mol.wt. 3000) can be measured within the rat brain parenchyma by image analysis using microdensitometrical (discrimination) and morphometrical procedures, so that total volume of spread and overall specific mean grey value (intensity) can be determined. The biotinylated dextran is a marker for the extracellular space and a streptoavidineHRP staining procedure was used with DAB as chromogen (see Fig. 2). The PMA was found to profoundly reduce the immunoreactivity for the adherens junction proteins cadherin and 13-catenin but not for a-catenin between the ependymal cells as well as between the plexus choroideus epithelial cells (Fig. 3). Fur-

401 TABLE 1. Time-course and dose-response for spread of biotinylated dextran (mol wt 3000) microinjected into the neostriatum: microdensitometrical and morphometrical parameters. Volume Vo (mm3) Time (min)

2 30 60 120

Dose (p~g/~l)

1 10 30 100

Mean grey value (MGV)

16_+2

30+_3

43 _+6" 56-+3" 66-+ 15a

30 +-2 25+-1 21 -+2b

20 +-3

24 +-3

21-+4 50_+3 c 54_+8c

24+-2 32+22 28+-1

Fig. 2. The spread of biotinylated dextran (mol. wt. 3000; 100 nl, 10 Ixg/Ixl),30 min following intrastriatal injection, detected by the streptavidin-horseradish peroxidase/DAB technique. Two pictures from the same representative section are shown with (B) and without (A) the superimposed discrimination frame used for image analysis area determination (Zeiss/ Kontron IBAS-system).Note also the preferential spread within and especially around the fibre bundles of the capsula interna. Scale bar=500 txm.

Taken from Jansson et al. (1999b). Volume (VD) estimated from the individual specifically stained areas in the sections measured; mean grey value (MGV), the overall median of the mean grey values from the individual specifically stained areas in the sections measured. Data are presented as means + S.E.M. (n = 5 rats in each group). One way ANOVA followed by Fisher's PLSD post hoc comparison was used in the statistical analysis, p <0.05 was regarded as a statistically significant difference. "Different from 2 min; bDifferent from 2 and 30 min; CDifferent from 1 and 10 txg/ixl group; dDifferent from 1 and 10 Ixg/pl groups. Statistically different trends (p < 0.05) were found for V o in the case of the time course and the dose-response.

thermore, the occludin and claudin-5 but not Z O - 1 and claudin-1 immunoreactivities (IR) were markedly reduced by the PMA treatment in the epithelial cells of the choroidal plexus (Figs. 3-5) (Lippoldt et al., 2000). These morphological results clearly indicated that protein-kinase C (PKC) activation can through reduction in both adherens and tight junction molecules reduce barrier functions at the CSF-blood and brain-CSF interfaces. In agreement with the above morphological results PMA (i.v.t.) produced a reduction in the volume of intrastriatal biotinylated dextran spread (m.wt. 3000), which may at least in part be

explained by an increased clearance over the brainblood barrier and brain-CSF interfaces due to reduced barrier and interface functions, probably related in part to impaired junctional properties of the ependymal cells. The results certainly emphasize an important role of PKC in the brain clearance processes and thus in the regulation of the VT. In this way VT may also shift towards an endocrinelike VT involving CSF with i.a. an increased control of the subventricular zone containing e.g. the stem cells (Kuhn and Svendsen, 1999; Peretto et al., 1999). On the role of NO in the clearance over the brain-blood barrier NO is a wellknown lipid soluble VT signal in the nervous system, which therefore diffuses both intra- and extracellularly to reach its intracellular target, namely guanylylcyclase, leading to increased c G M P formation and altered protein phosphorylation levels. NO possesses a half-life in the order of 0.5-5 sec but in view of its fast diffusion it may diffuse for distances in the order of 100 ~m, modulating a volume that may contain up tO 10 6 synapses (Wood and Garthwaite, 1994). Nitric oxide synthase (NOS) is the enzyme forming

402

Fig. 3. Acute i.v.t. PMA-injections led to marked changes in adherens and tight junction marker immunoreactivities. The 13-catenin immunoreactivity was markedly reduced between the epithelial cells of the choroid plexus (a) arrows) compared to control injected animals (b). Cadherin immunoreactivity was also markedly reduced after acute PMA injection between the ventricular wall ependymal cells (c) thin arrows) but even more pronounced between the borders of the epithelial cells of the choroid plexus (c) thick arrows) when compared to the control animal (d). No change of immunoreactivity was seen in PMA-treated (e) compared to control injected animals (f) when the sections were stained with ZO-1. In contrast, occludin immunoreactivity was markedly reduced between the epithelial cells of the choroid plexus after acute PMA-treatment (g) arrows) compared to control injected animals (h). Note the patchy pattern of occludin immunoreactivity in g. Magnification: × 200.

403 NO through enxymatic oxidation of L-arginine to L-citrulline. There exist three different isoforms. The endothelial NOS and neuronal NOS are constitutive and calcium/calmodulin dependent enzymes. The inducible NOS is mainly formed in astroglia and microglia upon activation by inflammatory signals. NO has previously been shown to cause a number of actions on cardiovascular events, including capillary permeability, and on astroglial cells (Jansson et al., 1999b). Therefore, NO may alter the diffusion of other VT signals through effects on clearance over the brain-blood barrier and on extracellular space properties. In the present study we have therefore studied the effects of unspecific and neuronal specific NOS inhibitors on the diffusion parameters of the extracellular space using the tetramethylammon-

ium (TMA ÷) method (see Nicholson, Chapter 28, this volume; Sykov~i et al., Chapter 6, this volume) and the morphological method using biotinylated dextran (M.wt. 3000) spread (see above) (Jansson et al., 1999b). The non-specific NOS inhibitors N~-nitro-L arginine methylester (L-NAME) and NG-monomethyl-L-arginine acetate (L-NMMA) produced dose-related (10-100 and 30-200 mg/kg, respectively) reductions in the total volume of striatal spread of dextran (Fig. 6). In contrast, the neuronal specific NOS inhibitor 7-nitroindazole monosodium salt (50-100 mg/kg) did not influence the spread of dextran. The reduction in the volume of the dextran with LNAME and L - N M M A at the 30 min time-interval was 37 and 42% (maximal effect), respectively.

Fig. 4. (a) - (c): Claudin-1 immunoreactivity in the ventricular system of the rat brain. The pictures demonstrate the Claudin-1 punctate appearance in the ependymal cells (thin arrows) of the lateral ventricle (a) as well as staining between the choroid plexus epithelial cells (b) thick arrows). The choroidal capillaries exhibit weak Claudin-1 immunoreactivity(c) thin arrows). (d) and (e): Claudin-5 is another transmembrane protein found in tight junctions. The ependymal cells of the lateral ventricle are not stained for Claudin-5. In contrast, the epithelial cell border areas of the choroid plexus (d) thick arrows) are strongly immunoreactive for Claudin-5, but in contrast to Claudin-1 the appearance is patchy. Strong immunoreactivity is found between the capillary endothelial cells in the choroid plexus (e) thin arrows). Magnification: x 1000.

404

Furthermore, with L-NAME but not with LNMMA treatment (50 mg/kg) the specific mean grey value was reduced by 40%. Studies with the TMA ÷ method demonstrated that non-specific NOS inhibition as described above does not influence volume fraction and tortuosity of the ECS. Instead, the parameter that was altered by L-NAME (50 mg/kg) and L-NMMA (20 mg/kg) was the non-specific uptake parameter (k 1) as determined by the TMA ÷ method (see Nicholson et al., this volume). 13% (L-NAME) and 16% (L-NMMA) increases were observed which correlated with the decreased volume of dextran spread. The impact of the increase in k ~ after LNAME (50 mg/kg) on TMA ÷ diffusion is illustrated in Fig. 7 by showing isoconcentration spheres of TMA ÷, where TMA ÷ reaches 0.1 ~M 10 min after its application in the center of the

sphere. A reduction of the sphere is produced by the L-NAME treatment. As previously reported L-NAME reduced striatal blood flow and increased systemic mean arterial pressure (Jansson et al., 1999b). However, the effects on blood flow cannot explain the reduction in dextran volume spread, since it should rather increase the retention of dextran in the striatum. Also no leakage of blood proteins into the brain could be observed nor of Evans blue after NOS inhibition and after pharmacological treatment (phenylephrine) producing a similar increase of mean arterial pressure (MAP). Thus, the increases in MAP does not cause substantial disruption of the blood-brain barrier. Instead it seems likely that the changes in dextran spread and in non-specific uptake can be explained by increased brain capillary clearance from brain to blood through

Fig. 5. Acute i.v.t. PMA-injections led to an increase in the immunoreactive area and an altered cellular localization of Claudin-1. The immunoreactivity is found to spread into cytoplasmic compartments after PMA treatment (a) arrows) compared to the sham treated animal (b). In contrast, Claudin-5 immunoreactivity decreased markedly after PMA treatment (c) arrows) compared to the control animals (d) arrows). Magnification: x 200.

405

~NAME --

120q

i

100

,

.

.

80

~

~

60 40

0

10

50

1O0

Dose (mg/kg) Fig. 6. Dose-related effects of L-NAME on the spread of biotinylated dextran (mol.wt. 3000), following microinjection into the neostriatum. The total volume (VD) decreased dose-dependently and significantly following L-NAME as did the MGV (p < 0.05). LNAME was given 10 min prior to the dextran microinjection and 30 min after dextran the rats were killed. The graph shows the mean relative change compared to the respective control value _+ S.E.M. (n = 3-5 in each group). Statistical analysis was made with a oneway ANOVA followed by a PLSD test (*: p < 0.05). 100% for VD = 26 mm3; for MGV = 26). From Jansson et al., 1999b).

inhibition of endothelial NOS, neuronal NOS inhibition being without apparent effect. It seems possible that endothelial NO can normally restrict the transport of extracellular transmitters and modulators over the brain capillaries, while NO

A

may not effect diffusion of tracers in the CNS (Fig. 8). Endothelial NO may exert its effects on brain capillary clearance through control of protein phosphorylation processes in junctional proteins (Rubin and Staddon, 1999).

B

'1

II

II Fig. 7. Illustration of neostriatal ECS diffusion parameters, measured with the real-time iontophoretic TMA ÷ method following LNAME treatment (50 mg/kg, i.p.). Isoconcentration spheres are shown representing surfaces, where TMA ÷ reaches an extracellular concentration of 0.1 txM 10 min after its application in the center of the sphere. Mean values of the ECS diffusion parameters (a, h and Kl) before and after L-NAME treatment as well as parameters typical for TMA ÷ measurements were used. The isoconcentration sphere after L-NAME treatment is smaller than the isoconcentration sphere before the treatment (Jansson et al., 1999b).

406

Interleukin 113 as a long-distance endocrine-like VT signal

IL-1 [3 is a very effective messenger in the immune system and is formed in relation to local tissue injury. In the brain its formation leads to fever, CRH release, inhibition of food intake and to the development of sickness behaviour, involving e.g. sleep promotion (Dinarello, 1994; Alheim and Bartfai, 1998). In view of its powerful actions on many neuronal-glial networks once formed and released from activated astroglia, microglia and endothelial cells it seems likely that IL-113 operates as a VT signal in brain that through diffusion and flow may reach and influence the many complex cellular networks involved in the above functions. In line with this view IL-1 [3 extracellular levels are normally kept under rigorous control by the existence of soluble endogenous type I IL-1 receptor (R) antagonists and soluble and membrane

bound type II IL-1R lacking the signalling component and acting as decoy receptors for IL-1 [3. In a series of experiments (Jansson et al., 1997; Jansson et al., 1999a) striatal microinjections of IL-1 [3 have therefore been given in the halothane anaesthetised rat in doses of 5-100 ng in 100 nl and its possible diffusion analysed together with local and distant effects on microglia, FOS immunoreactivity and cfos mRNA levels. The evidence suggests that IL-1 [3 microinjected into the neostriatum of one side can increase its volume several fold over a period of 3 hr in spite of its high m.wt (18 kDa); in this way indications for long-distance diffusion of IL-113 was obtained. Also, not only local microglia responses (Andersson et al., 1992) were observed (3 h) but also microglia activation in the paraventricular hypothalamic nucleus (PVH) bilaterally, where also hypertrophic astroglial cells were found. Furthermore, c-fos mRNA levels were only weakly

OFs

GFs

Ts ~utocrine and oaracrine YT

paracrine YT ~signals

Is~ creased '~ vels of VT~

IIiOlre&se(

perrneabi

NO from eNOS present

)S inhibited

Fig. 8. The concentration of volume transmission (VT) signals present in the extracellular space (ECS) may be regulated by mechanisms controlling the brain to capillary permeability. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) could be one important factor influencing VT signals in the ECS. An inhibition of eNOS may increase the capillary permeability for biotinylated dextran (M.W. 3000). GFs: growth factors, Ts: transmitters: K+: potassium (Jansson, 1999b).

407

increased locally at the site of IL-1 [3 injections but strongly increased in distant regions such as the PVH, suggesting IL- 1[3 CSF signalling. In line with this view sleep has been found to be linked to CSF levels of IL-I[3 (Fang et al., 1998). As indicated in Fig. 9, the activation of IL-1 type I receptors probably also leads to formation of prostaglandins in the meninges, in the ependymal cells, and in the plexus choroideus that contribute to the activation of FOS mechanisms in the cerebral cortex and in the PVH through diffusion via CSF

into these brain areas. The results (Jansson et al., 1997; Jansson et al., 1999a) give rise to the hypothesis that IL-l[3 formed upon injury due, e.g. to inflammation in a local complex cellular network can via long distance diffusion and flow involving also CSF signalling (asynaptic endocrine-like VT, see Agnati and Fuxe, Chapter 1, this volume) exert its global actions on brain with production of fever, CRH release, sleep and and sickness behaviour etc. (for details, see (Jansson et al., 1999a)).

Involvement of CSF signaling in the action of striatal IL-113 on PVH IL-18

:horoideus Type 1 IL-1R1

1 ',activation 2 IL c(

CRF relase

itary gland

Fig. 9. Putative involvement of CSF signalling in the actions of IL-l[3 on the paraventricular hypothalamic nucleus, following its microinjection in the central dorsal striatum. Diffusing IL-1 [3 may reach the ventricles over the brain-CSF interface. Thus, it may go with the CSF flow to the plexus choroideus where it may activate synthesis and release of prostaglandins that in turn via CSF can activate the paraventricular nucleus of the hypothalamus (PVH). CSF IL-1 [3 may also directly effect the PVH through the existence of IL-1 receptors of type I in the microvessels of the PVH. The FOS activation will subsequently lead to the release of CRH (Jansson et al., 1999a).

408

¢

M

k

Fig. 10. Somatodendritic internalization of [3-endorphin after its intraventricular injection (dose: 0.5 nmol in 5 txl; time: 10 min) in the rat into large unidentified paraventricular nerve cell body systems (Agnati et al., 1992). Scale bar: 100 txm; M = medial close to third ventricle. L = lateral.

Internalisation of intracerebrally injected 13endorphin and porcine galanin (1-29) into discrete nerve cell populations of the brain. Possible involvement in CSF signalling

13-endorphin In 1986 Agnati et al. (Agnati et al., 1986), noticed the topographic transmitter-receptor mismatches for 13-endorphin IR nerve terminals and Ix and opioid receptors in several brain regions which led to the suggestion that [3-endorphin was a VT signal. The potential involvement of 13-endorphin in CSF signalling was indicated by the demonstration of high 13-endorphin CSF levels after long distance running which correlated with euphoric actions (Radosevich et al., 1989). In 1992 it was found that intraventricularly injected 13-endorphin in the rat was taken up and intemalised into the somadendritic regions of discrete nerve cell populations of e.g. the paraventricular hypothalamic and preoptic regions and of the hippocampal formation (Fig. 10) (Agnati et al., 1992). Subsequently, it was possible to demonstrate that intraventricular 13-endorphin accumulates in dopamine and cAMP responsive phosphoprotein (DARPP-32) ir tanycytes that may

represent potential transport pathways for CSF 13endorphin to reach distant opioid receptors (Bjelke and Fuxe, 1993). Bjelke (Bjelke, 1994) then demonstrated with intrastriatal [3-endorphin injections that 13-endorphin was internalised into the cytoplasm of DARPP-32 IR negative nerve cell bodies, giving evidence that striatal interneurons (lacking DARPP-32 IR) were selectively involved in this transient process with clearance within 30 min. It was then speculated that these events could represent a type of transcellular signalling, possibly involved in the regulation of gene transcription, but also with potential release of 13-endorphin and/or its fragments into ECF from the dendritic processes and soma of these interneurons to activate striatal opioid receptors in the surround (Fuxe et al., 1994). Strong evidence for 13-endorphin as a CSF signal came from the paper of Duggan's group (MacMillan et al., 1998) demonstrating that upon electrical stimulation of the arcuate nucleus (rich in 13endorphin cells) sharp rises of CSF 13-endorphin level develop together with the appearance of 13endorphin IR in regions such as the cerebral cortex, where very few 13-endorphin ir terminals exist (see Duggan, Chapter 24, this volume). [3-endorphin therefore appears to be a CSF and ECF signal highly suited for long distance signalling with ability to globally affect brain function via the ~z and ~ opioid receptor populations.

Galanin In previous work intraventricular injections of galanin or local microinjections of galanin (1.5 nmol/rat) into the ventral hippocampus have demonstrated the appearance of galanin IR in small to medium sized neurons of the hippocampal formation with rapid labelling of the somatodendritic region through a possible internalisation (Misane et al., 1998; Sch6tt et al., 1998). Recently, this work has been continued with a more detailed analysis of the above galanin internalisation phenomenon in hippocampal nerve cells (Jansson et al., 2000). In this study it could be demonstrated that both intraventricular and intrahippocampal microinjections of galanin (1-29) led to the selective and strong labelling of the same discrete hippocampal nerve cell population (Fig.

409 11). The labelling of these neurons occurs only through internalisation at the somatodendritic level with the dominant localisation in the cytoplasm of these nerve cells. The labelling was transient and disappeared between 20 to 60 min after injection (Fig. 12). A double immunolabelling analysis showed that the intemalisation of galanin (1-29) was mainly restricted to GABA interneurons costoring NPY and/or somatostatin IR (Fig. 13). The galanin intemalisation (1.5 nmol/rat) in these nerve cells appeared to be receptor-mediated, since it was partly blocked by the galanin receptor antagonist M35 (0.5 nmol) as evaluated following i.v.t. injections. Finally a labelling of the same hippo-

campal subpopulation was observed after i.v.t. fluo-galanin (1.5 nmol/rat) indicating that rapid activation of gene expression for galanin was not the explanation for the phenomenon observed. These results open up the possibility that CSF galanin signals can be internalised by this GABA/ NPY/somatostatin interneuronal population where it may exert nuclear actions (gene regulation), possibly explaining the long-lasting behavioural actions of galanin (Ogren et al., 1992). Fanre et al. (Faure et al., 1995) have obtained evidence that neurotensin upon somatodendritic internalisation shows targeting for perinuclear regions. Nevertheless, the exogenous galanin IR was mainly

Fig. 11. Appearance of galanin-IR nerve cell bodies in ventral hippocampus 20 min following i.v.t, injection of 1.5 nmol porcine galanin. The small round galanin-IR nerve cell bodies were present mainly in the CA1 and CA3 regions of the ventral hippocampus. Arrowheads show the correspondingnerve cells in panels A and B. The epithelial cells of the plexus choroideushave become strongly galanin-IR. CAI: CAI field of Ammon's horn. Bregma=-47 mm. Scale= 100 ~m (A) and 50 Ixm (B) (Jansson et al., 2000).

410

Fig. 12. Time course for the appearance of galanin-IR nerve cell bodies in the dorsal hippocampus (CA1 field) on the injected side following unilateral i.v.t, injection of porcine galanin (1.5 nmol/rat). After control injection of aCSF (20 min), only a few punctate galanin IR terminals were found(A).At 10 min (B) or 20 min (C) followinggalanin injections,galanin-IR developedin many smallto medium-sizedrounded cell bodies and some pyramidal-likecells, mainly in the pyramidal and stratum oriens layers. At 60 min, very few if any cell bodies remained galanin-IR. Bregma=-3.6 mm. Scale bar = 50 txm (Jansson et al., 2000). located in the cytoplasm and it must be considered that N-terminal biological active galanin fragments could be rapidly formed extra-and intracellularly from the GABA interneurons to activate the high affinity galanin N-terminal fragment binding sites in the dorsal hippocampal region (Hedlund et al., 1992). This could also contribute to the long lasting actions of galanin seen in vivo.

Conclusions (1) Evidence laas been presented that protein kinase C may be an important regulator of the tight and adherens junctions of the brain-CSF and CSF-blood interfaces as well as of endocrine-like VT. (2) Endothelial NO may be a regulator of the clearance of VT signals over the brain-blood barrier.

(3) Brain interleukin-113, when formed, may be a long distance endocrine-like VT signal, involving also CSF signalling, regulating in this way many neuronal-glial networks such as those for fever, food intake, sleep-wakefulness and sickness behaviours. (4) Evidence has been obtained that exogenous [3endorphin and galanin (1-29) locally and in CSF can undergo rapid somatodendritic internalisation into discrete populations of interneuronal populations of the neostriatum and hippocampus, underlining their potential role in CSF signalling. These results support the possibility that at least in these specific interneurons 13-endorphin and galanin and/or their active fragments can act as transcellular signals by effects on gene regulation and/or by rapid dendritic and somatic release into the

411

Fig. 13. Characterization of the galanin-IR nerve cell bodies appearing in the dorsal hippocampus, 10 minutes following i.v.t, injection of porcine galanin (1.5 nmol/rat) using double-labelling immunocytochemistry. In the first row galanin-IR nerve cell bodies with dendrites are shown (A-D). In panel E, some of the nerve cells are co-labelled with somatostatin-IR (arrows in A and E). The galaninIR nerve cells were sometimes located close to 5-HTzA-IR dendritic processes (arrowheads in B and F) and occasionally the galanin-IR nerve cell bodies were 5-HTzA-IR (arrow in C and G). Some galanin-IR nerve cell bodies (D), were glutamic acid decarboxylase-IR. Bregma =-3.6 mm. Scale bar = 50 p~m (Jansson et al., 2000).

surrounding neuropil to activate their respective high affinity peptide receptors. Overall the present paper indicates that clearance and internalisation processes are important components of VT regulation.

Acknowledgement This work has been supported by a grant (04X715) from the Swedish Medical Research Council.

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